Bacterial strain in the prevention and / or treatment of inflammatory diseases

Abstract

The present invention relates to a new bacterial strain belonging to the genus Christensenella, a composition comprising it and its uses, in particular as a drug in the treatment and / or prevention of diseases such as metabolic diseases and / or inflammatory diseases.

Description

Title of the invention: Bacterial strain in the prevention and / or treatment of inflammatory diseases. Technical field

[0001] The invention relates to a new bacterial strain belonging to the genus Christensenella, a composition comprising it and its uses, in particular as a drug in the treatment and / or prevention of diseases such as metabolic diseases and / or inflammatory diseases.

[0002] State of the art

[0003] Our gut microbiota, or "intestinal flora," consists of a collection of bacteria, viruses, parasites, and non-pathogenic fungi, representing 10¹² to 10¹⁴ microorganisms present in the digestive tract of each individual, which is 2 to 10 times more than the number of cells in our body. Consequently, its role has been increasingly studied in recent years. In 2012, a microbiologist identified and cultured a new species from human feces: Christensenella minuta, belonging to the Gram-negative Clostridiales. (Morotomi et al., Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposai of Christensenellaceae fam. nov., International Journal of Systematic and Evolutionary Microbiology (2012), 62, 144-149).

[0004] It is now accepted by the scientific community that the human gut microbiota is linked to the development of certain so-called "non-communicable" diseases associated with microbiota dysbiosis, such as obesity, diabetes, metabolic diseases, inflammatory bowel diseases, and depression. However, the prevalence of these diseases continues to rise, without any satisfactory treatment.

[0005] Inflammatory bowel disease (IBD) encompasses diseases related to chronic intestinal inflammation. Crohn's disease and ulcerative colitis are the main examples. These are recent, multifactorial diseases of unknown origin. In a number of cases, the onset of these diseases may be linked to a genetic predisposition, the environment, or the microbiota. These diseases all share the characteristic of presenting with intestinal inflammation, which induces a modification of the microbiota and thus perpetuates the inflammation.

[0006] In Crohn's disease, inflammation is most often concentrated in the ileum and colon. Ulcerative colitis, on the other hand, more specifically affects the distal end of the digestive tract. that is to say the colon and the rectum. Its incidence has remained stable, at 4.4 cases per 100,000 inhabitants in France, while that of Crohn's disease increased from 5.3 to 7.6 cases per 100,000 inhabitants in France between 1988 and 2014.

[0007] These are debilitating diseases for which no curative treatment is available and whose prevalence is increasing sharply in Western countries. These diseases are characterized by flare-ups with remission periods of varying lengths. The goal of current treatments is to prolong the duration of remissions as much as possible and thus improve the quality of life of patients despite lifelong treatment.

[0008] Despite progress in understanding and diagnosing these pathologies, modern medicine does not have a satisfactory curative solution, which causes major public health problems, particularly among adolescents whose prevalence has increased sharply in recent years.

[0009] There is therefore a strong medical need for a product capable of acting on the intestinal microbiota, particularly in the prevention and treatment of chronic inflammatory bowel diseases.

[0010] An objective of the present invention is therefore to provide a solution that is simple, effective and economical for restoring a healthy intestinal microbiota and thus treating intestinal microbiota dysbiosis, in particular treating chronic inflammatory diseases, preferably avoiding the administration of lifelong treatment and thus reducing the prevalence of these diseases. Summary of the invention

[0011] To meet this objective, the inventors identified, among the multitude of bacteria present in the intestinal microbiota and among the already known strains belonging to the species Christensenella minuta, a new specific bacterial strain of Christensenella minuta deposited with the Leibniz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, in French German Collection of Microorganisms and Cell Cultures GmbH) under the number DSM 33715, particularly suitable in the prevention and / or treatment of intestinal dysbiosis, preferentially chronic inflammatory bowel diseases.

[0012] Thus, the invention relates to a bacterial strain of Christensenella minuta filed under 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% ANI (Average Nucleotide Identity) identity with the nucleotide sequence of the genome of the bacterial strain DSM 33715.

[0013] The invention also relates to the supernatant of the bacterial strain culture 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 being preferably a pharmaceutically acceptable excipient when it concerns a product intended for use as a medicinal product for human or veterinary use.

[0014] Finally, the invention is particularly suitable for using said bacterial strain, said supernatant, or said composition according to the invention as a medicinal product. Indeed, the present invention is particularly suitable for the prevention and / or treatment of so-called "non-communicable" diseases associated with dysbiosis of the gut microbiota, or diseases associated with dysbiosis of the gut microbiota, in particular diseases such as metabolic diseases, inflammatory diseases, and others that will be detailed below.

[0015] Preferably, the invention aims to prevent and / or treat chronic inflammatory bowel diseases, more preferably Crohn's disease, or ulcerative colitis.

[0016] Finally, 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 to maintain and / or 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 in a healthy subject.

[0017] Other features and advantages will become apparent from the detailed description of the invention, the examples and figures that follow. Brief description of the Figures

[0018] [Fig.1A] represents an image of a Christensenella minuta bacterium according to the invention produced by transmission electron microscopy in negative staining (magnification: 36kX).

[0019] [Fig.1B] represents a MALDI spectrum characteristic of a pure culture of a Christensenella minuta strain according to the invention.

[0020] [Fig.2A] represents the immunomodulatory effect of the bacterium DSM 33715 on PBMCs (3 healthy donors tested)

[0021] [Fig.2B] represents the anti-inflammatory effect of the DSM 33715 bacterium on a THP-1 cell line differentiated into MO-type macrophages by treatment with PMA (Phorbol 12-myristate 13-acetate).

[0022] [Fig.3] represents the immunomodulatory effect of the bacterium DSM 33715 and its floating on the IL-8-producing HT-29 colon adenocarcinoma cell line after TNF-α stimulation.

[0023] [Fig.4A] represents the anti-inflammatory effect of DSM 33715 on the NF-κB inflammatory signaling pathway. Activation of the NF-κB pathway by DSM 33715 in HT-29 cells transfected with a reporter system and stimulated by TNF-α, measured by luciferase activity and normalized by Renilla luminescence.

[0024] [Fig. 4B] represents the anti-inflammatory effect of DSM 33715 on the JAK / STAT inflammatory signaling pathway. Activation of the JAK / STAT pathway by DSM 33715 in HT-29 cells transfected with a reporter system and stimulated by ITFN-γ, measured by luciferase activity and normalized by Renilla luminescence.

[0025] [Fig.5] represents the immunomodulatory effect of the DSM 33715 supernatant on the HT-29 colon adenocarcinoma cell line producing IL-8 after stimulation with TNF-a, in the presence of an anti-inflammatory agent used in therapy, 5-ASA or Budesonide.

[0026] [Fig.6] represents the anti-inflammatory effect of DSM 33715 associated or not with Tofacitinib on the modulation of the ITFN-γ-induced JAK / STAT inflammatory signaling pathway. The JAK / STAT pathway is activated in HT-29 cells transfected with a reporter system and stimulated by ITFN-γ, as measured by luciferase activity and normalized by Renilla luminescence.

[0027] [Fig.7] represents the effect of the DSM 33715 strain on barrier integrity Intestinal by measurement of transepithelial electrical resistance. The PBS-Glycerol + / - TNF-α (control) condition corresponds to the bacterial resuspension medium.

