CN113631174A - Cristiparous bacteria for the prophylaxis and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer - Google Patents

Abstract

The present invention relates to a bacterium of the family of the Cristensenellaceae, in particular the genus of the genus Cristensenella or a composition containing said bacterium for use in the prevention and/or treatment of chronic inflammatory diseases and/or cancer in humans or animals.

Description

Cristiparous bacteria for the prophylaxis and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer

The present invention relates to the prevention and treatment of chronic inflammatory diseases, inflammatory gastrointestinal diseases and/or cancer with specific bacteria of the intestinal microbiota and compositions thereof.

Inflammation is the body's normal defense process against attacks. Which makes it possible to combat and eliminate external factors at the origin of said attack. This inflammation, which is considered acute, is reversible and has only a limited duration, ranging from a few minutes to a few days. However, in some cases, the inflammatory process can go wrong and inflammation can become chronic. The agents involved in the inflammatory process become dangerous rather than contributing to the body's defenses and lead to pathological conditions that are often severe and disabling. Thus, a chronic inflammatory disease is a disease whose pathophysiology is directly linked to inflammation, which is of autoimmune and/or autoinflammatory origin.

All organs may be affected by chronic inflammation, such as the digestive system (crohn's disease, ulcerative colitis, autoimmune gastritis, etc.), liver (hepatitis, NAFLD, non-alcoholic steatohepatitis, etc.), pancreas (pancreatitis), lung (asthma), skin (atopic dermatitis, such as psoriasis), nervous system (multiple sclerosis), joints (multiple arthritis). In addition, chronic inflammation is also present in other situations involving other mechanisms, such as cancer in particular.

Chronic inflammation and in particular chronic inflammatory diseases affect a large proportion of the population in different proportions depending on the condition concerned. Because these diseases, although slowly evolving, are disabling, painful and associated with a high risk of early death, the health impact is great.

Currently, the treatments proposed for these diseases are anti-inflammatory, immunomodulatory and/or immunosuppressive agents. Examples thereof include:

non-steroidal anti-inflammatory drugs for short-term use and not suitable for chronic pain, such as ibuprofen, naproxen and aspirin,

modulators of purine metabolism as immunomodulators, such as methotrexate and azathioprine,

biopharmaceuticals, such as anti-TNF-alpha, IL1 and IL6 receptor antagonists, or even

-sulfasalazine.

All these drugs have a very variable response rate due to the degradation of the molecule when they are administered and have significant side effects. In addition, immunomodulators interfere with the normal and healthy immune response to pathogens, rendering treated patients susceptible to infection. As regards methotrexate, in particular it is known that it has an adverse effect on sperm production and thus on fertility. Thus, most of these inflammatory diseases are not treated or not adequately treated.

Therefore, the research and exploration of new anti-inflammatory therapeutic strategies, which are easy to administer and without side effects, constitute the main focus of medical and biomedical research, in particular the development of effective treatments of chronic inflammatory diseases, gastrointestinal diseases and diseases caused by chronic inflammation.

This is the object of the present invention, which is intended to achieve this object using specific bacteria of the human intestinal microbiota, i.e. bacteria of the family kristestoniaceae (christenseellaceae), preferably the genus kristevensis (christenseella). These bacteria may optionally be combined with other bacteria and in particular bacteria of the species Akkermansia muciniphila (Akkermansia muciniphila).

Cristesyniaceae, in particular Cristesynebacteria, have been studied and described. In particular, this is the case for the small kristensenesella (christensella minuta), the mosaic rrinsella (christensella massilisensis) and the tesserella timonensis. Specifically, the small kriging senecium was first described in 2012. In 2014, a study showed that cremastra parva is the most inherited taxa among a population of uk twins, and the presence of cremastra parva was associated with a low body mass index. This correlation between cretinismus and low body mass index was then observed in ten studies published since 2014 in different geographical populations.

The advantage of reconstituting the minimal cretinism group in individuals exhibiting inflammatory bowel disease associated with a deficiency of cretinism in the gut microbiota is disclosed in application WO 2016/156527. However, none of the above bacteria has proven to be directly associated with these conditions in humans.

Furthermore, applications WO2018/162738 and WO2018/162726 teach that the use of specific bacteria belonging to the family of claimanthrisciaceae, which are species defined as being particularly distant from the species of the genera cricetris and in particular cretinismus and telangiectasias, makes it possible to specifically combat overweight and obesity by intervening on, for example, the presence of visceral fat and intestinal permeability.

Ackermanella viscophila, belonging to the family Verrucomicrobiaceae and the genus Ackermansia (Akkermansia), is the bacterium first identified in 2004 and accounts for approximately 1 to 3% by number of the total number of bacterial genera of the intestinal microbiota of healthy adults (Derrien et al, 2004,54: 1469-.

