WO2016139217A1 - Composition comprenant un microbiote intestinal humain cultivé en conditions anaérobies - Google Patents

Composition comprenant un microbiote intestinal humain cultivé en conditions anaérobies Download PDF

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WO2016139217A1
WO2016139217A1 PCT/EP2016/054359 EP2016054359W WO2016139217A1 WO 2016139217 A1 WO2016139217 A1 WO 2016139217A1 EP 2016054359 W EP2016054359 W EP 2016054359W WO 2016139217 A1 WO2016139217 A1 WO 2016139217A1
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composition
cultivation
dietary fibers
fibers
anaerobic
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PCT/EP2016/054359
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English (en)
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Jonatan SANTAS GUTIÉRREZ
Jordi CUÑÉ CASTELLANA
Elisabet LÁZARO MALLÉN
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Ab-Biotics, S.A.
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Publication of WO2016139217A1 publication Critical patent/WO2016139217A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • TITLE COMPOSITION COMPRISING ANAEROBICALLY CULTIVATED HUMAN INTESTINAL MICROBIOTA
  • the present invention relates to a method for preparing a composition comprising human intestinal microbiota, wherein the method comprises the step of anaerobic cultivation of a fecal sample from a healthy donor in a cultivation medium, wherein the cultivation is done as a continuous cultivation process and the cultivation medium comprises dietary fibers.
  • the obtained composition may e.g. be used for the treatment of Clostridium difficile infection.
  • the intestinal habitat of an individual contains 300-500 different species of bacteria, and the number of microbial cells within the gut lumen is about 10 times larger than the number of eukaryotic cells in the human body.
  • intestinal microbiota is involved and regulates many metabolic processes.
  • alterations of the microbiota equilibrium are the underlying cause of not only gastrointestinal conditions but also many other metabolic and auto-immune diseases (e.g. obesity, allergies, autism, etc).
  • Clostridium difficile an anaerobic, Gram-positive bacterium, is a major cause of antibiotic-associated diarrhea and challenges healthcare infection control measures by producing highly infectious and resistant spores.
  • C. difficile colonization leading to a spectrum of outcomes ranging from asymptomatic carriage, severe diarrhea, pseudomembranous colitis or even death.
  • Current first line treatments for C. difficile disease are vancomycin or metronidazole, although in 20-35% of these cases recurrent disease (relapse or reinfection) follows the cessation of antibiotic therapy.
  • Recurrent C. difficile disease is associated with a pathological imbalance within the resident intestinal microbial community, or "dysbiosis", so therapies that restore a healthy microbiota are viewed as promising alternatives.
  • Fecal bacteriotherapy (alternatively termed fecal transplantation), which relates to the administration of homogenized feces from a healthy donor, has been investigated as an alternative therapy for recurrent C. difficile disease in humans (Nood et al. 2013).
  • Fecal transplantation is a known method, but not without risk. Until now fecal material from donors of close relatives has been used. A main problem with this procedure is the risk of transmission of genes resistant to certain antibiotics. The transfer of such genes may have serious consequences for the patient when antibiotic treatment is needed in the future. Another main problem with this therapy is the risk of
  • the article of Kim et al (201 1 ) essentially relates to testing of different fecal microbiota culture conditions (i.e. different cultivation media/conditions) in order to obtain a cultivated sample with a microbiota that as best as possible corresponds to the fecal microbiota.
  • the article reads: "We tested a variety of conditions for the human intestinal microbiota growth in short-term in vitro batch cultures. The combination of DGGE, real-time PCR, and pyrosequencing was sufficient to compare communities of intestinal microbiota in the different cultures.
  • low-concentration carbohydrate medium supplemented with 1 % fecal supernatant and inoculated with a fecal suspension to a final concentration of 3% performed best in maintaining a metabolically active diverse population of bacteria over the 18 hour incubation.
  • the article further reads: "The dominant phyla
  • WO2013171515A1 relates to identification of bacteria present in fecal samples that could be suitable for use in bacteriotherapy of C. difficile related diseases.
  • This document describes mice related experimental data. To identify candidate bacteria they passaged mice healthy feces overnight in nutrient broth at 37°C to reduce the community complexity and to enrich for readily culturable bacteria and the documents says that results confirmed the presence of culturable bacteria within the microbiota of healthy mice that can suppress C. difficile infection as effectively as whole fecal bacteriotherapy.
  • Actinobacteria and Proteobacteria were tested individually and in different compositions and the document says that they rationally identified simple mixture (termed “MixB") consisting of six readily culturable intestinal bacterial strains that can cure C. difficile infection in mice. Based on further analysis, they determined that the MixB mixture includes three previously described species, Staphylococcus warneri, Enterococcus hirae, Lactobacillus reuteri, and three novel species, Anaerostipes sp. nov, Bacteroidetes sp. nov. and Enterorhabdus sp. Nov.