[0028] [Fig.8A] is a histogram representing the average length of the colons at the end of the experiment. The data are represented by the mean + / - SEM in centimeters for length and in grams for colon weight.

[0029] [Fig.8B] represents colon inflammation at the macroscopic level calculated by the Wallace score.

[0030] [Fig. 8C] represents inflammation at the histological level calculated by the Ameho score. The data shown are the means + / - SEM. MGG staining of colon sections for histological scoring (Ameho)

[0031] [Fig. 8D] represents the levels of IL-1 [3] and Lipocalin-2 in the colon. The data shown correspond to the mean + / - SEM in ng / mg of protein. Detailed description of the invention

[0032] Definition

[0033] For the purposes of this invention, "bacteria" means a unicellular microorganism capable of reproducing by cell division. Bacteria are classified by family, genus, and species. Each bacterial species comprises a diversity of bacterial strains. The bacterial strain according to the invention belongs to the family Christensenellaceae, the genus Christensenella, and the species minuta. Therefore, "bacterial strain" or "strain" for the purposes of this invention means not only a specific bacterial strain but also all bacteria derived from, obtained from, or corresponding to the bacterial strain and having the same metabolic functions; for example, at least one bacterium taken from a colony derived from the strain. Therefore, "bacteria according to the invention" for the purposes of this invention also means a bacterial strain according to the invention.

[0034] For the purposes of this invention, "derived bacterial strain" or "mutant strain" means a bacterial strain having a high degree of similarity to the bacterial strain filed under DSM 33715. Preferably, the strain comprises a nucleotide sequence having at least 99.90% ANI identity with the nucleotide sequence of 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% ANI identity.

[0035] By "supernatant" in the sense of the invention, we mean the culture supernatant of the bacterial strain according to the invention, possibly comprising cellular compounds of said strain and / or cellular debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0036] By "prevention" in the sense of the invention, we mean the reduction to a lesser degree of the risk or 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.

[0037] For the purposes of this invention, "treatment" means a reduction in disease progression, stabilization, reversal or regression, or even an interruption or inhibition of the progression of intestinal microbiota dysbiosis and associated diseases, more preferably chronic inflammatory diseases, for example Crohn's disease or ulcerative colitis. In the context of this invention, these terms also apply to one or more symptoms of the diseases described in this invention.

[0038] For the purposes of this invention, a "physiologically acceptable medium" is defined as a medium that is compatible with the organism of the individual to whom the composition is to be administered. This could be, for example, a non-toxic solvent such as Water, buffer, saline solutions. In particular, said medium is compatible with oral administration.

[0039] For the purposes of this invention, an "acceptable excipient" is any compound that facilitates the formulation of the composition and does not alter the nature of the biological activity of the active ingredient. An acceptable excipient may be a solvent, buffer, saline solution, plasticizer, lubricant, dispersion medium, absorption retardant, flow agent, or isotonic agent. Preferably, these are pharmaceutically acceptable excipients chosen according to the desired pharmaceutical form and route of administration, from among 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, etc., administration.

[0040] For the purposes of this invention, "disease associated with dysbiosis of the gut microbiota" means diseases associated with dysbiosis of the gut microbiota such as certain metabolic diseases, for example diseases related to obesity including obesity, diabetes, NASH, hepatic or pancreatic steatosis; inflammatory diseases, in particular chronic inflammatory bowel diseases, for example Crohn's disease, ulcerative colitis, diverticulitis, gastritis, pancreatitis, or irritable bowel syndrome; but also chronic diseases associated with dysbiosis, cardiac and vascular diseases, cancers, for example cancers related to metabolism.

[0041] For the purposes of this invention, "gut microbiota dysbiosis" refers to an imbalance in the gut microbiota that could promote the initiation, maintenance, or severity of inflammation. For example, in approximately 5% of patients with Crohn's disease, a family of Escherichia coli (AIEC) is found that adheres more strongly to the cells of the intestinal wall and is more invasive than the usual strains, thus facilitating a local inflammatory response. Potential causes of dysbiosis could be dietary (a high-fat, high-sugar diet, lacking fiber, which limits beneficial short-chain fatty acid-producing bacteria), infectious (acute episodes of infectious gastroenteritis), or environmental (repeated antibiotic treatments, insufficient exposure to pathogens during childhood).

[0042] For the purposes of this invention, "anti-inflammatory agent" means a molecule, an active ingredient, a medicinal product, or a pharmaceutical composition intended to combat inflammation. In particular, it refers to a group of medicinal products intended to treat an inflammatory reaction and the resulting diseases, such as chronic inflammatory bowel diseases. Examples of anti-inflammatory drugs include corticosteroids (or glucocorticoids or steroidal anti-inflammatory drugs) and non-steroidal anti-inflammatory drugs.

[0043] By "healthy subject", in the sense of the invention, we mean a subject who is not ill and not suffering from any 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.

[0044] Bacterial strain or supernatant according to the invention

[0045] The inventors discovered the bacterial strain Christensenella minuta, registered under DSM number 33715, then isolated it from the 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 increased production of IL-10, an anti-inflammatory cytokine; and a decrease in certain signaling pathways such as the NF-κB pathway or the JAK / STAT pathway. Results in a preclinical model of TNBS-induced colitis in rats confirm the anti-inflammatory effects of the strain, particularly on chronic inflammatory bowel diseases.

[0046] The inventors have also demonstrated that said bacterial strain DSM 33715 restores or strengthens, and thus preserves, the integrity of the intestinal barrier. Finally, a synergistic effect has been established when the strain is used in combination with clinically used anti-inflammatory agents such as Pentasa (5-ASA), Tofacitinib, or Budesonide, thus allowing for the administration of lower doses of such anti-inflammatory agents, which are known for their toxicity and adverse effects in patients.

[0047] The present invention therefore relates to a bacterial strain of Christensenella minuta filed under the number DSM 33715, hereinafter referred to as strain DSM 33715, having the 16S rDNA sequence SEQ ID NO: 1.

[0048] 16S ribosomal RNA (16S rRNA) is the ribosomal RNA that constitutes the small subunit of prokaryotic ribosomes. 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 allows the reconstruction of the evolutionary history of organisms, particularly prokaryotes and bacteria.

[0049] The percentage of 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 well-known heuristic search method. It makes it possible to find similar regions between two or more nucleotide or amino acid sequences and to perform an alignment of these homologous regions.

[0050] However, the 16S rDNA sequence does not always allow differentiation between two strains of the same species that may have different properties, for example, anti-inflammatory properties. Therefore, a person skilled in the art, by virtue of their 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 Séquence d'ADN Codante), but also the identification of genes specific to said strain of interest.

[0051] For the purposes of this invention, "ANI" means the average percentage of nucleotide identity calculated from a pairwise comparison of all genome sequences shared between the two bacterial strains. According to the general knowledge of those skilled in the art, genomic DNA can be extracted from a pure bacterial culture of said strain of interest deposited with the DSMZ, followed by DNA sequencing using various well-known methods, for example Sanger, Roche 454, Illumina, Oxford Nanopore. The sequenced genome is then assembled using bioinformatics, and the resulting sequences are analyzed. Finally, the genomes of interest are compared pairwise to calculate the ANI.

[0052] According to one variant, 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% ANI identity with the nucleotide sequence of the bacterial strain DSM 33715.

