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• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; 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
• • 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; 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
  • C12N1/205—Bacterial isolates
• • 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
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

• the invention relates to a new bacterial strain belonging to the genus Christensenella, a composition comprising it and its uses, in particular as a medicament in the treatment and/or prevention of diseases such as metabolic diseases and/or inflammatory diseases.
• intestinal microbiota or "intestinal flora” is made up of a set of bacteria, viruses, parasites and non-pathogenic fungi, i.e. 10 12 to 10 14 microorganisms present in the digestive tract of each individual, which represents 2 10 times more than the number of cells present in our body. Therefore, its role has been increasingly studied in recent years.
• a microbiologist identified and cultured a new species from human faeces: Christensenella minuta, belonging to the gram-negative Clostridiales. (Morotomi et al., Description of Christensenella minuta gen. nov., sp.
• IBD Inflammatory bowel disease
• IBD chronic inflammatory bowel disease
• Crohn's disease and ulcerative colitis are the main ones.
• These are recent, multifactorial diseases whose origin is unknown. In a certain number of cases, the appearance of these diseases could be linked to a genetic predisposition, to the environment, but also to the microbiota.
• These diseases all have the particularity of presenting an inflammation of the intestine which induces a modification of the microbiota and then maintains the inflammation.
• An objective of the present invention is therefore to provide a solution which is simple, effective and economical for restoring a healthy intestinal microbiota and therefore treating dysbiosis of the intestinal microbiota, in particular treating chronic inflammatory diseases, preferably by avoiding the administration of lifelong treatment and thus reduce the prevalence of these diseases.
• the inventors have identified, among the multitude of bacteria present in the intestinal microbiota and among the strains already known belonging to the species Christensenella minuta, a new bacterial strain of specific Christensenella minuta deposited with the Leibniz DSMZ Institute (Deutsche Sammlung von Mikro expn und Zellkulturen GmbH, in French German Collection of Microorganisms and Cell Cultures GmbH) under the number DSM 33715, particularly suitable for the prevention and / or treatment of intestinal dysbiosis, preferentially chronic inflammatory bowel diseases.
• the invention relates to a bacterial strain of Christensenella minuta deposited under the number DSM 33715 and having for 16S rDNA sequence, the sequence SEQ ID NO: 1, as well as any strain comprising a nucleotide sequence having at least 99 90% identity of ANI (Average Nucleotide Identity - Average Nucleotide Identity) with the nucleotide sequence of the genome of the bacterial strain DSM 33715.
• ANI Average Nucleotide Identity
• the invention also relates to the supernatant of the culture of the bacterial strain according to the invention but also to a composition comprising (i) at least the bacterial strain according to the invention and/or (ii) the supernatant of the culture according to the invention and (iii) at least one acceptable excipient.
• Said acceptable excipient preferably being a pharmaceutically acceptable excipient in the case of a product intended to be used as a medicament for human use or veterinary use.
• the invention is particularly suitable for using said bacterial strain, said supernatant or said composition according to the invention as a medicament.
• the present invention is particularly suitable for the prevention and/or treatment of so-called “non-transmissible” pathologies associated with dysbiosis of the intestinal microbiota or diseases associated with dysbiosis of the intestinal microbiota, in particular diseases such as diseases metabolic, inflammatory and other diseases which will be detailed below.
• the invention aims to prevent and/or treat chronic inflammatory bowel diseases, more preferably Crohn's disease, or ulcerative colitis.
• the invention also relates to the non-therapeutic use of the bacterial strain according to the invention, and/or of the supernatant according to the invention or of the composition according to the invention for maintaining and/or strengthening the intestinal microbiota and/or support the diversity of the intestinal microbiota and/or promote the increase of beneficial bacteria in the intestinal microbiota in a healthy subject.
• Figure IA shows an image of a bacterium Christensenella minuta according to the invention produced by transmission electron microscopy in negative staining (magnification: 36kX).
• Figure IB shows a characteristic MALDI spectrum of a pure culture of a Christensenella minuta strain according to the invention.
• Figure 2A represents the immunomodulatory effect of the DSM 33715 bacterium on PBMCs cells (3 healthy donors tested)
• FIG. 2B represents the anti-inflammatory effect of the bacterium DSM 33715 on a THP-1 line differentiated into MO type macrophages by treatment with PMA (Phorbol 12-myristate 13-acetate).
• Figure 3 shows the immunomodulatory effect of the DSM 33715 bacterium and its supernatant on the colon adenocarcinoma line HT-29 producing IL-8 after stimulation with TNF- ⁇ .
• Figure 4A shows the anti-inflammatory effect of DSM 33715 on the NF-0B inflammation signaling pathway. Activation of the NF-0B pathway by DSM 33715 in HT-29 cells transfected with a reporter system and stimulated with TNF- ⁇ , measured by luciferase activity and normalized by Renilla luminescence.
• Figure 4B shows the anti-inflammatory effect of DSM 33715 on the JAK/STAT inflammation signaling pathway. Activation of the JAK/STAT pathway by DSM 33715 in HT-29 cells transfected with a reporter system and stimulated with IFN- ⁇ , measured by luciferase activity and normalized by Renilla luminescence.
• Figure 5 represents the immunomodulatory effect of the supernatant of DSM 33715 on the colon adenocarcinoma line HT-29 producing IL-8 after stimulation with TNF-a, in the presence of an anti-inflammatory agent used in therapy, 5-ASA or Budesonide.
• FIG. 6 represents the anti-inflammatory effect of DSM 33715 associated or not with Tofacitinib on the modulation of the signaling pathway of JAK/STAT inflammation induced by IFN- ⁇ -
• the JAK/STAT pathway is activated in HT-29 cells transfected with a reporter system and stimulated with IFN- ⁇ , measured by luciferase activity and normalized by Renilla luminescence.
• Figure 7 represents the effect of the DSM 33715 strain on the integrity of the intestinal barrier by measuring the transepithelial electrical resistance.
• the PBS-Glycerol +/- TNF-a condition (control) corresponds to the bacteria resuspension medium.
• Figure 8A is a histogram representing the average length of the colons, at the end of the experiment. The data is represented by the mean +/- SEM in centimeters for the length and in grams for the weight of the colon.
• Figure 8B represents the inflammation of the colon at the macroscopic level calculated by the Wallace score.
• Figure 8C represents the inflammation at the histological level calculated by the Ameho score. Data shown are means +/- SEM. MGG staining of colon sections for histological score evaluation (Ameho)
• Figure 8D shows the levels of I L-ip (panel A) and Lipocalin-2 (panel B) in the colon.
• the data represented correspond to the mean +/- SEM in ng/mg of protein.