  • WO2013053836A1 also relates to identification of bacteria present in fecal samples that could be suitable for use in bacteriotherapy of C. difficile related diseases (see e.g. page 9, lines 30-34).
  • This document describes human related experimental data.
  • Working Example 1 describes that anaerobically re-cultivated human intestinal microbiota were obtained by anaerobic cultivation of a stool (feces) sample (from a healthy donor) in a cultivation medium. The microbiota obtained from the cultivated feces sample were analyzed and it was found that it contained anaerobic bacteria of the following four Phyla: Bacterioidetes, Firmicutes, Proteobacteria and
  • Actinobacteria i.e. the four phyla that constitute the majority of the mammalian intestinal microbiota as mentioned in e.g. WO2013171515A1 and Kim et al discussed above.
  • Working Example 3 describes that the cultured fecal sample of Example 1 was given rectally to 32 patients, wherein at least one had Clostridium Difficile infection and out of the 32 patients were 22 patients cured.
  • One problem to be solved by the present invention may be seen as related to the provision of an improved method for cultivation of a fecal sample to thereby be able to obtain a cultivated composition with a microbiota that as best as possible corresponds to the natural fecal microbiota of a healthy donor.
  • composition with a microbiota that as best as possible corresponds to the natural fecal microbiota of a healthy donor may be useful for e.g. treatment of Clostridium difficile infection.
  • the solution is based on that an improved "copy" of the fecal microbiota may be obtained by:
  • (b) use of a cultivation medium that comprises dietary fibers and/or pre-treated dietary fibers.
  • an improved "copy" of the fecal microbiota relates to a cultivated composition with a microbiota that corresponds better/closer to the natural fecal microbiota than a less good "copy” of the fecal microbiota.
  • a first aspect of the invention relates to a method for preparing a composition comprising anaerobic human intestinal microbiota, wherein the method comprises the steps of:
  • the cultivation medium of step (i) comprises dietary fibers and/or pre-treated dietary fibers, in a concentration of from 0.5 to 100 g/L of the cultivation medium, wherein the pre-treatment is with an acid solution, with enzymes and/or with bile salts.
  • human healthy donor relates herein to a human donor that does not have a herein significant relevant disease.
  • a human healthy donor is a person that does not have a clinical relevant Clostridium difficile infection. Preferred requirements of a human healthy donor are discussed further details below.
  • dietary fibers is well known and understood by the skilled person in the present context.
  • dietary fibers relates herein to the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine, with complete or partial fermentation in the large intestine.
  • Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances.
  • obligate and facultative anaerobic bacteria is well known and understood by the skilled person in the present context.
  • Obligate or strict anaerobes are microorganisms that are killed by normal atmospheric concentrations of oxygen (21 % 0 2 ).
  • Some species, called facultative anaerobes are able to grow either with or without free oxygen.
  • a facultative anaerobe is an organism that is capable of switching from respiration with oxygen to fermentation or anaerobic respiration if oxygen is absent.
  • the method of the invention allows the growth of both obligate and facultative anaerobic bacteria.
  • a second aspect of the invention relates to a composition
  • a composition comprising anaerobic human intestinal microbiota, wherein the composition comprises obligate and factultative anaerobic bacteria of at least three of the following four Phyla:
  • a third aspect of the invention relates to a composition of the second aspect and/or herein described embodiments thereof, for use in the treatment of a condition related to intestinal dysbiosis, such as e.g. for use in the treatment of Clostridium difficile infection.
  • the third aspect be formulated as a method for treating a condition related to intestinal dysbiosis (such as e.g. Clostridium difficile infection), comprising administering to a human or animal a composition of the second aspect and/or herein described embodiments thereof.
  • a condition related to intestinal dysbiosis such as e.g. Clostridium difficile infection
  • a fourth aspect of the invention relates to a composition of the second aspect and/or herein described embodiments thereof, for use in the treatment of antibiotic- associated diarrhea (AAD), C. d/ ' ff/ ' c/Ve-associated diarrhea (CDAD),
  • AAD antibiotic- associated diarrhea
  • CDAD C. d/ ' ff/ ' c/Ve-associated diarrhea
  • the fourth aspect be formulated as a method for treating antibiotic- associated diarrhea (AAD), C. d/ ' ff/ ' c/Ve-associated diarrhea (CDAD),
  • First aspect - A method for preparing a composition comprising anaerobic human intestinal microbiota
  • step (i) of the method of the first aspect reads:
  • Bacterioidetes, Firmicutes, Proteobacteria and Actinobacteria there is in this step (i) proliferation of the anaerobic bacteria of at least the following four Phyla: Bacterioidetes, Firmicutes, Proteobacteria and Actinobacteria.