[0053] Preferably 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% ANI identity with the nucleotide sequence of bacterial strain DSM 33715.

[0054] Such a strain is therefore a bacterial strain of Christensenella minuta derived from the bacterial strain filed under number DSM 33715 allowing to maintain or improve the described capabilities of strain DSM 33715 within the framework of the present invention.

[0055] Said derived strain can thus be produced naturally or intentionally, by mutagenesis methods known in the prior art. By way of example, mutagenesis methods that can be implemented within the framework of the present invention include the growth of the original microorganism in the presence of mutagenic or stress-producing agents, or by genetic engineering aimed at modifying specific or non-specific genes, such as targeted mutagenesis or random mutagenesis. According to one aspect of the invention, the strain derived from the DSM 33715 strain of C. minuta is a genetically modified mutant.

[0056] According to a particularly preferred embodiment, the invention relates to the Christensenella minuta strain filed under DSM number 33715 with The Leibniz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, in French Collection allemande de micro-organismes et de cultures cellulees GmbH). The said strain has the 16S rDNA sequence, the sequence SEQ ID NO: 1.

[0057] Another object of the invention relates to the culture supernatant obtained from the bacterial strain DSM 33715 and / or the derived strain. Preferably, the culture supernatant comprises at least one of the following constituents selected from a bacterial cell compound, bacterial cell debris, a metabolite and / or one or more molecules secreted by the strain and / or the derived strain, or combinations thereof.

[0058] Thus, cellular compounds and / or cellular debris can be cell wall components, nucleic acids, membrane components, proteins, lipids, etc. Metabolites or secreted molecules can be any molecule produced or modified by the bacterium due to its metabolic activity during growth or its use in technological processes (for example, but not limited to, food or drug production processes). For example, metabolites or molecules can be proteins, amino acids, enzymes, lipids, nucleic acids, etc. "Metabolite and / or molecule(s) secreted by the strain and / or the derived strain" means a molecule produced and exported or released outside the bacterium by the bacterium.

[0059] Thus the invention also relates to the culture supernatant obtained from the bacterial strain according to the invention.

[0060] Composition

[0061] The 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.

[0062] Thus, the invention relates to 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.

[0063] In particular, the composition comprises at least one bacterial strain filed under DSM number 33715 and / or at least one strain having at least 99.5% identity with the 16S rDNA sequence of strain DSM 33715 and / or the culture supernatant obtained from said strains and at least one acceptable excipient and / or a physiologically acceptable medium.

[0064] According to one embodiment, the composition may comprise the bacterial strain according to the invention in any form producing the desired effects and efficacy described in this application. In particular, the strain may be in live (culturable or not), dead, semi-live, attenuated, or inactivated form. The dead, semi-live, attenuated, or inactivated form may be obtained by various techniques known to those skilled in the art. Examples include: irradiation, thermal inactivation or freeze-drying, in particular by heat, exposure to a suitable pH, UV, gamma rays, X-rays or high pressure.

[0065] The term "semi-alive" refers to a bacterium with low physiological activity whose ability to proliferate is reduced, either temporarily or permanently. The term "inactivated" refers to a bacterium that is no longer capable of proliferating, either temporarily or permanently. The term "dead" refers to a bacterium that is no longer capable of proliferating, either permanently or permanently. Dead or inactivated bacteria may have intact or ruptured cell membranes. Thus, the term "inactivated" also refers to extracts and lysates of bacteria obtained from these products.

[0066] The composition according to the invention may also include at least one additional compound. An additional compound may, in particular, be an ingredient, a molecule, an active ingredient, a microorganism, a bacterium, or a mixture of bacteria.

[0067] Preferably, the additional compound is a microorganism different from the bacterial strain according to the invention or from the derived strain. More preferably, the microorganism is a bacterium of the genus Christensenella. By way of example, a bacterium belonging to the species C. minuta, C. timonensis, C. massiliensis, and mixtures thereof may be cited.

[0068] According to one variant, the additional compound is a probiotic and / or a prebiotic. The prebiotic may be, for example, at least one prebiotic selected from galactooligosaccharides, fructooligosaccharides, inulins, arabinoxylans, beta-glucans, lactoglobulins and / or beta-caseins.

[0069] According to another variant, the additional compound may be: - at least one lactic acid-producing bacterium that creates an anaerobic environment favorable to Christensenellaceae, such as at least one bacterium chosen from the genera Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. and / or - at least one butyric acid-producing bacterium that creates a favorable environment for Christensenellaceae, such as a bacterium from the genera Ruminococcaceae and Lachnos piraeaceae, - at least one other organism that promotes the anaerobic conditions necessary for the survival of Christensenellaceae, such as at least one yeast selected from Saccharomyces spp. or microorganisms from the Methanobacteriaceae family, and / or - at least one bacterium associated with the Christensenellaceae ecosystem because they facilitate their survival in the intestine, such as at least one chosen bacterium among the bacteria of the phylum Firmicutes, Bacteroidetes, Actinobacteria, Tenericutes, and Verrucomicrobia, and / or - at least one polyphenol such as, for example, at least one polyphenol selected from quercetin, kaempferol, resveratrol, flavones (such as luteolin), flavan-3-ols or catechins, flavanones (such as naringin), isoflavones, anthocyanins, oligo-proanthocyanidins, and / or - at least one mineral and / or at least one vitamin and / or at least one nutritional agent, and / or - at least one pharmaceutical active ingredient preferably chosen from among non-steroidal anti-inflammatory drugs, antibodies directed against pro-inflammatory targets (such as anti-TNF-alpha or anti-IL6), antirheumatics, analgesics, antimicrobials, corticosteroids, anabolic steroids, antidiabetics, thyroid agents, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietin, immunoglobulins, immunosuppressants, growth hormones, hormonal drugs, estrogen receptor modulators, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta-agonists, inhaled steroids, leukotriene inhibitors,Cromoglycates or cromoglycidic acids, epinephrine, dornase alpha, cytokines, cytokine antagonists, Janus kinase inhibitors, immunomodulators, lipid-lowering agents, cholesterol-lowering agents, antihypertensives.

[0070] When the additional compound is a microorganism different from the strain according to the invention, the microorganism is preferably chosen from Akkermansia muciniphila, Faecalibacterium prausnitzii, Anaerobutyryricum hallii, Hafnia alvei, Roseburia intestinalis, Roseburia hominis, Roseburia faecis, Roseburia inulinivorans, Dysosmobacter welbionis, Oscillospira guillermondii, Lact iplanti bacillus plantarum, Lact icaseibacillus casei, Latilactobacillus sakei, Ligilactobacillus salivarius, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Levilactobacillus brevis, Bifidobacterium pseudolongum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium stercoris and Bifidobacterium thermophilum.

[0071] When the additional compound is a pharmaceutical active ingredient, it is preferably an active ingredient selected from 5-AS A, mesalamine, olsalazine, budesonide, tofacitinib, filgotinib, upadacitinib, infliximab, adalimumab, golimumab, certolizumab, vedolizumab and ustekinumab.

[0072] Preferably, the composition according to the invention is in any form acceptable for administration to a subject, preferably a human or non-human animal subject. Thus, the composition according to the invention is also intended for veterinary uses.

[0073] Preferably, the composition according to the invention is in solid, liquid or lyophilized form.