• Figure 9 represents the intensity of the inflammation and the lesions of the colon which were evaluated at the macroscopic level according to the Wallace score (panel A) and the intensity of the inflammatory lesions at the tissue level evaluated according to the histological score (panel B).
• bacteria within the meaning of the invention, is meant a unicellular microorganism capable of reproducing by cell division. Bacteria are classified by family, genus, species. Each bacterial species includes a diversity of bacterial strains.
• the bacterial strain according to the invention belongs to the Christensenellaceae family, the Christensenella genus and the minuta species.
• bacterial strain or “strain” within the meaning of the invention, is therefore meant a specific bacterial strain but also all the bacteria derived from the strain or obtained from the strain or corresponding to the bacterial strain and having the same functions metabolic, for example, at least one bacterium taken from a colony derived from the strain.
• bacteria(s) according to the invention within the meaning of the invention, is therefore also meant a bacterial strain according to the invention.
• derived bacterial strain or “mutant strain” or “derived strain” within the meaning of the invention, is meant a bacterial strain having a strong similarity with the bacterial strain deposited under the number DSM 33715.
• the strain comprises a nucleotide sequence having at least 99.90% identity of ANI with the nucleotide sequence of the bacterial strain DSM 33715, more preferably at least 99.91%, at least 99.92%, at least 99.93% , at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, or at least 99.99%
• ANL identity [ 0040]
• supernatant within the meaning of the invention is meant the culture supernatant of the bacterial strain according to the invention optionally comprising cellular compounds of said strain and/or cell debris of said strain, and/or metabolites and/or molecules secreted by said strain.
• prevention within the meaning of the invention, is meant the reduction to a lesser degree of the risk or the probability of occurrence of a given phenomenon, that is to say, in the context of the present invention, dysbiosis of the intestinal microbiota and associated diseases, more preferably chronic inflammatory diseases, for example Crohn's disease or ulcerative colitis.
• treatment within the meaning of the invention, is meant a reduction in the progression of the disease, a stabilization, an inversion or regression, or even an interruption or inhibition of the progression of a dysbiosis of the intestinal microbiota and the associated diseases, more preferably chronic inflammatory diseases, for example Crohn's disease or ulcerative colitis. In the context of the invention, these terms also apply to one or more symptoms of said diseases of the present invention.
• physiologically acceptable medium within the meaning of the invention is meant a medium which is compatible with the body of the individual to whom said composition is to be administered. It can be, for example, a non-toxic solvent such as water, buffer, saline solutions. In particular, said medium is compatible with oral administration.
• acceptable excipient within the meaning of the invention is meant any compound making it possible to facilitate the shaping of the composition and not modifying the nature of the biological activity of the active principle.
• An acceptable excipient can be a solvent, buffer, saline solution, plasticizer, lubricant, dispersing medium, agents delaying absorption, flow agent, isotonic agent.
• they are pharmaceutically acceptable excipients which are chosen according to the pharmaceutical form and the desired mode of administration, from the usual excipients known to those skilled in the art and suitable for human and/or veterinary use. The excipient will thus be chosen according to the route of administration, for example suitable for oral, intravenous, intramuscular, topical administration, etc.
• disease associated with a dysbiosis of the intestinal microbiota within the meaning of the invention, is meant the diseases associated with a dysbiosis of the intestinal microbiota such as certain metabolic diseases, for example diseases linked to obesity whose obesity, diabetes, NASH, hepatic or pancreatic steatosis; inflammatory diseases, in particular inflammatory bowel diseases, eg Crohn's disease, colitis ulcer disease, diverticulitis, gastritis, pancreatitis, or irritable bowel syndrome; but also chronic diseases associated with dysbiosis, heart and vascular diseases, cancers, for example metabolism-related cancers.
• certain metabolic diseases for example diseases linked to obesity whose obesity, diabetes, NASH, hepatic or pancreatic steatosis
• inflammatory diseases in particular inflammatory bowel diseases, eg Crohn's disease, colitis ulcer disease, diverticulitis, gastritis, pancreatitis, or irritable bowel syndrome
• chronic diseases associated with dysbiosis heart and vascular
• dysbiosis of the intestinal microbiota is meant within the meaning of the invention an imbalance of this microbiota which could favor the initiation, the maintenance or the severity of the inflammation.
• a family of Escherichia coli (AIEC) more adherent to the cells of the intestinal wall and more invasive than the usual strains, which facilitate a local inflammatory reaction.
• Potential causes of dysbiosis could be dietary (fatty and sugary diet, lacking fiber, which limits bacteria producing beneficial short-chain fatty acids), infectious (acute episodes of infectious gastroenteritis), or environmental (antibiotic treatments repeated, insufficient exposure to pathogens during childhood).
• anti-inflammatory agent within the meaning of the invention a molecule, an active principle, a medicament, a pharmaceutical composition intended to combat inflammation.
• a group of drugs intended to treat an inflammatory reaction and the resulting diseases such as inflammatory bowel diseases.
• anti-inflammatory drugs of corticosteroids (or glucocorticoids or steroidal anti-inflammatory drugs) and non-steroidal anti-inflammatory drugs.
• health subject within the meaning of the invention, is meant a subject who is not sick and not suffering from disease, in particular not suffering from a disease chosen from among chronic diseases and/or obesity and/ or metabolic diseases and/or inflammatory diseases and/or cancers.
• the inventors discovered the bacterial strain of Christensenella minuta deposited under number DSM 33715, then isolated it from feces of a healthy human donor and demonstrated, in in vitro models, anti-inflammatory effects , in particular an immunomodulatory effect such as the induction of higher production of IL-10, an anti-inflammatory cytokine; and a decrease in certain signaling pathways such as the NF-0B pathway or the JAK/STAT pathway.
• Results on a pre-clinical model of colitis induced by TNBS in rats confirm the anti-inflammatory effects of the strain, in especially in chronic inflammatory bowel disease.
• said bacterial strain DSM 33715 makes it possible to restore or strengthen and therefore preserve the integrity of the intestinal barrier.
• anti-inflammatory agents used clinically such as Pentasa (5-ASA), Tofacitinib or Budesonide, thus making it possible to consider the administration lower doses of such anti-inflammatory agents known for their toxicity and their undesirable effects in patients.
• the present invention therefore relates to a bacterial strain of Christensenella minuta deposited under the number DSM 33715 hereinafter referred to as strain DSM 33715 having for 16S rDNA sequence, the sequence SEQ ID NO: 1.
• 16S ribosomal RNA (16S rRNA) is the ribosomal RNA constituting the small subunit of the ribosomes of prokaryotes.
• the genes encoding this RNA are called 16S rDNA.