  • Anaerobic cultivation as such is well known to the skilled person and it is therefore not believed to be necessary to discuss this in great details herein.
  • - anaerobic cultivation may e.g. be made by fluxing nitrogen or using oxygen scavengers.
  • Fecal sample can be obtained from a pool of volunteers or a single volunteer. It may be preferred that the fecal sample is from a single human healthy donor. More preferably, the fecal sample is obtained from a donor selected from a plurality of donors by using different criteria for assessing its suitability based on their biographic information, clinical history or dietary habits. In particular, the donor is selected by using characteristics according to their biographic information, clinical history and dietary habits.
  • the fecal sample provided by the donor can be directly used 'as is' for cultivation as described in the first step of the first aspect of the invention. Alternatively, the fecal sample can be treated or processed prior to is use for cultivation. Non-limiting examples of processing include blending, filtering, shieving, and/or freezing.
  • the sample can be treated for displacing undesired organisms.
  • the cultivation medium of the method of the first aspect is characterized by that it comprises dietary fibers and/or pre-treated dietary fibers in a certain defined amount. Beside the presence of the essential dietary fibers - the other ingredient of the cultivation medium may e.g. be ingredients present in a suitable cultivation medium as described in the prior art. Growth media comprises a nutritive media
  • the nutritive media contains a source of inorganic compounds or minerals, vitamins, a carbon source and a protein source.
  • growth media is composed by potato starch (3 g/L), glucose (0.4 g/L), yeast extract (3 g/L), peptone (1 g/L), mucin (4 g/L) and cysteine (0.5 g/L) in distilled water.
  • WO2013053836A1 described a suitable cultivation medium comprising pre-reduced sterilized bacteriological peptone yeast based medium (Difco, USA), with addition of 1.25 % (w/v) freeze dried hen yolk
  • a suitable cultivation medium comprising low-concentration carbohydrate medium (LCM) supplemented with 1 % fecal supernatant and inoculated with a fecal suspension to a final concentration of 3%.
  • LCM low-concentration carbohydrate medium
  • step (a) the cultivation in step (i) is done in as a continuous cultivation process in a continuous anaerobic fermenter using a retention time between 5 and 168 hours;
  • the cultivation medium of step (i) comprises dietary fibers and/or pre-treated dietary fibers, in a concentration of from 0.5 to 100 g/L of the cultivation medium, wherein the pre-treatment is with an acid solution, with enzymes and/or with bile salts.
  • the pre-treatment of the dietary fibers is done in a way that mimics the conditions of the gastrointestinal tract.
  • the pre-treatment is with an acid solution that mimics the conditions of the gastrointestinal tract, with enzymes that mimics the conditions of the gastrointestinal tract and/or with bile salts that mimics the conditions of the gastrointestinal tract.
  • the concentration of dietary fibers and/or pre-treated dietary fibers is maintained within the concentration of from 0.5 to 100 g/L of the cultivation medium during at least 90% (more preferably at least 95% and even more preferably at least 99%) of the period of time of cultivation in step (i) of the first aspect.
  • the retention time (RT) in the fermenter is in a range between 12 to 86 h, more preferably between 12-76 h, and more preferably, RT is 24 h.
  • the cultivation is done for a period of time from 12h to 30 days.
  • the cultivation time is between 24h and 21 days.
  • pH is in a range of from 5.5 to 7.5 (preferably wherein pH is in a range of from 6.0 to 7.0).
  • the fibers in item (b) are in a concentration of from 1 to 20 g/L of the cultivation medium, more preferably are in a concentration of from 5 to 6 g/L of the cultivation medium.
  • the dietary fibers are at least of the types of dietary fibers selected from the group consisting of: hemicelluloses (arabinoxylans, xyoglucans, glucomannans, galactomannans, beta-glucans), pectins (homogalacturonans, rhamnogalacturonans (RG) type I and type II, pectic galactans, arabinogalactans, arabinans),
  • maltodextrins psyllium, agar, celluloses, resistant starch, n-acetyl-glucosamines, polyfructans (inulin, levan), gums (carrageenan, alginate, xhantan gum, guar gum, partially hydrolyzed guar gum ("PHGG”), dextrins, fructooligosaccharides (FOS), galactooligosaccharides (GOS), mucilage, iso-malto oligosaccharides, lignin, cutin, hyaluronans, chondroitin sulfate and suberin.