Decreased numbers of Ackermansia muciniphila have been reported in stool samples from obese individuals and patients with inflammatory gastrointestinal disease (Png et al, gastroenterology (2010; 105: 2420-2428)). Akkermansia muciniphila is associated with a healthier metabolic state and better clinical outcome after calorie-restricted intervention in overweight or even obese adults.

Surprisingly, and in accordance with the present invention, bacteria of the genus cretinismus and in particular cretinismus, Maricitess and Timocross can act on inflammatory diseases, in particular inflammatory gastrointestinal diseases and chronic inflammatory diseases, as well as inflammatory markers of cancer origin, when administered to a human or animal. In addition, the inventors have surprisingly determined the synergistic anti-inflammatory properties resulting from the use of bacteria of the genus cretinistonia in combination with bacteria of the genus akkermansia, in particular the well-known New Generation Probiotic (NGP) muciniphilic akkermansia from the family verrucomicrobiaceae.

The invention therefore relates to a bacterium of the genus crewsonia for use in the prophylaxis and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer in humans or animals.

Advantageously, such bacteria, when administered to a human or animal exhibiting chronic inflammation, are capable of acting on molecules overproduced during chronic inflammation, such as kynurenine, interleukin 6, interleukin 8, TNF α, interleukin 1b, lipocalin or fecal calprotectin, and/or molecules produced by stimulation of the production of the anti-inflammatory cytokine interleukin 10.

The present invention also relates to a composition comprising at least one bacterium of the genus cremastesynsis, alone or in combination with at least one further bacterium, preferably at least one further bacterium of the genus akkermansia, for use in the prevention and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer in humans or animals.

Such a composition comprising at least one of these bacteria (at least one bacterium of the genus cremastra, alone or in combination with at least one further bacterium of the genus akkermansia) and/or a supernatant thereof in a physiologically acceptable medium is also targeted per se.

Other features and advantages will appear from the following detailed description and the accompanying drawings of the invention.

Drawings

FIG. 1 shows the production of interleukin-8 (IL-8) (in pg/mL) (ordinate) in HT-29 cells stimulated by TNF- α in the presence of Klebsiella parvum, Klebsiella remobilis, Ackermanella mucophila, Klebsiella parvum + Ackermanella mucophila, and Klebsiella remobilis + Ackermanella mucophila (abscissa).

FIG. 2 shows IL-8 secretion by HT29 line gut cells after co-incubation in the presence of supernatant of a strain of Clastis minutissima (DSMZ: DSM22607) and stimulation by TNF-alpha. Dunn non-parametric statistical test (Dunn's nonparametric statistical test): p < 0.05; p < 0.01; p < 0.001. IL-8: interleukin 8; TNF- α: tumor necrosis factor alpha. The average of 4 experiments was repeated in triplicate.

FIG. 3 shows transepithelial resistance measurements on Caco-2 lineage intestinal cells after co-incubation in the presence of Microcoris parvulus bacteria (DSMZ: DSM22607) and after stimulation with TNF- α. Dunne non-parametric statistical test: p < 0.05; p < 0.01; p < 0.001. IL-8: interleukin 8; TNF- α: tumor necrosis factor alpha. The average of 3 experiments was repeated in triplicate.

FIG. 4 shows the effect of Klebsiella parvula (DSM 22607) on a model of inflammation induced by DNBS. (A) The treated groups were monitored for weight loss starting on the day of injection (OJ). (B) A significant reduction in macroscopic score indicating a reduction in inflammatory severity after DSM22607 and 5ASA treatment. DSM22607 is effective in reducing myeloperoxidase activity (C) and increasing the immunomodulatory effect of the anti-inflammatory cytokine interleukin-10 (IL-10) (D) in the spleen. Nonparametric analysis of variance followed by dunn statistical test: p < 0.05; p < 0.01; p < 0.001. DNBS: dinitrobenzenesulfonic acid.

FIG. 5 presents a summary of the anti-inflammatory effects of Klebsiella parvula on a DNBS-induced model of inflammation. (A) Reduction in macroscopic score indicating reduction in severity of inflammation following treatment with different species of cremastra parvula. (B) The immunomodulating effect of different species of Klebsiella parvula by reducing myeloperoxidase activity (C). Nonparametric analysis of variance followed by dunn statistical test: p < 0.05; p < 0.01; p < 0.001. DNBS: dinitrobenzenesulfonic acid.