• the 16S rDNA sequence is widely used in phylogeny because of its highly conserved structure which makes it possible to reconstruct the evolutionary history of organisms and in particular prokaryotes and bacteria.
• the percentage identity of the 16S rDNA sequence between two bacterial strains, more particularly between two strains of Christensenella minuta can be determined by the so-called BLAST method which is a heuristic research method well known to those skilled in the art. art. It makes it possible to find the similar regions between two or more sequences of nucleotides or amino acids, and to achieve an alignment of these homologous regions.
• the 16S rDNA sequence does not always make it possible to differentiate between two strains of the same species which nevertheless have different properties, for example anti-inflammatory properties. Consequently, the person skilled in the art with his general knowledge is able to characterize a bacterial strain according to other parameters such as the calculation of phylogenetic distance based on the complete genome (ANI), the size of the genome, the number of CDS (Coding DNA Sequence or in French Coding DNA Sequence), but also the identification of genes specific to said strain of interest.
• ANI complete genome
• CDS Coding DNA Sequence
• French Coding DNA Sequence French Coding DNA Sequence
• ANI is meant in the sense of the invention the average percentage identity of the nucleotides calculated from the two-by-two comparison of all the genome sequences shared between the two bacterial strains.
• the genomic DNA can be extracted from a pure bacterial culture derived from said strain of interest, followed by DNA sequencing according to various well-known methods, for example Sanger, Roche 454, Illumina, Oxford Nanopore then the genome sequenced is assembled by bioinformatics and the sequences obtained are analyzed. Finally, the genomes of interest are compared two by two to calculate the ANI.
• the invention also relates to a bacterial strain derived from Christensenella minuta, characterized in that it comprises a nucleotide sequence having at least 99.90% identity of ANI with the nucleotide sequence of the bacterial strain DSM 33715.
• said strain according to the invention comprises a nucleotide sequence having at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% identity of ANI with the nucleotide sequence of the bacterial strain DSM 33715.
• Such a strain is therefore a bacterial strain of Christensenella minuta derived from the bacterial strain deposited under the number DSM 33715 making it possible to maintain or improve the described capacities of the strain DSM 33715 in the context of the present invention.
• Said derived strain can thus be produced naturally or intentionally, by mutagenesis methods known in the state of the art.
• the mutagenesis methods that can be implemented in the context of the present invention are the growth of the original microorganism in the presence of mutagenic or stress-producing agents, or by genetic engineering aimed at modifying genes specific or not such as directed mutagenesis or random mutagenesis.
• the strain derived from the DSM 33715 strain of C. minuta is a genetically modified mutant.
• the invention relates to the strain of Christensenella minuta deposited under number DSM 33715 with the Leibniz DSMZ Institute (Deutsche Sammlung von Mikroorganisationn und Zellkulturen GmbH, in French German Collection of Microorganisms and Cell Cultures GmbH).
• Said strain has the 16S rDNA sequence, the sequence SEQ ID NO:1.
• the culture supernatant obtained from the bacterial strain DSM 33715 and/or the derived strain comprises at least one of the constituents chosen from a bacterial cell compound, a bacterial cell debris, a metabolite and/or a molecule(s) secreted by the strain and/or the derived strain, or combinations thereof.
• the cellular compounds and/or cellular debris can be wall components, nucleic acids, membrane components, proteins, lipids, etc.
• the metabolites or molecules secreted can be any molecule produced or modified by the bacterium due to its metabolic activity during its growth, its use in technological processes (for example, but not limited to, production of food or medicine).
• the metabolites or molecules can be proteins, amino acids, enzymes, lipids, nucleic acids, etc.
• metabolite and/or one or more molecule(s) secreted by the strain and/or the derived strain is meant a molecule produced and exported or released outside the bacterium by the bacterium.
• the invention also relates to the culture supernatant obtained from the bacterial strain according to the invention.
• strain(s) according to the invention or derived strain(s) and/or the culture supernatant described above and the bacteria derived from said strains are advantageously administered in a composition.
• the invention relates to a composition
• a composition comprising: (i) at least one bacterial strain according to the invention and/or (ii) a supernatant according to the invention and (iii) at least one acceptable excipient.
• the composition comprises at least one bacterial strain deposited under the number DSM 33715 and/or at least one strain having at least 99.5% identity with the 16S rDNA sequence of the DSM 33715 strain and / or the culture supernatant obtained from said strains and at least one acceptable excipient and/or a physiologically acceptable medium.
• the composition may comprise the bacterial strain according to the invention in any form producing the expected effects and the efficacy described in the present application.
• the strain can be in living form (cultivable or not), dead, semi-living, attenuated or inactivated.
• the dead, semi-living, attenuated or inactivated form can be obtained by various techniques known to those skilled in the art. Examples include the following techniques: irradiation, thermal inactivation or lyophilization, in particular by heat, exposure to an appropriate pH, UV, gamma rays, X-rays or high pressure.
• semi-living is meant a bacterium with low physiological activity whose ability to proliferate is reduced, temporarily or permanently.
• inactivated designates a bacterium which is no longer capable, temporarily or permanently, of proliferating.
• dead designates a bacterium which is no longer capable, definitively, of proliferating.
• Dead or inactivated bacteria may have intact or ruptured cell membranes.
• inactivated also designates the extracts and lysates of bacteria obtained.
• composition according to the invention may also comprise at least one additional compound.
• An additional compound may in particular be an ingredient, a molecule, an active principle, a microorganism, a bacterium or a mixture of bacteria.
• the additional compound is a different microorganism from the bacterial strain according to the invention or from the derived strain.
• the microorganism is a bacterium of the genus Christensenella.
• the additional compound is a probiotic and/or a prebiotic.
• the prebiotic may, for example, be at least one prebiotic chosen from galactooligosaccharides, fructooligosaccharides, inulins, arabinoxylans, beta-glucans, lactoglobulins and/or beta-caseins.