  • PHGG partially hydrolyzed guar gum
  • At least 50% of the fibers in item (b) of claim 1 are pre-treated dietary fibers.
  • at least 70% of the fibers in item (b) of claim 1 are pre-treated dietary fibers or hat at least 90% of the fibers in item (b) of claim 1 are pre-treated dietary fibers.
  • composition of this step (ii) comprises anaerobic bacteria of at least the following four Phyla: Bacterioidetes, Firmicutes, Proteobacteria and
  • the composition of step (ii) is a composition, wherein the following bacterial species together comprise more than 1 % preferably more than 20%, and more preferably more than 50% of the total bacterial content of the composition;
  • the composition of step (ii) is a composition, which comprises one or more of the above mentioned bacteria species - preferably, it comprises 10 or more of the above mentioned bacteria species.
  • the method of the invention comprises suitable conditions that maximize the level of biodiversity of the original sample, thereby obtaining an improved "copy" of the human fecal microbiota.
  • the level of biodiversity of the obtained composition comprising anaerobic human intestinal microbiota is not lower to 5% of that in the original fecal sample and preferably, a 10%, 20%, 30%, 40% and 50%.
  • step (ii) of the first aspect may be done according to the art in relation to the particular composition/product of interest (e.g. a freeze-dried composition).
  • the in step (ii) obtained composition is obtained by harvesting the bacteria from the cultivation medium.
  • - harvesting generally involves a centrifugation step and/or a filtration step wherein one removes significant amount of the supernatant of the cultivation medium and isolates/harvest the bacteria cell to get a composition of isolated/harvested cells.
  • the in step (ii) obtained composition is a dried composition, preferably a freeze-dried composition into a powder, or equivalent.
  • the composition may a frozen solution, fresh from the culture or a live microencapsulated culture dosage.
  • the composition further comprises a cryoprotectant, a lyoprotectant, or a preservative.
  • the resulting product can be further processed or formulated by reconstituting the frozen or freeze-dried powder in a liquid, wherein optionally the liquid is a sterile saline.
  • Suitable cryoprotectants are triols (e.g glycerol), sulfoxides (i.g.dimethylsulfoxides)), diols and derivatives (e.g. polyethylene glycol, ethylene glycol, diethylene glycol), polyalcohols (e.g. mannitols, sorbitols, dulcitol), monosaccharides (glucose, xylose), trisaccharides and polysaccharides (e.g. dextran, dextrin), amides (e.g. acetamide, methylacetamide, dimethylfomamide, succinimide), heterocyclyc compounds (e.g.
  • triols e.g glycerol
  • sulfoxides i.g.dimethylsulfoxides
  • diols and derivatives e.g. polyethylene glycol, ethylene glycol, diethylene glycol
  • polyalcohols e.g. mannito
  • cryoprotectants are between 1 to 20% (v/v). In particular the concentration of these cryoprotectans is between 5 to 10%. Cooling rate is betwen 0.5 to 5°C/min. In particular, cooling rate is 1 °C/min to minimize cellular damage due to osmotic imbalance and ice crystal formation. Frozen samples are stored at -80°C. In another embodiments, frozen samples are stored at -20°C.
  • the in step (ii) obtained composition is a pharmaceutical composition or a medical device composition comprising acceptable excipients.
  • Suitable acceptable excipients are well known to the skilled person.
  • pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are well-known and readily available.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like.
  • the in step (ii) obtained composition is free from the following microbes: hepatitis viruses A, B and C, cytomegalo virus, Epstein-Barr virus, human immunodeficiency virus (HIV), Calici- and Rotavirus, Salmonella, Shigella
  • Campylobacter, Yersinia, and protozoan cysts are also free from material selected from Extended spectrum- and Metallo-beta-lactamases, metabolites of pharmaceutical active substances, and xenobiotics.
  • the in step (ii) obtained composition is having a concentration of viable bacteria cells, which is in the range of 10 4 to 10 13 cfu (colony forming units) per gram (dry matter) of the composition.
  • the in step (ii) obtained composition is having a concentration of viable bacteria cells, which is in the range of 10 6 to 10 11 cfu (colony forming units) per gram (dry matter) of the composition.
  • Second aspect A composition obtained by a method of the first aspect
  • a second aspect of the invention relates to a composition comprising anaerobic human intestinal microbiota, wherein the composition comprises obligate and factultative anaerobic bacteria of at least three of the following four Phyla: Bacterioidetes, Firmicutes, Proteobacteria and Actinobacteria and wherein the composition is obtained by a method of the first aspect and/or any herein described embodiments thereof.