FIG. 6 shows the effect of supernatants of Klebsiella parvula strains (DSMZ: DSM22607, Klebsiella parvula 1(C.minuta 1) and Klebsiella parvula 2) on the proliferation of HT-29 cells (colon adenocarcinoma). RLU: relative luminescence units. Dunnett test (Dunnett's test), wherein the control group was GAM group (which represents the mons Anaerobic Broth (Gifu Anaerobic Broth) medium). Code: p <0.05, p <0.01, p <0.001, p < 0.0001.

Definition of

Within the meaning of the present invention, the term "bacterium" is also understood to mean the term "bacterial strain". Bacterial strains are understood to be bacteria of a given strain belonging to a particular species, for example cretini. For example, mention may be made of a bacterial strain or the bacterium cremastasonia micranthum DSM 22607. Thus, in the context of the present invention, the two terms may be used interchangeably to designate one or several bacteria.

Within the meaning of the present invention, "overproduction of interleukin 6" is understood to mean an overproduction of interleukin 6 relative to the production in a healthy human or animal body without inflammation.

Within the meaning of the present invention, "overproduction of interleukin 8" is understood to mean an overproduction of interleukin 8 relative to the production in a healthy human or animal body without inflammation.

Within the meaning of the present invention, "insufficient production of interleukin 10" is understood to mean an insufficient production of interleukin 10 relative to the production in a healthy human or animal body without inflammation.

Within the meaning of the present invention, "chronic inflammatory disease" is understood to mean in particular any chronic disease whose slow and gradually evolving pathophysiology is directly linked to inflammation of autoimmune and/or autoinflammatory origin.

Within the meaning of the present invention, "inflammatory gastrointestinal disease" is understood to mean in particular inflammatory bowel disease, more particularly inflammatory bowel disease of the colon. The colonic inflammatory bowel disease may specifically be selected from the group consisting of Crohn's Disease (CD), Ulcerative Colitis (UC) and pouchitis, specifically CD and UC. CD and UC are two diseases characterized by inflammation of the inner wall of a portion of the digestive tract, which is associated with hyperactive digestive immune system.

Within the meaning of the present invention, "prevention" or "prevention" is understood to mean a reduction to a lesser extent of the risk or probability of occurrence of a given phenomenon, namely chronic inflammation, gastrointestinal inflammation and pathologies resulting therefrom, such as cancer, in particular intestinal inflammation, in the context of the present invention.

Within the meaning of the present invention, "treatment" or "treatment" is understood to mean a slowing of the progression of the disease, a stabilization, a reversal or a regression of the inflammatory chronic disease, the inflammatory gastrointestinal disease, the cancer or even an interruption or inhibition of its progression. In the context of the present invention, these terms also apply to one or more symptoms of the diseases described herein.

Within the meaning of the present invention, an "absence of klebsiella in the gut microbiota" is understood to mean a human or an animal that: the crewsonia bacteria in its gut microbiota account for at least 0.01%, in particular at least 0.1% and preferably at least 0.5% of the total number of bacterial genera detected in its gut microbiota. The abundance of the klebsiella collected in the stool can be measured, for example, by quantitative sequencing tests, Fluorescence In Situ Hybridization (FISH), qPCR (quantitative PCR) or by metagenomic analysis (relative quantification) well known to the person skilled in the art.

Within the meaning of the present invention, "physiologically acceptable medium" is understood to mean a medium compatible with the organism of the individual to which the composition is to be administered. The physiologically acceptable medium may be, for example, a non-toxic solvent such as water. In particular, the medium is compatible with oral administration.

Within the meaning of the present invention, a "microbiota" is understood to mean all microorganisms that colonize on an individual and with which said individual coexists: most are bacteria, but also viruses, fungi, yeasts and protozoa. The combination of microbiota varies according to the surface of colonization: thus, a distinction is made between skin microbiota, vaginal microbiota, urinary microbiota, respiratory microbiota, ENT (otorhinolaryngology) microbiota and intestinal microbiota (previously known as intestinal microbiota, which is by far the most important 100 trillion pathogens). Thus, "gut microbiota" is understood to mean all microorganisms, in particular bacteria, residing in the gut of a given individual.

According to the official definition of the World Health Organization (WHO), "body mass index" (BMI) is understood to mean an indicator of health risks associated with overweight and underweight. BMI is calculated by dividing an individual's weight (in kilograms) by the square of its height (in meters). BMI values are associated with a particular body type according to the classification given by the WHO (https:// www.who.int/features/objects/features/en /).