• the additional compound can be:
• At least one bacterium producing lactic acid which makes it possible to create an anaerobic environment favorable to Christensenellacées, such as at least one bacterium chosen from bacteria of the genus Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. and or
• At least one bacterium producing butyric acid which makes it possible to create an environment favorable to Christensenellacées such as a bacterium of the genera Ruminococcaceae and Lachnospiraceae,
• At least one other organism favoring the anaerobic conditions necessary for the survival of Christensenellacées such as at least one yeast chosen from Saccharomyces spp. or microorganisms of the Methanobacteriaceae family
• bacterium associated with the Christensenellaceae ecosystem because they facilitate their survival in the intestine, such as at least one bacterium chosen from bacteria of the phylum Firmicutes, Bacteroidetes, Actinobacteria, Tenericutes, and Verrucomicrobia, and/or
• At least one polyphenol such as for example at least one polyphenol chosen from quercetin, kaempferol, resveratrol, flavones (such as luteolin), flavan-3-ols or catechins, flavanones (such as naringinin), isoflavones, anthocyanidins, oligo-proanthocyanidins, and/or
• non-steroidal anti-inflammatories antibodies directed against pro-inflammatory targets (such as anti-TNF-alpha or anti-IL6), antirheumatics, analgesics, antimicrobials, corticosteroids, anabolics steroids, antidiabetics, thyroid agents, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietin, immunoglobulins, immunosuppressants, growth hormones, hormonal drugs, modulators of estrogen receptors, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, anti-depressants, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications , beta-agonists, inhaled steroids, leukotriene inhibitors, cro moglycates or cromoglycidic acids,
• pro-inflammatory targets such as anti-TNF-
• the microorganism is preferably chosen from Akkermansia muciniphila, Faecalibacterium prausnitzii, Anaerobutiryricum hallii, Hafnia alvei, Roseburia intestinalis, Roseburia hominis, Roseburia faecis, Roseburia inulinivorans, Dysosmobacter welbionis, Oscillospira guillermondii, Lactiplantibacillus plantarum, Lacticaseibacillus casei, Latilactobacillus sakei, Ligilactobacillus salivarius, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Levilactobacillus brevis, Bifidobacterium pseudoIongum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animal
• the additional compound is a pharmaceutical active ingredient
• it is preferably an active ingredient chosen from 5-ASA, mesalamine, olsalazine, budesonide, tofacitinib, filgotinib, upadacitinib, infliximab, adalimumab, golimumab, certolizumab, vedolizumab and ustekinumab.
• the composition according to the invention is in any form acceptable for administration to a subject, preferably a human or non-human animal subject.
• the composition according to the invention is also aimed at veterinary uses.
• composition according to the invention is in solid, liquid or freeze-dried form.
• composition when in liquid form, it may in particular comprise bacterial strains according to the invention and/or the derived strain and/or a physiologically acceptable culture medium for said bacteria which makes it possible to preserve them, such as, for example, preferably anaerobic Columbia agar medium enriched with sheep's blood, or an equivalent medium containing no product derived from animal origin.
• bacterial strains according to the invention and/or the derived strain and/or a physiologically acceptable culture medium for said bacteria which makes it possible to preserve them, such as, for example, preferably anaerobic Columbia agar medium enriched with sheep's blood, or an equivalent medium containing no product derived from animal origin.
• the bacterial strains according to the invention and/or the derived strain may be present in freeze-dried form, and may also comprise excipients such as, for example, microcrystalline cellulose. , lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum, calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin , galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, mannitol, sorbitol and/or glycerol.
• excipients such as, for example, microcrystalline cellulose. , lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amy
• the composition according to the invention is in a form suitable for oral, nasal, parenteral, rectal, sublingual, ocular, auricular, intramuscular, intravenous, inhaled or cutaneous administration.
• composition can be in any suitable form.
• the composition according to the invention may be in a form chosen from a powder, powder capsule, capsule, tablet, lozenge, granules, emulsion, suspension, suppository, inhaler and syrup.
• the composition according to the invention may be in a gastro-resistant form, for example a coated tablet containing microencapsulated bacteria.
• Said composition can thus be provided with an enteric coating that is resistant to gastric juice, in order to ensure that the bacterium or bacteria included in said composition can cross the stomach. The release of the bacterium(ies) may thus occur for the first time in the upper intestinal tract.
• the composition comprises 10 4 to 10 12 colony forming units (CFU) of bacteria per daily dose of composition to be administered preferably 10 6 to 10 12 colony forming units (CFU) of bacteria per daily dose of composition to be administered.
• CFU colony forming units
• this corresponds to a daily dose of bacteria to be administered, regardless of the weight of the person or the animal.
• this dose is administered all at once.
• the useful composition comprises 10 5 to 10 11 CFU or 10 7 to 10 11 CFU of bacteria per daily dose to be administered, even more preferentially 10 9 CFU.
• Said bacteria are a mixture of bacteria corresponding to or originating from the bacterial strain DSM 33715 and/or corresponding to the derived strain.
• CFU Cold Forming Unit
• UFC Coldy Forming Unit
• the daily dose is measured per gram or milliliter of the composition according to the final invention.
• the composition according to the invention comprises a mixture of bacteria, said mixture comprising at least live bacteria
• the composition preferably comprises at least 1% of live bacteria (by number), more preferably at least 10% live bacteria (by number), even more preferably at least 50% live bacteria (by number).
• the live bacteria are preferably a mixture of bacteria corresponding to the bacterial strain DSM 33715 and/or corresponding to the derived strain having at least 99.90% identity of ANI with the nucleotide sequence of the bacterial strain DSM 33715
• the composition according to the invention including it may in particular comprise a content of between 0.1 and 99% by weight, in particular from 5 to 95% by weight, in particular from 10 to 90% by weight and more particularly from 15 to 85% by weight, relative to the total weight of the composition.
• a composition according to the invention may comprise a content of between 0.1 and 99% by weight, in particular from 5 to 95%, in particular from 10 to 90% by weight, more particularly from 15 to 85% by weight, of supernatant relative to the total weight of the composition.
• the composition when the composition is intended for therapeutic use, the composition may comprise at least one pharmaceutically acceptable excipient.
• the composition according to the invention is preferably a pharmaceutical composition.
• the invention therefore also relates to a pharmaceutical composition
• a pharmaceutical composition comprising one or more ingredients included in the composition according to the invention and according to any one of the embodiments described above.
• Said pharmaceutical composition according to the invention has at least one application in improving the physical, physiological or psychological well-being of a sick subject, which results in an improvement in the general state of health of said subject or reduced risk of disease.
• Said pharmaceutical composition is a medicine.
• composition according to the invention may be in the form of a food product, a drink, a nutraceutical, a food additive, a food supplement or a dairy product.
• composition according to the invention when included in a food supplement for oral administration, it may be in the form of capsules, capsules, soft capsules, tablets, sugar-coated tablets, pills , pastes, lozenges, gums, drinkable solutions or emulsions, syrup or gel.
• a food supplement according to the present invention may further comprise a sweetener, a stabilizer, an antioxidant, an additive, a flavoring agent and/or a colorant.
• the formulation thereof is carried out by means of the usual methods for producing sugar-coated tablets, capsules, gels, hydrogels for controlled release, emulsions, tablets or capsules.
• a composition according to the present invention can also be in the form of a nutritional composition.
• a nutritional composition according to the present invention may be in the form of a yoghurt, a cereal bar, a breakfast cereal, a dessert, a frozen food, a soup, a pet food, a suspension liquid, powder, tablet, gum or candy.