  • the composition comprises anaerobic bacteria of at least the following four Phyla: Bacterioidetes, Firmicutes, Proteobacteria and Actinobacteria. It may be preferred that the composition is a pharmaceutical composition, or a medical device composition comprising acceptable excipients.
  • the composition comprising anaerobic human intestinal microbiota is a mixture of different the in step (ii) obtained compositions.
  • a third aspect of the invention relates to a composition of the second aspect and/or herein described embodiments thereof, for use in the treatment of a condition related to intestinal dysbiosis - preferably, for use in the treatment of Clostridium difficile infection.
  • a fourth aspect of the invention relates to a composition of the second aspect and/or herein described embodiments thereof, for use in the treatment of antibiotic-associated diarrhea (AAD), C. d/ ' ff/ ' c/Ve-associated diarrhea (CDAD), pseudomembranous colitis or toxic megacolon.
  • AAD antibiotic-associated diarrhea
  • CDAD C. d/ ' ff/ ' c/Ve-associated diarrhea
  • pseudomembranous colitis or toxic megacolon.
  • compositions comprising microorganisms may vary significantly depending on the characteristics or physiological conditions of the patient (i.e. illnesses, physic characteristics, etc) as well as dietary habits. This have different consequences that can compromise the efficacy of the treatment.
  • the microbial composition can be administered to the patient and improve the microbial ecosystem, but turn to its original status of 'dysbiosis' in a short period of time, liming the effect of the treatment.
  • microbial compositions administered to the human subject may not be enough efficient to shift the composition of the intestinal microbiota with an acute condition to a 'healthy status'.
  • the composition is administrated to a human person, who is instructed to consume within at latest 24 hours after the administration of the pharmaceutical/microbial composition a food product, food or dietary supplement, food ingredient and/or medical food. More particularly the human person is instructed to consume from 0.001 to 10 g/kg body weight of the person of dietary fibers or indigestible carbohydrates.
  • the human person is instructed to consume from 0.05 - 1 .0 g/kg weight of the person of dietary fibers, more preferably the human person is instructed to consume from 0.1 -0.5 body weight of the person of dietary fibers.
  • the human person is instructed to consume the dietary fibers within at latest 2 hours after the administration of the composition.
  • the human person is instructed to consume the dietary fibers daily for at least two weeks.
  • the dietary fibers the human person is instructed to consume are fibers that consist essentially of the same fibers as the fibers of item (b) of the method of the first aspect.
  • Said dietary fibers are preferably food grade and are formulated as pharmaceutical composition, food supplement composition or medical food composition comprising acceptable excipients.
  • composition comprising anaerobic human intestinal microbiota may e.g. be administrated to a human by a device selected from a naso-duodenal tube, gastroscope, colo/sigmoideoscope, and enema, or in freeze dried form in a suitable galenic preparation, e.g. gastric acid resistant capsule, nano- encapsulation or suppository.
  • a device selected from a naso-duodenal tube, gastroscope, colo/sigmoideoscope, and enema
  • a suitable galenic preparation e.g. gastric acid resistant capsule, nano- encapsulation or suppository.
  • the composition comprising anaerobic human intestinal microbiota as described herein is applied to the human gastro-intestinal tract at least one time per day during the course of a number of days, e.g. 1 -30 days, depending on the nature, cause and severity of the problem.
  • composition comprising anaerobic human intestinal microbiota as described herein can preferably be administered according to the following non limiting examples: in the form of a solution through the oral or the nose cavity or through the colon with e.g. a naso-duodenal tube, gastroscope, colo/sigmoideoscope, enema or in freeze dried form in a suitable galenic preparation, e.g. a gastric acid resistant capsule, nano-encapsulated or a suppository.
  • delivery is performed by intubation, by elongation, by looping (alpha maneuver), and by accordionization (dither-torquing).
  • compositions which facilitates the administration or supports the effect of the invention may be added to the composition.
  • the treatment with the invention may or may not be preceded by an antibiotic treatment and/or motility reducing substances.
  • Example 1 This example is a non-limiting example describing suitable possible relevant embodiments of the present invention.
  • SELECTION OF DONORS Candidates are asked to complete questionnaires including biographic information, clinical history and dietary habits, and provide fecal and blood samples.
  • Inclusion criteria include: men or female with average body mass index
  • fecal material is processed to determine the complete microbiome composition by genetic tools such as
  • Fecal microbial biodiversity of donor candidates is compared to that of patients suffering from a condition associated to low biodiversity of the intestinal microbiome such as Inflammatory Bowel Disease (IBD) or Irritable Bowel Syndrome (IBS).