[0074] When the composition is in liquid form, it may include bacterial strains according to the invention and / or the derived strain and / or a physiologically acceptable culture medium for said bacteria which allows them to be preserved, such as preferably Columbia agar anaerobic medium enriched with sheep blood, or an equivalent medium not containing any product derived from animal origin.

[0075] When the composition according to the invention is in solid form, the bacterial strains according to the invention and / or of the derived strain may be present in lyophilized form, and may also include excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, ramylum, calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin, galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, mannitol, sorbitol and / or glycerol.

[0076] According to another preferred embodiment, the composition according to the invention is in a form suitable for administration by oral, nasal, parenteral, rectal, sublingual, ocular, auricular, intramuscular, intravenous, inhaled or cutaneous route.

[0077] The composition can be in any suitable form. Thus, the composition according to the invention can be in a form selected from a powder, microencapsulated powder, capsule, tablet, lozenge, granules, emulsion, suspension, suppository, inhaler and a syrup.

[0078] According to a particularly suitable embodiment, the composition according to the invention may be in a gastro-resistant form, for example, a coated tablet containing microencapsulated bacteria. The composition may thus be provided with a gastro-resistant coating, i.e., resistant to gastric juices, in order to ensure that the bacterium(s) included in the composition can pass through the stomach. The release of the bacterium(s) can thus occur for the first time in the upper intestinal tract.

[0079] In a particularly preferred manner, the composition comprises 104 to 1012 colony-forming units (CFU) of bacteria per daily dose of composition to be administered preferably 106 to 1012 colony-forming units (CFU) of bacteria per daily dose of composition to be administered.

[0080] Preferably, this corresponds to a daily dose of bacteria to be administered, regardless of the weight of the person or animal. Preferably, this dose is administered in a single administration. More preferably, the effective composition comprises 10⁵ to 10¹¹ CFU or 10⁷ to 10¹¹ CFU of bacteria per daily dose to be administered, and even more preferably 10⁹ CFU.

[0081] Said bacteria are a mixture of bacteria corresponding to or derived from the bacterial strain DSM 33715 and / or corresponding to the derived strain.

[0082] The term CFU (Colony Forming Unit) is a unit used to count the number of bacteria capable of forming a colony during propagation, i.e., viable bacteria. It should be understood that non-viable bacteria may also be present in the compositions and that, in general, they should not have a negative effect on the properties of the live bacteria in the composition; on the contrary, they may have an effect on themselves.

[0083] Preferably, the daily dose is measured per gram or millilitre of the composition according to the final invention.

[0084] According to another preferred object, when 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% live bacteria (by number), more preferably at least 10% live bacteria (by number), even more preferably at least 50% live bacteria (by number).

[0085] 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% ANI identity with the nucleotide sequence of the bacterial strain DSM 33715.

[0086] In the case of the implementation of a supernatant of any of the aforementioned bacterial strains, the composition according to the invention including it may in particular include 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.

[0087] Thus, 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.

[0088] When the composition is intended for therapeutic use, the composition may include at least one pharmaceutically acceptable excipient.

[0089] When the composition includes such a pharmaceutically acceptable excipient, the composition according to the invention is preferably a pharmaceutical composition.

[0090] The invention therefore also relates to a pharmaceutical composition comprising one or more ingredients included in the composition according to the invention and according to any of the embodiments described above.

[0091] The pharmaceutical composition according to the invention has at least one application in improving the physical, physiological, or psychological well-being of a sick subject, resulting in an improvement in the subject's general health or a reduction in the risk of illness. The pharmaceutical composition is a medicinal product.

[0092] According to another aspect, the composition according to the invention may be in the form of a food product, a beverage, a nutraceutical, a food additive, a food supplement or a dairy product.

[0093] When the composition according to the invention is included in a food supplement for oral administration, it may be in the form of capsules, softgels, softgels, tablets, coated tablets, pills, pastes, lozenges, gums, oral solutions or emulsions, syrup or gel.

[0094] 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 coloring agent. Its formulation is carried out using the usual processes for producing coated tablets, capsules, gels, controlled-release hydrogels, emulsions, tablets, or caplets.

[0095] A composition according to the present invention may also be in the form of a nutritional composition. Such a nutritional composition according to the present invention may be in the form of a yogurt, a cereal bar, breakfast cereal, dessert, frozen food, soup, pet food, liquid suspension, powder, tablet, gum, or candy.

[0096] A nutritional composition according to the present invention may further comprise at least one ingredient selected from: antioxidants, fish oils, DHA, EPA, vitamins, minerals, phytonutrients, a protein, a lipid, probiotics, prebiotics and combinations thereof.

[0097] Use

[0098] 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 medicinal product.

[0099] In particular, 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 drug.

[0100] Use as a medicinal product in the context of the present invention refers to human or veterinary use, where said medicinal product is administered to a human subject or a non-human animal subject. Human subject is also understood to mean an adult, a child, or a baby, including a newborn or an infant.

[0101] Preferably, the composition according to the invention is intended to be used 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.

[0102] More preferably, the composition according to the invention is intended to be used in the prevention and / or treatment of chronic inflammatory bowel diseases, in particular a disease selected from Crohn's disease, ulcerative colitis, ulcerative colitis, diverticulitis, esophagitis, gastritis, pancreatitis, gastroduodenal ulcer, irritable bowel syndrome.

[0103] According to one embodiment, the invention also relates to the composition according to the invention for its use as a medicinal product, in the prevention and / or treatment of at least one disease selected from: - Metabolic diseases, chosen from non-insulin-dependent diabetes, gestational diabetes, NASH, hepatic steatosis, pancreatic steatosis, hyperlipidemia, hypercholesterolemia, infertility related to overweight, urinary incontinence related to overweight, - Other chronic metabolic diseases, including thyroiditis, - cardiac and vascular diseases, selected from atherosclerosis, thrombopathies, acute pericarditis and chronic constrictive pericarditis, arterial hypertension, vasculitis - diseases of the liver and bile ducts, selected from hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, cirrhosis, hepatic encephalopathy, and gallstones - Joint diseases related to excess weight, chosen from osteopenia, osteoporosis, osteoarthritis, and inflammation of the vertebral discs - neurodegenerative diseases, chosen from 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 microbiota dysbiosis, chosen from hepatocellular carcinomas, cancers of the digestive tract such as esophageal, stomach and colorectal cancer, pancreatic carcinoma, neuroendocrine tumors (NETs) of the gastro-enteropancreatic system, liver tumors, tumors of the gallbladder and bile ducts, kidney tumors, glioblastomas, lymphomas, multiple myeloma, chronic myeloid leukemia, chronic myeloproliferative diseases, lung carcinomas - autoimmune diseases, chosen from insulin-dependent diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, systemic lupus erythematosus, and polyendocrine autoimmune syndrome - atopic dermatological diseases, chosen from eczema and psoriasis - Chronic inflammatory bowel diseases, chosen from Crohn's disease, ulcerative colitis, diverticulitis, esophagitis, gastritis, pancreatitis, peptic ulcer, irritable bowel syndrome - respiratory function disorders, chosen from asthma, cystic fibrosis, chronic obstructive pulmonary disease and chronic bronchitis, interstitial lung disease and pulmonary fibrosis, sleep apnea syndrome (OSAS) - pneumonias, chosen from infectious pneumonias, influenza pneumonia and avian influenza, severe acute respiratory syndrome (SARS), Pneumocystis pneumonia - Infectious diarrhea, chosen from Clostridium difficile infection, EHEC infection, Salmonella gastroenteritis, Campylobacter enteritis, foodborne illnesses caused by enterotoxin-producing bacteria, cholera, yersiniosis, shigellosis, cryptosporidiosis, and listeriosis - Food allergies, chosen from celiac disease, lactose intolerance, and bile salt malabsorption syndrome - Inflammatory nephrological conditions or those related to microbiota dysbiosis, chosen from urethritis, chronic renal failure, and urolithiasis - Other inflammatory disorders, chosen from multiple sclerosis, lymphangitis - Neurological diseases linked to microbiota dysbiosis, chosen from anorexia, bulimia, depression, bipolar syndrome, autism, schizophrenia, Tourette syndrome.