• a nutritional composition according to the present invention may also comprise at least one ingredient chosen from: antioxidants, fish oils, DHA, EPA, vitamins, minerals, phytonutrients, a protein, a lipid, probiotics , prebiotics and combinations thereof.
• the invention therefore relates to the bacterial strain according to the invention and/or the derived strain, the supernatant according to the invention, more particularly the composition according to the invention for its use as a medicament.
• the invention makes it possible to provide an effective quantity of bacterial strains according to the invention or of the derived strain, for its use as a medicament.
• medicament in the context of the present invention refers to human or veterinary use, said medicament is then administered to a human subject or a non-human animal subject.
• human subject it is also meant an adult or a child or a baby, including a newborn or an infant.
• the composition according to the invention is intended for use in the prevention and/or treatment of dysbiosis of the microbiota, in particular of the intestinal microbiota and/or diseases associated with dysbiosis of the intestinal microbiota and/ or chronic diseases and/or obesity and/or metabolic diseases and/or inflammatory diseases and/or cancers.
• composition according to the invention is intended for use in the prevention and/or treatment of chronic inflammatory bowel diseases, in particular of a disease chosen from Crohn's disease, ulcerative colitis, ulcerative-hemorrhagic recto-colitis, diverticulitis, esophagitis, gastritis, pancreatitis, ulcer gastroduodenal, irritable bowel syndrome.
• chronic inflammatory bowel diseases in particular of a disease chosen from Crohn's disease, ulcerative colitis, ulcerative-hemorrhagic recto-colitis, diverticulitis, esophagitis, gastritis, pancreatitis, ulcer gastroduodenal, irritable bowel syndrome.
• the invention also relates to the composition according to the invention for its use as a medicament, in the prevention and/or treatment of at least one disease chosen from:
• Metabolic diseases chosen from non-insulin-dependent diabetes, gestational diabetes, NASH, hepatic steatosis, pancreatic steatosis, hyperlipidemia, hypercholesterolemia, infertility linked to overweight, urinary incontinence linked to overweight,
• vascular diseases chosen from among atherosclerosis, thrombopathies, acute pericarditis and chronic constrictive pericarditis, arterial hypertension, vasculitis
• liver and bile ducts chosen from hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, cirrhosis, hepatic encephalopathy, vesicular lithiasis
• osteopenia chosen from osteopenia, osteoporosis, osteoarthritis, inflammation of the vertebral discs
• Alzheimer's disease Parkinson's disease
• motor neuron diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and Kennedy's disease
• cancers linked to metabolism and/or dysbiosis of the microbiota chosen from hepatocarcinomas, cancers of the digestive tract such as esophagus, stomach and colorectal cancer, pancreatic carcinoma, neuroendocrine tumors (NET) of the gastroenteropancreatic system, hepatic tumors, tumors of the gallbladder and bile ducts, kidney tumors, glioblastomas, lymphomas, multiple myelomas, chronic myeloid leukemia, chronic myeloproliferative diseases, lung carcinomas
• hepatocarcinomas cancers of the digestive tract such as esophagus, stomach and colorectal cancer
• pancreatic carcinoma neuroendocrine tumors (NET) of the gastroenteropancreatic system
• hepatic tumors tumors of the gallbladder and bile ducts
• kidney tumors glioblastomas
• lymphomas multiple myelomas
• chronic myeloid leukemia chronic myelop
• autoimmune diseases chosen from insulin-dependent diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, systemic lupus erythematosus, polyendocrine autoimmune syndrome
• atopic dermatological diseases chosen from eczema, psoriasis - chronic inflammatory bowel diseases, chosen from Crohn's disease, ulcerative-hemorrhagic recto-colitis, diverticulitis, esophagitis, gastritis, pancreatitis, peptic ulcer, irritable bowel syndrome
• - respiratory function disorders chosen from asthma, cystic fibrosis, chronic obstructive pulmonary diseases and chronic bronchitis, interstitial lung diseases and pulmonary fibrosis, sleep apnea syndrome (OSAS)
• - pneumonia chosen from infectious pneumonia, influenza pneumonia and avian flu, severe acute respiratory syndrome (SARS), Pneumocystis pneumonia
• Infectious diarrhoea chosen from Clostridium difficile infection, EHEC infection, Salmonella gastroenteritis, Campylobacter enteritis, food poisoning by enterotoxin-producing bacteria, cholera, yersiniosis, shigellosis, cryptosporidiosis, listeriosis
• nephrology linked to a dysbiosis of the microbiota, chosen from urethritis, chronic renal failure, urolithiasis
• the invention also relates to a non-therapeutic use of the composition according to the invention for maintaining and/or strengthening the intestinal microbiota and/or supporting the diversity of the intestinal microbiota and/or promoting the increase in beneficial bacteria in the intestinal microbiota and/or promote weight loss in a healthy subject.
• the composition aims to strengthen the intestinal microbiota and/or support the diversity of the intestinal microbiota and/or promote the increase of beneficial bacteria in the intestinal microbiota and/or promote weight loss in a healthy subject
• the composition is in the form of a food product, a drink, a nutraceutical, food additive, food supplement or dairy product.
• the invention is now illustrated by examples of useful bacteria according to the invention, methods for culturing these bacteria, examples of compositions containing them and test results demonstrating their effectiveness, presented solely as an indication. 'drawing.
• Example 1 Characterization of C. minuta according to the invention filed under number DSM 33715.
• the bacterial strain C. minuta DSM 33715 can be characterized according to the following methods.
• the native bacteria of the DSM 33715 strain (10 ⁇ L of culture medium dosed at 10 9 CFU/mL) were deposited on ionized carbon grids (Delta Microscopy, Toulouse, France) and a negative staining was carried out with Nano-Tungsten (Nano-W, Nanoprobes, LFG Distribution, France).
• the oxidase activity was determined using oxidase test strips (Sigma-Aldrich) by evaluating the color change after adding a drop of liquid bacterial culture.
• the catalase activity was determined at two concentrations of FbCh (3 and 15%), added to a colony of DSM 33715, the catalase status being assessed visually by the formation of bubbles.
• Bile and pH tolerance were evaluated in liquid modified GAM (HyServe) medium.
• media containing 0-2-4-6 and 8% Oxgall (Difco Laboratories) were used, corresponding to 0-20-40-60 and 80% bile, respectively.
• pH tolerance media at pH 4-5-6-7 and 8 were used. All the media were degassed in the anaerobic chamber before inoculation with 10 ⁇ l of a McFarland 0.5 bacterial suspension. The optical density at 600 nm was taken at Oh, 24h, 48h, 72h and 96h.
• the antibiotic resistance tests were carried out using the minimum concentration (MIC) according to the reference method of dilution in agar medium of the CLSI (Clinical and laboratory Standards Institute).