  • Candidates are considered valid donors if the biodiversity of the fecal samples provided is higher that of fecal samples obtained from IBD and/or IBS. Presence of bacterial species (e.g.
  • Faecalibacterium prasnutzii, Akkermansia sp.) considered as biomarker of healthy microbiome is also an inclusion criteria.
  • Exclusion criteria include family history of autoimmune, metabolic diseases, alcohol or drug abuse or intestinal cancer or inflammatory bowel disease; any gastrointestinal illness or gastrointestinal complaints (abdominal discomfort, regularly loose stools, or constipation); use of antibiotic, proton pump inhibitors or immunosuppressive agents within the preceding 90 days, increased risk for (contracting) infectious diseases in the phase between screening and donation of feces (such as a recent visit to a tropical area in the last three months, risky sexual behavior defined as a new sexual contact in the last six months, recent needle stick accident, receiving blood products, or getting a tattoo); a general illness or use of medication that could be excreted in feces and pose a potential risk for recipients.
  • Candidates are also excluded by presence of potentially transmissible diseases, human immunodeficiency virus, sexually transmitted diseases and hepatitis B or C as determined by blood analyses.
  • Blood samples are screened for antibodies to HIV; human T- cell lymphotropic virus types 1 and 2 (HTLV-1 and II); hepatitis A, B, and C; cytomegalovirus; Epstein-Barr virus; Treponema pallidum; Strongyloides stercoralis; and Entamoeba histolytica. If donors tested positive for one of the above mentioned pathogens, they are excluded.
  • a resolved EBV or CMV infection is not an exclusion criterion.
  • a donor has antibodies against Hepatitis A (IgG), but is IgM negative and does not visit a tropical country in the past six months, he is not considered at risk for Hepatitis A and therefore not excluded. Donors with a resolved Hepatitis B virus infection are excluded.
  • IgG Hepatitis A
  • Donor fecal samples are processed to culture with the aim of screening for enteric pathogens (Salmonella, Shigella, Yersinia enterocolitica and
  • Campylobacter species including Blastocystis hominis and Dientamoeba fragilis, triple feces test for ova and parasites; Giardia sp.
  • C. difficile toxins A and B which are considered an exclusion criteria.
  • Donors with Blastocystis hominis or Dientamoeba fragilis in their stool are excluded.
  • donors fill out a second questionnaire the day before donation, concerning their stool frequency and pattern, general health, use of antibiotics and sexual behavior. This is to screen for any acute
  • a single-vessel pH-controlled continuous anaerobic fermenter is used for industrial propagation of intestinal microbiome of the donors.
  • Fecal samples from healthy donors are used for preparing a fecal suspension for inoculating the system.
  • Fecal samples are collected in anaerobic conditions.
  • Fecal samples can be further cryopreserved or directly processed for propagation of the microbiome.
  • a first step for propagation of microbiome includes mixing the fecal material with an appropriate buffer or saline solution in a final concentration between 5-50%, under sterile or semi- sterile conditions.
  • the concentration of the fecal suspension range between 10-30%. In particular, the concentration is 20%.
  • the buffer used for preparing the suspension is sodium phosphate buffer (0.1 M, pH 7.0).
  • the saline solution is 0.9%.
  • the slurry can be filtered to remove larger particles.
  • the propagation of the microbiome is conducted under an oxygen free atmosphere (anaerobic). In particular, anaerobic conditions are created by flushing nitrogen to the fermentation vessel.
  • the pH of the vessel is controlled during the propagation of the microbiome. In particular, the pH is controlled in a range between 2 and 8. In particular, the pH is controlled in a range of 5.5 and 7.5. In particular, the pH is controlled in a range between 6.0 to 7.0. In particular, pH is controlled by adding an acidic or a basic dilution. In particular, acid is HCI and base is NaHC0 3 .
  • Liquid growth media is pumped continuously via peristaltic pumps to feed the system.
  • Growth media consists in a nutritive media (NM) supplemented with a previously selected Synthetic Colonic Fraction (SFC).
  • NM nutritive media
  • SFC Synthetic Colonic Fraction
  • NM contains a source of inorganic compounds or minerals, vitamins, a carbon source and a protein source.
  • growth media is composed by potato starch (3 g/L), glucose (0.4 g/L), yeast extract (3 g/L), peptone (1 g/L), mucin
  • Fermentation is conducted by adjusting different factors or parameters or
  • PIM Propagated Intestinal Microbiome
  • SCFA Short Chain Fatty Acids
  • lactate lactate
  • ammonium Suitable methods for determining these metabolic products are well known by the skilled in the art.