[0104] According to a final object, the invention also relates to a non-therapeutic use of the composition according to the invention to maintain and / or 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.

[0105] Preferably, when the composition aims to strengthen the gut microbiota and / or support the diversity of the gut microbiota and / or promote the increase of beneficial bacteria in the gut microbiota and / or promote weight loss in a healthy subject, then the composition is in the form of a food product, a beverage, a nutraceutical, a food additive, a food supplement or a dairy product.

[0106] The invention is now illustrated by examples of useful bacteria according to the invention, methods of culturing these bacteria, examples of compositions containing them and test results demonstrating their effectiveness, presented solely by way of illustration. Examples

[0107] Example 1: Characterization of C. minuta according to the invention filed under number DSM 33 715.

[0108] The bacterial strain C. minuta DSM 33715 can be characterized according to the following methods.

[0109] Native bacteria of strain DSM 33715 (10 pL of culture medium dosed at 109 CFU / mL) were deposited on ionized carbon grids (Delta Microscopy, Toulouse, France) and negative staining was performed with Nano-Tungsten (Nano-W, Nanoprobes, LFG Distribution, France).

[0110] Observation of grid-mounted bacteria was performed using a transmission electron microscope (Talos F200S G2 - Thermo Fisher Scientific - Eindhoven) at 200kV, equipped with a 4K*4K One View camera (Gatan, Paris, France). Sample preparation and image acquisition were carried out at the Bordeaux Imaging Center (member of France-Biolmaging, ANR-10-INBS-04).

[0111] Oxidase activity was determined using oxidase test strips (Sigma-Aldrich) by evaluating the color change after adding a drop of liquid bacterial culture.

[0112] Catalase activity was determined at two concentrations of H2O2 (3 and 15%), added to a colony of DSM 33715, catalase status being assessed visually by bubble formation.

[0113] Gram staining was carried out following the classical procedure.

[0114] Bile and pH tolerance were evaluated in modified GAM (HyServe) liquid medium. For bile tolerance, media containing 0, 2, 4, 6, and 8% Oxgall (Difco Laboratories) were used, corresponding to 0, 20, 40, 60, and 80% bile, respectively. For pH tolerance, media with pH values ​​of 4, 5, 6, 7, and 8 were used. All media were degassed in the anaerobic chamber before inoculation with 10 µL of a 0.5 McFarland bacterial suspension. Optical density at 600 nm was measured at 0, 24 h, 48 h, 72 h, and 96 h.

[0115] Antibiotic resistance tests were performed using minimum inhibitory concentration (MIC) according to the CLSI (Clinical and Laboratory Standards Institute) agar dilution reference method. Stock solutions of each antibiotic were prepared and sterilized by 0.2p filtration: ampicillin (Sigma-Aldrich), tetracycline (Sigma-Aldrich), chloramphenicol (Sigma-Aldrich), clindamycin (Sigma-Aldrich), meropenem (USP), piperacillin (USP), tazobactam (USP), and ceftriaxone (USP). Brucella agar (BD BBL) supplemented with 5 pg / ml hemin (Sigma-Aldrich), 1 pg / ml vitamin K1 (Sigma-Aldrich), and 5% (v / v) hemolyzed (laked) sheep blood (Hemostat) was used as the test medium. Agar plates with different antibiotic dilutions were prepared and degassed in the anaerobic chamber before inoculation. The DSM 33715 inoculum was resuspended at the standard 0.5 of McFarland in peptone water.Two µl of the inoculum were spotted onto the surface of the agar (each spot corresponding approximately to 10⁵ CFU / ml). A triplicate was prepared for each antibiotic concentration. The plates were then incubated for 6 days at 37°C under anaerobic conditions. MIC values ​​were determined according to CLSI guidelines and were interpreted as susceptible, intermediate, or resistant, based on CLSI anaerobic breaking points.

[0116] Characterization results

[0117] 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-sporulating, negative for oxidase and catalase activity, and is Gram-negative. DSM 33715 is negative for the majority of enzymes tested, with the exception of glucose acidification, xylose, rhamnose, and arabinose, which is consistent with the description of Christensenella minutaïaite by Morotomi et al. It exhibits the following enzymatic activities: 3-Glucosidase, α-Arabinosidase, Glutamic Acid Decarboxylase, as well as Esterase, Acid Phosphatase, and Napthol-AS-BL Phosphatase. Resistance to bile acids was observed up to 80 g / L of Oxgall, corresponding to 80% bile. DSM 33715 also exhibits growth in media with a pH ranging from 6 to 9. Finally, the DSM 33715 strain shows resistance to ampicillin and tetracycline.

[0118] Figure IA shows an image of a Christensenella minuta bacterium according to the invention obtained by transmission electron microscopy in negative staining (magnification: 36kX). The average dimensions of a bacterium of strain DSM 33715 are: length = 1.27 ± 0.28 mm; width = 0.507 ± 0.04 mm; with a membrane thickness of 29.4 nm (average obtained from 41 bacteria). The strain has a short shape with tapered ends, occurring in singlets, pairs, or rosettes.

[0119] The strain according to the invention can also be characterized by the analysis of a MALDI spectrum.

[0120] Thus, 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 employing the standard method recommended by the manufacturer.

[0121] MALDI identification 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. Thus, [Fig. 1B] represents the MALDI spectrum characteristic of a pure culture of Christensenella minuta according to the invention (DSM 33715). This makes it possible to confirm the belonging of said strain to the species C. minuta and thus to validate the purity of the culture.

[0122] Example 2: Comparison between the strain according to the invention (DSM 33715) and C. minuta strains of the prior art.

[0123] The inventors also compared the strain according to the invention (DSM 33715) to known strains of C. minuta such as the reference strain filed under DSM 22607, the strain filed under DSM 32891 and the strain filed under DSM 33407 according to several parameters such as the 16S rDNA sequence, the genome size in base pairs (bp), the phylogenetic distance based on the whole genome (ANI), the coding sequence number (CDS) and examples of strain-specific genes.

[0124] The results are presented in Tables 1 to 3 below.