• Stock solutions of each antibiotic were prepared and sterilized by filtration at 0.2 ⁇ : ampicillin (Sigma-Aldrich), tetracycline (Sigma-Aldrich, Chloramphenicol (Sigma-Aldrich), clindamycin (Sigma-Aldrich), meropenem (USP) , piperacillin (USP), tazobactam (USP), and ceftriaxone (USP) Brucella agar medium (BD BBL) supplemented with 5 ⁇ g/ml hemin (Sigma-Aldrich), 1 ⁇ g/ml vitamin Kl (Sigma-Aldrich) Aldrich) and 5% (v/v) hemolyzed (laced) sheep blood (Hemostat) was used as test medium.Agar plates with different MIC
• the inoculum of DSM 33715 was resuspended in McFarland's standard 0.5 in peptone water. 2 ⁇ l of the inoculum were deposited on the surface of the agar (each spot corresponding approximately to 10 5 CFU/ml). triplicate is carried out by concentration of antibiotic. The dishes were then incubated for 6 days at 37°C under anaerobic conditions. eu. MIC values were determined according to CLSI guidelines and were interpreted as susceptible, intermediate, or resistant, based on CLSI anaerobe breakpoints.
• the DSM 33715 strain is strictly anaerobic with an exponential growth phase between 20 and 40 h and a stationary phase beginning at 40 h, non-motile, non-spore forming, negative for oxidase and catalase activity and is gram negative. DSM 33715 is negative for the majority of the enzymes tested, except for the acidification of glucose, xylose, rhamnose and arabinose, which is consistent with the description of Christensenella minuta made by Morotomi et al.
• DSM 33715 also shows growth in a medium with a pH ranging from 6 to 9. Finally, the DSM 33715 strain shows resistance to ampicillin and tetracycline.
• FIG. 1A represents the image of a bacterium Christensenella minuta according to the invention produced by transmission electron microscopy in negative staining (magnification: 36kX).
• the stump has a short shape with the ends tapered, occurring in singlet, pairs or rosettes.
• the strain according to the invention can also be characterized by the analysis of a MALDI spectrum.
• a pure culture of the DSM 33715 strain is incubated in an agar medium and subjected to matrix-assisted laser desorption-ionization (MALDI) analysis using a MALDI Biotyper instrument (Bruker) and using the standard method recommended by the manufacturer.
• MALDI matrix-assisted laser desorption-ionization
• FIG. 1 Identification by MALDI is a known method for identifying a bacterial colony at the genus and species level. This is characteristic of the molecular composition of the surface of said bacterium.
• Figure IB represents the characteristic MALDI spectrum of a pure culture of Christensenella minuta according to the invention (DSM 33715). This makes it possible to confirm that said strain belongs to the species C. minuta and thus to validate the purity of the culture.
• Example 2 Comparison between the strain according to the invention (DSM 33715) and strains of C. minuta of the prior art.
• the inventors also compared the strain according to the invention (DSM 33715) with known strains of C. minuta such as the reference strain deposited under number DSM 22607, the strain deposited under number DSM 32891 and the strain filed under DSM number 33407 according to several parameters such as 16S rDNA sequence, genome size in base pair (bp), phylogenetic distance based on the whole genome (ANI), coding sequence number (CDS ) and examples of strain-specific genes.
• DSM 33715 known strains of C. minuta
• known strains of C. minuta such as the reference strain deposited under number DSM 22607, the strain deposited under number DSM 32891 and the strain filed under DSM number 33407 according to several parameters such as 16S rDNA sequence, genome size in base pair (bp), phylogenetic distance based on the whole genome (ANI), coding sequence number (CDS ) and examples of strain-specific genes.
• the strain according to the invention (DSM 33715) has the same 16S rDNA sequence as the three other strains of C. minuta already known, despite different metabolic properties and anti-inflammatory potential.
• the size of the genome, the whole of the genome, the number of coding sequences and certain genes specific to said strain make it possible to characterize and differentiate between the strains.
• the strain according to the invention has the largest genome and a greater number of coding sequences than the strains already known.
• Example 2 Anti-inflammatory effect of the strain according to the invention.
• the inventors studied the pro- and anti-inflammatory immunomodulatory effect of the DSM 33715 strain.
• the study protocol is as follows.
• PBMCs from 3 healthy donors were seeded in 24-well plates at 1 ⁇ 10 6 with 11 ⁇ l/well in RPMI medium (Gibco) supplemented with 2% fetal calf serum (FCS, Gibco) and incubated at 37°C/5% CO2 for 24 hours in the presence of the DSM 33715 bacterium at an MOI of 50 or bacterial medium-Glycerol as a control. After 24 h, the cell culture supernatants were collected and stored at ⁇ 80° C. until quantification of IL-10 by ELISA. The IL-10 assay was performed by specific BioLegend ELISA, according to the manufacturer's instructions. Absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech.
• the THP-1 cells (ECACC) were seeded at 5 ⁇ 10 5 cells/well in 24-well plates in RPMI medium (Gibco) supplemented with 10% FCS (Gibco) in the presence of 100 ng/ml of PMA ( Enzo Life Sciences) for 48 hours, in order to differentiate monocytic cells into MO type macrophages.
• the cells were then washed with PBS-1X (Gibco) and cultured for an additional 24 hours in RPMI medium with 2% FCS.
• the cells were then stimulated for 24 hours with the bacterium DSM 33715 at an MOI of 50 or with PBSIX-Glycerol as a control. After 24 h, the cell culture supernatants were collected and stored at ⁇ 80° C.
• IL-10 assay was performed by specific BioLegend ELISA, according to the manufacturer's instructions. Absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech.
• the chemokine IL-10 is an anti-inflammatory cytokine.
• the values represent the secretion of IL-10 induced by the strain according to the invention (DSM 33715).
• DSM 33715 induces a production of IL-10 greater than a reference strain of C. minuta DSM 22607 (FIG. 2A).
• the induction of production of IL-10, an anti-inflammatory cytokine, by DSM 33715 was also demonstrated by the THP-1 cell model differentiated into macrophage type MO by treatment with PMA (FIG. 2B) .
• the DSM 33715 strain thus exhibits a greater anti-inflammatory immunomodulatory effect than the reference strain of C. minuta and therefore has a high therapeutic potential in inflammatory diseases.
• Example 3 Anti-inflammatory effect of the strain according to the invention.
• the inventors studied the anti-inflammatory effect of the DSM 33715 strain on a colon adenocarcinoma line HT-29 producing IL-8 after TNF- ⁇ stimulation.