  • a threshold of variability is fixed according to inter-assay variability of the analytic methods used and it is assumed that stability of the microbial communities is reached when the variation between different time intervals of the measured fermentation products is lower than this threshold. Samples at the steady state are taken for determining the evolution and biodiversity of the microbial communities by molecular microbial analysis.
  • composition of bacterial community samples at steady state are studied using metagenomic analyses such as high-throughput sequencing pyrosequencing methods.
  • metagenomic analyses such as high-throughput sequencing pyrosequencing methods.
  • the DNA is extracted with a
  • DNA isolation kit DNA library, sequences, amplification and basic analysis is performed by using an automated sequencing system. Data analysis is further processed by bioinformatic methods.
  • a first step involves pre- filtering of the data obtained, including the removal of redundant, low-quality sequences and sequences of probable eukaryotic origin (i.e. human origin). Methods available for the removal of contaminating eukaryotic genomic DNA sequences may include Eu-Detect and DeConseq.
  • the pyrosequencing reads are compared based upon homology with sequences already available in sequence database such as BLASTN for taxonomic profiling.
  • Community diversity is determined by using biodiversity index including richness and evenness parameters.
  • the biodiversity index is Shannon's biodiversity index.
  • the biodiversity index is Simpson's biodiversity index.
  • the multifactorial design is a screening design, which compared to a full factorial design allows to screen large number of factors and factor levels in an efficient manner, that is, with the least number of observation necessary.
  • the screening design is a Plackett-Burman desing. In particular, these factors are: A) Retention time (RT), B) the concentration of SCF used in the growth media (SCF-c), and C) the number of components constituting the SCF used (SCF-n).
  • RT is in a range between 5 to 168 h. In particular, RT is in a range between 12 to 86 h. In particular, RT is in a range between 12-76h. In particular, RT is 24h. In particular, the fermentation time is in a range between 12 h and 30 days. In particular, the fermentation time is between 24 h and 21 days.
  • the concentration of SCF ranges from 0.5 to 100 g/L. In particular, the concentration of SCF, ranges from 1 to 20 g/L. In particular, SCF is in a concentration selected from 5 or 6 g/L.
  • SCF is obtained from the chemical and enzymatic digestion of a Proimplantation Medical Food (PMF).
  • the acid hydrolysis is performed at 37°C for 30 min by using a solution adjusted to pH 2 with HCI and containing peptone at a concentration of 10 mg/l of pepsin.
  • the hydrolysis with pancreatic juice is performed at 37°C for 2 hours with a solution containing 12.5 g of NaHC0 3 , 6 g of Oxgal and 1 .9 g of pancreatin).
  • the composition of the SCF is influenced by the initial composition of the PMF.
  • PMF is composed by different ingredients selected from a group of compounds or polymers with potential capacity to influence or modulate the composition of the microbiota.
  • these ingredients are selected from the group of dietary fibers such as indigestible carbohydrates; micronutrients such as isoflavones; proteins and lipids.
  • the ingredients of the PMF are selected from the group of dietary fibers. The selection of these dietary fibers is performed according to a method that comprises the following steps.
  • dietary fibers are described according to their chemical structure according to the parameters: a) type of different forming units (i.e. monosaccharide
  • composition b) linkage types (i.e. linkages alpha or beta); c) branch chain composition (i.e. lengths and distribution of links and units).
  • characterization allows to predict the number of A) different families of structurally-related catalytic enzymes necessary for their degradation and b) the number of different degradation enzymes belonging to each family.
  • the dietary fibers are divided in 4 groups (Group A, B, C and D) wherein these groups A are:
  • Group A Including soluble dietary fibers whose digestion requires one or more enzymes from the family of Glycoside hydrolase enzymes (Family GH).
  • Group B Including soluble dietary fibers whose digestion requires one or more enzymes from the family of polysaccharide lyases enzymes (Family PL).
  • Group C Including soluble dietary fibers whose digestion requires one or more enzymes from the family of carbohydrate esterase enzymes (Family CE).
  • Group D Including insoluble dietary fibers.
  • one dietary fiber may be present in different groups.
  • PEB Index is calculated according to the following formula:
  • PEBi F ——— -T—
  • F is the number of different families of enzymes needed to degrade a given fiber; E, the total number of all enzymes required, and e F the number of enzymes of each particular family of enzymes.
  • This raking is performed scoring each fiber according to their maximum solubility in aqueous media and their viscosity at this concentration, wherein a maximum solubility and minimum viscosity are desirable characteristic for the dietary fibers.
  • this score can be determined by an index providing a value based on this characteristics (SV Index), wherein SV Index can be determined by using the following formula.
  • S is the maximum solubility of the fiber in liquid medium
  • [n] the intrinsic viscosity of the fiber.