[0125] [Tables 1] 16S rDNA Genome size (bp) ANI (% genome homology) CDS (Number of Coding Sequences) Example of specific genes DSM22607 SEQ ID NO: 1 2944440 99.84% 2765 nd DSM 33715 SEQ ID NO: 1 2959549 2773 nd

[0126] [Tables2] 16S rDNA Genome size (bp) ANI (% genome homology) CDS (Number of Coding Sequences) Example of specific genes DSM 32891 SEQ ID NO: 1 2841506 99.37% 2663 gene encoding RibZ DSM 33715 SEQ ID NO: 1 2959549 2773 gene encoding a beta-mannanase

[0127] [Tables3] 16S rDNA Genome size (bp) ANI (% genome homology) CDS (Number of Coding Sequences) Example of specific genes DSM 33407 SEQ ID NO: 1 2791808 99.69% 2595 Gene encoding PknD DSM 33715 SEQ ID NO: 1 2959549 2773 Gene encoding TrxB

[0128] Thus, the strain according to the invention (DSM 33715) has the same 16S rDNA sequence as the three other known strains of C. minuta, despite different metabolic properties and anti-inflammatory potential. However, the genome size, the entire genome, the number of coding sequences, and certain genes specific to said strain allow for the characterization and differentiation of the strains from one another. In particular, the strain according to the invention has the largest genome and a greater number of coding sequences than the known strains.

[0129] Example 2: Anti-inflammatory effect of the strain according to the invention,

[0130] In the present study, the inventors investigated the pro- and anti-inflammatory immunomodulatory effect of the DSM 33715 strain.

[0131] The study protocol is as follows.

[0132] PBMCs from 3 healthy donors (Lonza) were seeded in 24-well plates at 1xlO6 cells / well in RPMI medium (Gibco) supplemented with 2% fetal bovine serum (FBS, Gibco) and incubated at 37°C / 5% CO2 for 24 h in the presence of DSM 33715 bacteria at an MOI of 50 or bacterial-glycerol medium as a control. After 24 h, the cell culture supernatants were collected and stored at -80°C until IL-10 quantification by ELISA. IL-10 was The analysis was performed using a BioLegend specific ELISA, according to the manufacturer's instructions. The absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech.

[0133] THP-1 cells (ECACC) were seeded at 5 x 10⁵ cells / well in 24-well plates in RPMI medium (Gibco) supplemented with 10% SVF (Gibco) in the presence of 100 ng / ml PMA (Enzo Life Sciences) for 48 h, in order to differentiate monocytic cells into MO-type macrophages. The cells were then washed with PBS-IX (Gibco) and cultured for an additional 24 h in RPMI medium with 2% SVF. The cells were then stimulated for 24 h 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 until IL-10 quantification by ELISA. IL-10 was measured using a BioLegend specific ELISA, according to the manufacturer's instructions. Absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech.

[0134] Statistics: one-way ANOVA followed by Dunett's multiple comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0135] Results

[0136] To evaluate the immunomodulatory effect of strain DSM 33715, PBMCs were incubated for 24 hours in the presence of DSM 33715 bacteria at an MOI of 50. The chemokine IL-10 is an anti-inflammatory cytokine. The values ​​represent the IL-10 secretion induced by the strain according to the invention (DSM 33715). For 2 of the 3 donors, DSM 33715 induced IL-10 production higher than that of a reference strain of C. minuta DSM 22607 ([Fig. 2A]).

[0137] 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 MO-type macrophage by PMA treatment ([Fig.2B]).

[0138] The DSM 33715 strain thus exhibits a greater anti-inflammatory immunomodulatory effect than the reference strain of C. minutait. It therefore possesses strong therapeutic potential in inflammatory diseases.

[0139] Example 3: Anti-inflammatory effect of the strain according to the invention,

[0140] In the present study, the inventors investigated the anti-inflammatory effect of the DSM 33715 strain on an IL-8-producing HT-29 colon adenocarcinoma cell line after TNF-a stimulation.

[0141] HT-29 cells (ECACC) were seeded in 24-well plates at 3 x 0.5 cells / well in McCoy's 5A medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and incubated at 37°C / 5% CO2 for 24 h. The cells were washed with PBS-1X (Gibco) and cultured for a further 24 h in medium McCoy's 5A with 2% FBS. Cells were then stimulated for 6 hours with 5 ng / ml of TNF-α in the presence of 50 bacteria per 1 HT-29 cell (MOI 50) of DSM 33715 or 10% supernatant, or PBSIX-Glycerol or bacterial culture medium as controls. DSM 22607 was the reference strain. 5-ASA (20 mM) was used as an anti-inflammatory agent in the control. After 6 hours, cell culture supernatants were collected and stored at -80°C until IL-8 quantification by ELISA. IL-8 was measured using a BioLegend specific ELISA, according to the manufacturer's instructions. Absorbance at 460 nm was read using the FluoStar Omega microplate reader, BMG Labtech. Six independent experiments were performed in duplicate. Statistics: one-way ANOVA followed by Dunett's multiple comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0142] Results

[0143] To evaluate the immunomodulatory effect of strain DSM 33715, HT-29 cells were stimulated with the pro-inflammatory cytokine TNF-α and the bacterium DSM 33715 at an MOI of 50 at the beginning of the stationary phase of growth or of its supernatant, for 6 hours. The chemokine IL-8 is secreted by HT-29 cells in a TNF-α-induced inflammatory state (Ctrl). The values ​​represent the normalized average percentage relative to the control + TNF-α (PBS-Glycerol for the bacterium and bacterial culture medium for the supernatant), representing the TNF-α-induced IL-8 secretion, i.e., 100%. In the presence of DSM 33715 or its supernatant, 1TL-8 secretion is reduced by 50% compared to the control (p<0.0001, Dunnett's multiple comparisons test). Thus, the DSM 33715 strain and its supernatant exhibit an anti-inflammatory immunomodulatory effect.

[0144] Example 4: Anti-inflammatory effect of the strain according to the invention,

[0145] In the present study, the inventors investigated the anti-inflammatory effect of the DSM 33715 strain on the NF-kB and JAK7 STAT inflammatory signaling pathways.

[0146] HT-29 cells at a density of 3 x 10⁵ cells / well, in a 24-well plate, were transfected according to a reverse transfection protocol with 200 ng of pRelA-luc vector (NF-κB signaling pathway) or pGL4 luc2P GAS-RE vector (ITFN-γ-dependent JAK / STAT signaling pathway) and 10 ng of pRL-TK vector (Promega), using X-tremeGENE HP DNA transfection reagent at a 3:1 ratio (Roche). Briefly, the reagent / DNA transfection complexes were prepared in a final volume of 50 µl of serum-free and antibiotic-free McCoy's 5A medium. The appropriate amount of each plasmid was added and homogenized, and then the transfection agent was added (3:1). The transfection complexes were gently mixed and incubated at room temperature for 15 minutes. After incubation, the transfection complexes were added to Freshly resuspended HT-29 cells were brought to the appropriate density in McCoy's 5A medium supplemented with 10% FBS. The cell / transfection complex mixtures were then 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 2% FBS medium for 12 hours after washing with PBS IX. After 12h of serum deprivation, the medium was removed and replaced with 2% FBS medium + / - 5 ng / ml of TNF-α (NF-κB reporter system) or 100 pg / ml of IFN-γ (JAK7 STAT reporter system) 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-κB pathway and 1 pM Tofacitinib for the JAK / STAT pathway).After 6 hours of coincubation, the cells were washed with PBS IX and lysed in 50 µl of passive lysis buffer (Promega) for 15 min at room temperature with orbital shaking. The protein lysates were then transferred to microtubes. A luciferase reporter assay (Dual-Luciferase Reporter Assay, Promega) was performed with minor modifications to the manufacturer's instructions. Briefly, 2 x 20 µl of lysate were transferred to a white 96-well plate, and 50 µl of LAR II reagent were added. The Firefly luciferase activity was read. Then, 50 µl of Stop&Glo reagent were added, and the Renilla activity was read using 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.