• the HT-29 cells (ECACC) were seeded in 24-well plates at 3x10 5 cells/well in McCoy's 5A medium (Gibco) supplemented with 10% fetal calf serum (FCS, Gibco) and incubated at 37° C/5% CO2 for 24 hours. The cells were washed with PBS-1X (Gibco) and cultured for an additional 24 h in McCoy's 5A medium at 2% FCS. The cells were then stimulated for 6 hours with 5 ng/ml of TNF-a in the presence of 50 bacteria per 1 HT-29 cell (MOI 50) of DSM 33715 or 10% of supernatant or PBSIX-Glycerol or culture medium bacterial as controls (ctrl).
• DSM 22607 is the reference strain.
• 5-ASA (20 mM) is an anti-inflammatory agent used in control.
• the cell culture supernatants were collected and stored at ⁇ 80° C. until quantification of IL-8 by ELISA.
• the IL-8 assay was performed by specific BioLegend ELISA, according to the manufacturer's instructions. Absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech. 6 independent experiments were carried out in duplicate.
• the HT-29 cells were stimulated with the pro-inflammatory cytokine TNF-a and the DSM 33715 bacterium at an MOI of 50 at the start of the stationary phase of growth or of its supernatant, for 6 hours.
• the chemokine IL-8 is secreted by HT-29 in a TNF-a (Ctrl)-induced inflammatory state.
• the values represent the average normalized in percentage compared to the control + TNF-a (PBS-Glycerol for the bacteria and the bacterial culture medium for the supernatant), representing the secretion of IL-8 induced by stimulation with TNF-a, i.e. 100%.
• the secretion of IL-8 is reduced by 50% compared with the control (p ⁇ 0.0001, Dunnett's multiple comparisons test).
• the DSM 33715 strain and its supernatant exhibits an anti-inflammatory immunomodulatory effect.
• Example 4 Anti-inflammatory effect of the strain according to the invention.
• the inventors studied the anti-inflammatory effect of the DSM 33715 strain on the NF-0B and JAK/STAT inflammation signaling pathways.
• Transfection complexes were mixed gently and incubated at room temperature for 15 minutes. After incubation, the transfection complexes were added to freshly resuspended HT-29 cells at the appropriate density in McCoy's 5A medium supplemented with 10% FCS, then the cell/transfection complex mixtures were gently homogenized and plated. The plates were incubated for 24 hours in a humid atmosphere at 5% CO2 and the medium was then replaced with medium containing 2% FCS for 12 hours, after washing in PBS IX.
• the medium was removed and replaced with 2% FCS medium +/- 5 ng/ml of TNF-a (NF-0B reporter system) or 100 pg/ml of IFN- ⁇ (system JAK/STAT reporter) and 10% of DSM 33715 supernatant or bacterial culture medium as control or the specific inhibitor of the signaling pathway (5 pM BAY11-7082 for the NF-0B pathway and 1 pM Tofacitinib for the JAK/STAT).
• TNF-a NF-0B reporter system
• IFN- ⁇ system JAK/STAT reporter
• the cells were washed with PBS IX and lysed in 50 ⁇ l of passive lysis buffer (Promega) for 15 min at room temperature with orbital agitation, the protein lysates were then transferred into microtubes.
• a Luciferase reporter assay (Dual-Luciferase reporter assay, Promega) was performed with minor modifications to the manufacturer's instructions. Briefly, 2x20 ⁇ l of lysate was transferred to a white 96-well plate, and 50 ⁇ l of LAR II reagent was added. Firefly luciferase activity was read.
• the supernatant of DSM 33715 leads to a decrease in the activation of the NF-0B pathway induced by TNF- ⁇ by 25% and a decrease in the activation of the JAK/STAT pathway induced by IFN- y of 40%.
• these pathways are particularly relevant because they are hyperactive in patients with chronic inflammation such as Crohn's disease, but also certain cancers.
• the N F-KB pathway is involved in cancers of epithelial origin, such as breast cancer; in gastrointestinal cancers, such as colorectal cancer.
• Jak/STAT pathway is overactivated in mainly solid cancers, such as breast, lung, liver, head, neck and stomach cancer.
• Example 5 Anti-inflammatory effect of the strain according to the invention in combination with an anti-inflammatory agent.
• the inventors studied the anti-inflammatory effect of the DSM 33715 strain in combination with 5-ASA or Budesonide.
• the HT-29 cells (ECACC) were seeded in a 24-well plate at 3 ⁇ 10 5 cells/well in McCoy's 5A medium (Gibco) supplemented with 10% fetal calf serum (FCS, Gibco) and incubated at 37° C/5% CO2 for 24 hours.
• the cells were washed with PBS-1X (Gibco) and cultured for an additional 24 h in McCoy's 5A medium at 2% FCS.
• the cells were then stimulated for 6 hours with 5 ng/ml of TNF- ⁇ in the presence of 10% of stationary phase supernatant of DSM 33715 or of bacterial culture medium as control (ctrl), in the presence or not of agents anti-inflammatories.
• 5-ASA (10 mM) and budesonide (20 pM) are molecules used in the treatment of inflammatory diseases, such as rectocolitis and Crohn's disease.
• the cell culture supernatants were collected and stored at ⁇ 80° C. until quantification of IL-8 by ELISA.
• the IL-8 assay was performed by specific BioLegend ELISA, according to the manufacturer's instructions. Absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech. Statistics: Sidak's multiple comparisons test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
• the HT-29 cells were stimulated with the pro-inflammatory cytokine TNF-a and 10% of the DSM supernatant. 33715 in the presence or absence of 5-ASA or Budesonide, for 6 hours.
• the chemokine IL-8 is secreted by HT-29 in a TNF-a (Ctrl)-induced inflammatory state.
• the values represent the average normalized in percentage compared to the control + TNF-a, representing the secretion of IL-8 induced by stimulation with TNF-a, that is to say 100%.
• Example 6 Anti-inflammatory effect of the strain according to the invention in combination with a JAK inhibitor.
• the inventors studied the anti-inflammatory effect of DSM 33715 associated or not with Tofacitinib on the modulation of the signaling pathway of JAK/STAT inflammation induced by IFN- ⁇ .
• Transfection complexes were mixed gently and incubated at room temperature for 15 minutes. After incubation, the transfection complexes were added to freshly resuspended HT-29 cells at the appropriate density in McCoy's 5A medium supplemented with 10% FCS, then the cell/transfection complex mixtures were gently homogenized and plated. The plates were incubated for 24 hours in a humid atmosphere at 5% CO2 and the medium was then replaced with medium containing 2% FCS for 12 hours, after washing in PBS IX.