  • S can be determined by adding an incremental concentration of fiber to a fixed volume of distilled water and determining the maximum concentration reached until precipitation is observed.
  • the intrinsic viscosity [n] is determined by using Huggin's plot of the reduced viscosity versus concentration.
  • solubility and viscosity is determined at 37°C.
  • viscosity of candidates is determined when candidates are solubilized in NM.
  • ranked lists of PMF candidates are created by using the method described above, wherein each list corresponds to a group of fibers.
  • a global score herein referred as Synthetic Colonic Fraction Suitability Index (SCFSi) per each candidate can be assigned according to the following formula:
  • N-SCF PEBi + 1 - (— )
  • the factor N-SCF is defined as the number of fibers selected from each group, starting from the best ranked to the worst ranked.
  • N-SCF is a number ranging from 3 to 10, meaning that the SCF used for fermentation includes from the first 3 to 10 candidates of the ranked list of each group.
  • PMF is composed by different ingredients selected from a group of compounds or polymers with potential capacity to influence or modulate the composition of the microbiota.
  • these ingredients are selected from the group of dietary fibers such as indigestible carbohydrates; micronutrients such as isoflavones; proteins and lipids.
  • the ingredients of the PMF are selected from the group of dietary fibers.
  • Dietary fibers can be based on a carbohydrate polymer structure or a non- carbohydrate polymer structure. Suitable dietary fibers with a carbohydrate polymer structure are hemicelluloses (arabinoxylans, xyoglucans, glucomannans,
  • galactomannans beta-glucans
  • pectins homoogalacturonans, rhamnogalacturonans (RG) type I and type II, pectic galactans, arabinogalactans, arabinans), maltodextrins, psyllium, agar, celluloses, resistant starch, n-acetyl-glucosamines, polyfructans (inulin, levan), gums (carrageenan, alginate, xanthan gum, guar gum, partially hydrolyzed guar gum (“PHGG”), dextrins, fructooligosaccharides (FOS),
  • PHGG partially hydrolyzed guar gum
  • Suitable fibers with a non-carbohydrate polymer structure are insoluble fibers such as lignin, cutin, hyaluronans, chondroitin sulfate and suberin.
  • Target patients are ⁇ 18 years, and a microbiologically confirmed relapse of C. difficile infection after at least one course of adequate antibiotic therapy ( ⁇ 10 days of vancomycin ⁇ 125 mg q.i.d., or ⁇ 10 days metronidazole 500 mg t.i.d.).
  • C. difficile infection is defined as (i) diarrhea ( ⁇ 3 loose or watery stools per day for at least 2 consecutive days, or ⁇ 8 loose stools in 48 hours) and (ii) a positive C. difficile toxin stool test. The Meridian A/B toxin premier test is used.
  • Vancomycin 500 mg orally four times per day for 4 or 5 days. Vancomycin is discontinued on the day of infusion. Bowel lavage to wash out residual antibiotic and fecal material using a standard four liter macrogol electrolyte suspension (Klean-Prep) is followed by a light meal one day before PIM implantation. On the day of implantation, patients are sober and a nasoduodenal tube (which fitted on a 50 cc luer-lock syringe) is placed using an electromagnetic sensing device (CortrakTM) 2 , or through duodenoscopy. The position of the tube is confirmed by X-ray.
  • Mucosal biopsies are taken to rule out lymphocytic colitis in absence of obvious inflammatory bowel disease.
  • PIM is infused slowly with a 50 cc syringe (approximately 30 seconds per syringe) through the nasoduodenal tube.
  • the first 4 or 5 syringes are infused in about 10 minutes into the patient's terminal ileum and cecum. After a break of 10 minutes, the remaining 5 syringes are infused. Patients are allowed to drink during the procedure (to set them at ease).
  • the tube is flushed with tap water after infusing PIM, and left in situ for at least 30 minutes after implantation. Immediately post procedure, patients are maintained in the Trendelenburg position for 60 minutes.
  • Patients are instructed to consume the Proimplantation Medical food daily for at least two weeks. Patients are followed by a study nurse to obtain stool samples and closely monitor their clinical response.

Abstract

L'invention concerne un procédé de préparation d'une composition comprenant un microbiote intestinal humain anaérobie, le procédé comprenant l'étape de culture anaérobie d'un échantillon fécal provenant d'un donneur sain dans un milieu de culture, la culture étant effectuée sous la forme d'un procédé de culture continu et le milieu de culture comprenant des fibres alimentaires. La composition obtenue peut, par exemple, être utilisée pour le traitement d'une infection à Clostridium difficile.
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