[0147] Results

[0148] The supernatant of DSM 33715 leads to a 25% decrease in TNF-α-induced NF-κB pathway activation and a 40% decrease in IFN-γ-induced JAK7 STAT pathway activation. These pathways are particularly relevant because they are overactive in patients with chronic inflammation, such as Crohn's disease, as well as in certain cancers. The NF-κB pathway is involved in cancers of epithelial origin, such as breast cancer, and in gastrointestinal cancers, such as colorectal cancer. The JAK / STAT pathway is overactivated primarily in solid tumors, such as breast, lung, liver, head, neck, and stomach cancer.

[0149] Example 5: Anti-inflammatory effect of the strain according to the invention in combination with an anti-inflammatory agent.

[0150] In the present study, the inventors investigated the anti-inflammatory effect of the DSM 33715 strain in combination with 5-ASA or Budesonide.

[0151] HT-29 cells (ECACC) were seeded in 24-well plates at 3 x 0.5 cells / well in McCoy's 5A medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and incubated at 37°C / 5% CO2 for 24 h. The cells have The cells were washed with PBS-1X (Gibco) and cultured for an additional 24 hours in McCoy's 5A medium with 2% FBS. The cells were then stimulated for 6 hours with 5 ng / ml of TNF-α in the presence of 10% supernatant in the stationary phase of DSM 33715 or bacterial culture medium as a control (ctrl), with or without anti-inflammatory agents. 5-AS α (10 mM) and budesonide (20 pM) are molecules used in the treatment of inflammatory diseases, such as ulcerative colitis and Crohn's disease. After 6 hours, the cell culture supernatants were collected and stored at -80°C until IL-8 quantification by ELISA. IL-8 was measured using a BioLegend specific 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.

[0152] Results

[0153] To evaluate the immunomodulatory effect of the DSM 33715 strain in combination with an anti-inflammatory agent used in therapy, HT-29 cells were stimulated with the pro-inflammatory cytokine TNF-α and 10% of the DSM 33715 supernatant, in the presence or absence of 5-ASA or budesonide, for 6 hours. The chemokine IL-8 is secreted by HT-29 cells in a TNF-α-induced inflammatory state (Ctrl). The values ​​represent the normalized average percentage relative to the control + TNF-α, representing the TNF-α-induced IL-8 secretion, i.e., 100%.

[0154] In the presence of DSM 33715, IL-8 secretion is decreased by 28% compared to the control, and by 38% in the presence of 5-ASA. The combination of the supernatant and 5-ASA leads to a 64% decrease (Panel A). Therefore, there is an enhanced effect of the supernatant and 5-ASA in modulating inflammation.

[0155] In the presence of DSM 33715, IL-8 secretion is decreased by 32% compared to the control, and by 38% in the presence of budesonide. The combination of the supernatant and budesonide leads to a 67% decrease (Panel B). Therefore, there is an enhanced effect of the supernatant and budesonide in modulating TNF-α-induced inflammation.

[0156] Example 6: Anti-inflammatory effect of the strain according to the invention in combination with a JAK inhibitor.

[0157] In the present study, the inventors investigated the anti-inflammatory effect of DSM 33715 associated or not with Tofacitinib on the modulation of the ITFN-γ-induced JAK / STAT inflammation signaling pathway.

[0158] HT-29 cells at a density of 3x05 cells / well, in a 24-well plate, were transfected according to a reverse-transfection protocol, with 200 ng of pGL4 luc2P GAS-RE vector (ITFN-y dependent JAK / STAT signaling pathway) and 10 ng pRL-TK vector (Promega) was used, along with X-tremeGENE HP DNA transfection reagent at a 3:1 ratio (Roche). Briefly, the reagent / DNA transfection complexes were prepared in a final volume of 50 µL of serum-free, antibiotic-free McCoy's 5A medium. The appropriate amount of each plasmid was added and homogenized, followed by the addition of the transfection agent (3:1). The transfection complexes were gently mixed 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% FBS. The cell / transfection complex mixtures were then gently homogenized and plated. The plates were incubated for 24h in a humid atmosphere at 5% CO2 and the medium was then replaced with medium at 2% SVF for 12h, after washing in PBS IX.After 12 hours of serum deprivation, the medium was removed and replaced with 2% FBS + / - 100 pg / ml IFN-γ medium (JAK / STAT reporter system) and 10% DSM 33715 supernatant or bacterial culture medium as a control or specific signaling pathway inhibitor (5 pM BAY11-7082 for the NF-κB pathway and 1 pM Tofacitinib for the JAK / STAT pathway). After 6 hours of co-incubation, cells were washed with PBS IX and lysed in 50 µl of passive lysis buffer (Promega) for 15 min at room temperature with orbital shaking. The protein lysates were then transferred to microtubes. A luciferase reporter assay (Dual-Luciferase reporter assay, Promega) was performed with minor modifications to the manufacturer's instructions. Briefly, 2 x 20 µl of lysate were transferred to a white 96-well plate, and 50 µl of LARII reagent were added. Firefly luciferase activity was read.Then 50 µL of Stop&Glo reagent were added and the Renilla activity measurement was read using 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.

[0159] Results

[0160] The supernatant of DSM 33715 leads to a 40% decrease in ITFN-γ-induced JAK / STAT pathway activation, tofacitinib to a 49% decrease, and the combination of the supernatant with tofacitinib leads to a 64% decrease in ITFN-γ-induced JAK / STAT pathway activation. Therefore, there is an enhanced effect of the supernatant and tofacitinib in modulating ITFN-γ-mediated inflammation.

[0161] Example 7: Effect of the strain according to the invention in the protection of the intestinal epithelium.

[0162] In the present study, the inventors investigated the effect of the DSM 33715 strain on the integrity of the intestinal barrier by measuring transepithelial resistance.

[0163] Caco-2 cells (ECACC) were seeded at 2 x 10⁴ 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 bovine serum (FBS, Gibco) and incubated at 37°C / 5% CO₂. Transepithelial electrical resistance was checked daily from the seventh day of culture using an EV₀M₃ ohmmeter (WPI). The medium was replaced every 2 days for 8 to 10 days, until the optimal TEER was reached (2000 Q / cm²). Upon reaching the baseline TEER, cells were treated in the apical compartment with DSM 33715 or DSM 22607 at a MOI of 50 or with PBS IX / 10% Glycerol (control) for 3 hours. Then, 100 ng / ml of TNF-α was added or not to the basal compartment. The TEER was measured immediately before the addition of TNF-α (T0) and 6 hours (T6H) after the addition of TNF-α.

[0164] Results

[0165] To evaluate the ability of strain DSM 33715 to restore or strengthen the intestinal barrier, polarized monolayer Caco-2 cells were pretreated with DSM 33715 and then sensitized with TNF-α. TEER was compared to T0 (before TNF addition) and T6H. The values ​​represent the mean normalized to the control – TNF-α, representing baseline TEER. As expected, treatment of Caco-2 cells with TNF-α induced an increase in permeability (Ctrl + TNF-α). Strain DSM33715 is able to restore the epithelial barrier, returning it to baseline levels.