• the medium was removed and replaced with 2% FCS medium +/- 100 pg/ml IFN- ⁇ (JAK/STAT reporter system) and 10% DSM 33715 supernatant or medium of bacterial culture as a control or the specific inhibitor of the signaling pathway (5 pM BAY11-7082 for the NF-0B pathway and 1 pM Tofacitinib for the JAK/STAT pathway).
• the cells were washed with PBS IX and lysed in 50 ⁇ l of passive lysis buffer (Promega) for 15 min at room temperature with orbital agitation, the protein lysates were then transferred into microtubes.
• a Luciferase reporter assay (Dual-Luciferase reporter assay, Promega) was performed with minor modifications to the manufacturer's instructions. Briefly, 2x20 ⁇ l of lysate was transferred to a white 96-well plate, and 50 ⁇ l of LAR II reagent was added. Firefly luciferase activity was read. Then 50 ⁇ l of Stop&GIo reagent were added and the Renilla activity measurement was read with the FLUOstar Omega plate reader (BMG Labtech). The results were reported as the Firefly/Renilla ratio. Statistics: Sidak's multiple comparisons test. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001 [0165] Results
• the supernatant of DSM 33715 leads to a decrease in the activation of the JAK/STAT pathway induced by IFN- ⁇ by 40%, Tofacitinib to a decrease by 49% and the association of the supernatant with Tofacitinib leads to a decrease in the activation of the JAK/STAT pathway induced by IFN- ⁇ , by 64%. There is therefore an enhanced effect of supernatant and Tofacitinib in modulating IFN- ⁇ -mediated inflammation.
• Example 7 Effect of the strain according to the invention in the protection of the intestinal epithelium.
• the inventors studied the effect of the DSM 33715 strain on the integrity of the intestinal barrier by measuring the transepithelial resistance.
• the Caco-2 cells were seeded at 2 ⁇ 10 4 cells per well in 24-well plates containing inserts with a transwell polyester permeable membrane (Corning Life Science). The cells were cultured in DMEM medium supplemented with 20% fetal calf serum (FCS, Gibco) and incubated at 37° C./5% CO2. Transepithelial electrical resistance was checked daily from the seventh day of culture using an EVOM3 (WPI) ohmmeter. The medium was replaced every 2 days for 8 to 10 days, until the optimum TEER was reached (2000 Q.cm 2 ).
• FCS fetal calf serum
• the cells were treated in the apical compartment with DSM 33715 or DSM 22607 at an MOI of 50 or PBS IX / Glycerol 10% (control) for 3 hours, then 100 ng/ml of TNF-a was added or not in the basal compartment.
• the TEER was measured just before the addition of TNF-a (T0) and 6 h (T6H) after the addition of TNF-a.
• the polarized monolayer Caco-2 cells were pretreated with DSM 33715, then sensitized with TNF- ⁇ .
• TEER was compared to T0 (before addition of TNF) and T6H. Values represent mean normalized to control - TNF- ⁇ , representing basal TEER.
• treatment of Caco-2 with TNF-a induced an increase in permeability (Ctrl+TNF-a).
• the DSM33715 strain is able to restore the barrier epithelial, with a return to the basal level.
• Example 8 Effect of the strain according to the invention in an in vivo model of colitis.
• Sprague Dawley rats were anesthetized for 2 hours and received an intra rectal injection of TNBS (Trinitrobenzene sulfonic acid, 80 mg/kg dissolved in 40% ethanol).
• TNBS Trinitrobenzene sulfonic acid, 80 mg/kg dissolved in 40% ethanol.
• the animals were sacrificed 4 days after the injection of TNBS.
• DSM 33715 was administered orally at a dose of 10 9 CFU/ml, gavage starting 14 days before the induction of colitis and until the day of euthanasia.
• the animals of the "positive control" group received the Pentasa mixed in their feed, at a dose of 150 mg/kg.
• the anti-inflammatory effect of the products tested was evaluated at sacrifice, following a total treatment period of 18 days.
• the variations in weight were monitored throughout the entire period of the experiment. Colon length was assessed on the day of autopsy. Macroscopic and microscopic scores were performed by two different operators to validate the scores. The colon of each rat was examined to assess macroscopic lesions according to the Wallace score.
• the Wallace score is an assessment of gross lesions on a scale of 0 to 10, and this score is based on characteristics reflecting inflammation, such as hyperemia, bowel thickness, and extent of ulcerations .
• Inflammation was assessed in the colon by measuring the production of IL-ip (pro-inflammatory cytokine) (eBioscience kit), as well as the level of Lipocalin-2 (Lcn-2), marker of neutrophil infiltration (Clinisciences) by ELISA quantification. Briefly, one centimeter of distal colon was collected and homogenized in a Tris-HCl solution containing a cocktail of protease inhibitors (Sigma-Aldrich) using the Precellys homogenizer using ceramic beads (1.4 and 2.8mm). The samples obtained were centrifuged for 20 min and the supernatants collected and stored at -80° C., until their use for the ELISAs.
• IL-ip pro-inflammatory cytokine
• Figure 8A The inventors observed a significant decrease in the length of the colon in the group of rats treated with TNBS and having received the vehicle as treatment compared to the control group having received the vehicle, as expected. A significant increase in colon length was seen in the group of TNBS-treated rats given the DSM 33715 strain compared to the TNBS-Vehicle group. The DSM 33715 strain has a protective effect on the colon in the TNBS-induced colitis model.
• Figure 8B The intensity of the inflammation and lesions of the colon was evaluated at the macroscopic level according to the Wallace score. A significant increase in the Wallace score was observed in the rats of the TNBS-Vehicle group compared to the group of "healthy" rats, with a score of 7.00+/-0.41 vs 0.0+/-0 respectively. .0, p ⁇ 0.0001. This result indicates that the rats exhibited severe colitis 4 days after induction with an ulcerative or inflammatory site greater than 3 cm from the colon (corresponding to a score of 7, on a scale ranging from 0 to 10). A significant decrease in the score was observed in the TNBS-Pentasa group and the TNBS-DSM 33715 group.
• FIG. 8D The level of lipocalin-2, a marker of neutrophil infiltration, is significantly increased in colic rats (TNBS-Vehicle) compared to the healthy control, confirming the development of severe colitis. A decreasing trend is observed for Pentasa and a significant decrease in LCN-2 is observed in the group of TNBS rats treated with the DSM 33715 strain.
• the strain according to the invention has a certain therapeutic potential in intestinal dysbiosis and in particular in the prevention and/or treatment of inflammatory diseases.
• Example 9 Comparative test in an in vivo model of colitis
• the inventors studied the in vivo effect of the strain according to the invention and of the reference strain C. minuta (DSM22607) in a model of colitis induced by TNBS in the rat.
• the test was carried out according to the same protocol as that described in Example 8.

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