[0166] Example 8: Effect of the strain according to the invention in an in vivo model of colitis.

[0167] In the present study, the inventors investigated the in vivo effect of colitis induced by TNBS in rats. The trial was carried out by an accredited provider (Intestinal Biotech Development, Lille) according to government guidelines.

[0168] For the induction of colitis, Sprague Dawley rats were anesthetized for 2h and received an intrarectal injection of TNBS (Trinitrobenzene sulfonic acid, 80 mg / kg dissolved in 40% ethanol).

[0169] The animals were sacrificed 4 days after TNBS injection. DSM 33715 was administered orally at a dose of 10⁹ CFU / ml, with gavage starting 14 days before colitis induction and continuing until the day of euthanasia. The animals in the positive control group received Pentasa mixed into their diet at a dose of 150 mg / kg. The anti-inflammatory effect of the tested products was assessed at sacrifice, following a total treatment period of 18 days.

[0170] Weight changes were monitored throughout the experiment. Colon length was assessed on the day of autopsy. Macroscopic and microscopic scores were obtained 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 macroscopic lesions on a scale of 0 to 10, and this score is based on characteristics reflecting inflammation, such as hyperemia, intestinal thickness, and the extent of ulcerations.

[0171] The method is described by Wallace JL et al. Gastroenterology. 1992 Jan;102(l):18-27. PMID: 1309357 or Wallace JL et al. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology. 1989 Jan;96(l):29-36. PMID: 2535830.

[0172] For histological evaluation, based on the Ameho criteria, a section was taken 2 cm above the anal canal. The sections obtained were stained using the MGG (May-Grünwald Giemsa) staining technique. This classification on a scale of 0 to 6 takes into account the degree of inflammatory infiltrate, the presence of erosion, ulceration or necrosis, as well as the depth and extent of the lesions. The method is described by Ameho et al., 1997, Gut.

[0173] Inflammation was assessed in the colon by measuring IL-1[3] production (a pro-inflammatory cytokine) (eBioscience kit), as well as the level of Lipocalin-2 (Lcn-2), a 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 with ceramic beads (1.4 and 2.8 mm). The resulting samples were centrifuged for 20 min, and the supernatants were collected and stored at -80°C until use for ELISAs.

[0174] Results

[0175] [Fig. 8A]: The inventors observed a significant decrease in colon length in the TNBS-treated rat group receiving the vehicle treatment compared to the vehicle-treated control group, as expected. A significant increase in colon length was observed in the TNBS-treated rat group receiving 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.

[0176] [Fig. 8B]: The intensity of inflammation and colonic lesions was assessed macroscopically using the Wallace score. A significant increase in the Wallace score was observed in the TNBS-Vehicle group rats compared to the “healthy” group, with scores of 7.00 ± 0.41 vs. 0.0 ± 0.0, p < 0.0001. This result indicates that the rats presented with severe colitis 4 days after induction, with an ulcerative or inflammatory site greater than 3 cm. colon (corresponding to a score of 7 on a scale of 0 to 10). A significant decrease in score was observed in both the TNBS-Pentasa and TNBS-DSM 33715 groups. DSM 33715 improved macroscopically observed inflammatory lesions by 38% compared to the TNBS-vehicle group, with Wallace scores of 4.33 ± 0.33 vs. 7.00 ± 0.41, respectively (p = 0.049). This indicates that the treatment induces a significant anti-inflammatory effect.

[0177] [Fig. 8C]: A significant improvement in the histological score—intensity of inflammatory lesions—was observed in the group of TNBS rats that received Pentasa, a positive control, and the DSM 33715 strain, compared to the TNBS rats that received the vehicle. Pentasa improved the score by 13% and DSM 33715 by 36%.

[0178] [Fig. 8D]: Lipocalin-2 levels, a marker of neutrophil infiltration, are significantly elevated in colitis-infected rats (TNBS-Vehicle) compared to healthy controls, confirming the development of severe colitis. A downward 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.

[0179] As expected, a significant increase in ITL-1 [>] was measured in the colon of TNBS-Vehicle group rats, partially corrected by treatment with Pentasa, validating the model. Administration of DSM 33715 to colitis-affected rats showed a significant decrease in dTL-1 [>] levels, indicating an anti-inflammatory effect of DSM 33715 in the rat model of acute TNBS-induced colitis.

[0180] All of these results correlate to highlight the anti-inflammatory properties of the DSM 33715 strain, particularly compared to the reference treatment for this type of pathology, but also in comparison with the reference strain of C. minuta. Thus, the strain according to the invention has definite therapeutic potential in intestinal dysbiosis and, in particular, in the prevention and / or treatment of inflammatory diseases.

Claims

Demands

1. Bacterial strain of Christensenella minuta filed under DSM number 33715.

2. Composition comprising at least one bacterial strain according to claim 1 and at least one acceptable excipient.

3. Composition according to claim 2, said composition also comprises a culture supernatant of the bacterial strain according to claim 1.

4. Composition according to any one of claims 2 or 3, characterized in that the bacterium is live, dead, attenuated or inactivated.

5. Composition according to any one of claims 2 to 4, characterized in that it is in solid, liquid or freeze-dried form.

6. Composition according to any one of claims 2 to 5, said composition is in a form suitable for administration by oral, nasal, parenteral, rectal, sublingual, ocular, auricular, intramuscular, intravenous, inhaled or cutaneous routes.

7. Composition according to any one of claims 2 to 4, characterized in that it is in the form of a powder, microencapsulated powder, capsule, lozenge, tablet, lozenge, granules, emulsion, suspension, suppository, inhaler or syrup.

8. Composition according to any one of claims 2 to 7, characterized in that it comprises at least 50% live bacteria (by number).

9. Composition according to any one of claims 2 to 8, characterized in that the live bacteria represent between 105 and 1011 CFU.

10. Composition according to any one of claims 2 to 4, characterized in that it is in the form of a beverage, a food product, a nutraceutical, a food additive, a food supplement or a dairy product.

11. Composition according to any one of claims 2 to 9, also comprising at least one probiotic and / or at least one bacterium of the Christensenellaceae family and / or an anti-inflammatory agent.

12. Composition according to the preceding claim, also comprising a bacterium selected 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 pseudolongum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium stercoris and Bifidobacterium thermophilum.

13. Composition according to any one of claims 11 or 12, also comprising a pharmaceutical active ingredient selected from mesalamine, olsalazine, budesonide, tofacitinib, filgotinib, upadacitinib, infliximab, adalimumab, golimumab, certolizumab, vedolizumab and ustekinumab.

14. Composition according to any one of claims 2 to 9 and 11 to 13, for its use as a medicinal product in humans or animals.

15. Composition according to any one of claims 2 to 13, for its use in the prevention and / or treatment of microbiota dysbiosis.

16. Composition according to any one of claims 2 to 13, for its use in the prevention and / or treatment of a disease selected from chronic diseases, metabolic diseases, inflammatory diseases, respiratory function disorders, and cancers.

17. Composition for its use according to the preceding claim in the prevention and / or treatment of chronic inflammatory bowel diseases.

18. Composition for its use according to the preceding claim, in the prevention and / or treatment of a disease selected from Crohn's disease, ulcerative colitis, ulcerative colitis, diverticulitis, esophagitis, gastritis, pancreatitis, gastroduodenal ulcer, irritable bowel syndrome.

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