By "effective amount," "an amount effective to … …," "an amount effective for X of … …," and the like, is meant an amount of active ingredient that is effective to alleviate or to reduce to some extent one or more of the symptoms of a disease in need of treatment or to delay the onset of clinical markers or symptoms of a disease in need of prevention when the compound is administered. Thus, the amount refers to the amount of active ingredient that exhibits the following effects: (i) reversing the rate of progression of the disease; (ii) inhibit disease progression to some extent; and/or (iii) alleviate (or preferably eliminate) to some extent one or more symptoms associated with the disease. The effectiveness of the amount can be determined empirically by testing the relevant compound in known in vivo and in vitro model systems for the disease in need of treatment.

Detailed Description

The subject of the present invention is therefore the use of at least one bacterium of the family of the cryptomelaceae, preferably of the genus cremastra or of a composition comprising at least one such bacterium and/or at least a culture supernatant of said bacterium of the genus cremastra, for the prevention and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancers, in particular cancers of an inflammatory nature, in humans or animals. The bacterium may be any bacterial strain of one of the species of the genus creisteinsis. Preferably, the composition further comprises a further bacterium of the family verrucomicrobiaceae and/or a culture supernatant of said further bacterium, in particular said bacterium belongs to the genus akkermansia, more particularly to akkermansia muciniphila.

According to the inventionBacteria

The invention therefore relates to a bacterium of the family of the Klebsiella, in particular of the genus Klebsiella, for use in the prophylaxis and/or treatment of an inflammatory disease, preferably a chronic inflammatory disease and/or an inflammatory gastrointestinal disease and/or a cancer in a human or animal, in particular a human or animal having an overproduction of interleukin 6 and/or an overproduction of interleukin 8 and/or an insufficient production of interleukin 10, which human or animal has an inflammatory disease or a cancer.

According to the invention, when administered to a human or animal exhibiting chronic inflammation, gastrointestinal inflammation or cancer, the bacterium of the genus crenstein is capable of acting on molecules overproduced during chronic inflammation, gastrointestinal inflammation or cancer, in particular on interleukin 6, interleukin 8 and kynurenine, and also on TNF α, interleukin 1b, lipocalin or fecal calprotectin. As, the inventors have found that bacteria of the genus cretinissella are unexpectedly capable of reducing inflammation and cell proliferation, in particular gastrointestinal inflammation and more particularly chronic inflammation of the intestinal tract, both in vitro and in vivo. More specifically, the bacteria according to the invention are capable of significantly reducing the production of pro-inflammatory molecules such as interleukin 6(IL-6) and interleukin 8 (IL-8).

Indeed, in inflammatory diseases or cancers presenting an increase in one of these markers, such as interleukin 6, interleukin 8, kynurenine, TNF α, interleukin 1b, lipocalin or calprotectin, the decrease in said marker is indicative of a decrease in pro-inflammatory signaling pathways, i.e. the immune cells responsible for this production are stimulated less. Thus, the excessive immune response that causes inflammation is slowed down and the system gradually returns to normal.

According to one embodiment, the bacterium or bacteria useful according to the invention is/are administered to the human or animal in an amount effective to act on at least one of the inflammatory markers, that is, to reduce the production of at least one of the molecules in vivo. According to one suitable embodiment, the one or more bacteria may be present at 10 per day⁷To 10¹¹Colony forming unit (CFU) regardless of the body weight of the human or animal, preferably 10 daily⁹To 10¹²Dose of CFU, more preferably equal to 10⁹Dose of CFU. In particular, the dose comprises at least one bacterium selected from the group consisting of: thymus dioxetanensis, Microcritinas minutus, Maricitescens mosaicensis and/or mixtures thereof. Preferably, the dose is a single dose, i.e. one full administration or each preprandial dose, i.e. three times a day.

Preferably, the cretinidae family, in particular the cretinib genus bacteria, can be used for the treatment of at least one chronic inflammatory disease selected from: chronic inflammatory diseases of intestinal tract, chronic inflammatory diseases of liver, chronic inflammatory diseases of pancreas, polyarthritis, atopic dermatitis, and chronic obstructive pulmonary disease. Specifically, the bacterium has a preventive and/or therapeutic effect on at least one disease selected from the group consisting of: crohn's disease, ulcerative colitis, pouchitis, ulcerative colitis, celiac disease, autoimmune gastritis, hepatitis, non-alcoholic steatohepatitis, primary sclerosing cholangitis, pancreatitis, rheumatoid arthritis, psoriatic arthritis, psoriasis, eczema.

In addition, the family of Cristigmaceae, in particular the genus Cristigma, can be used for the treatment of at least one inflammatory gastrointestinal disease, such as inflammatory bowel disease, more particularly an inflammatory chronic colonic disorder. For the purposes of the present invention, the term "disorder" is understood to mean a disease or condition. The inflammatory bowel disease may in particular be selected from the group consisting of crohn's disease, ulcerative colitis and pouchitis, in particular from the group consisting of crohn's disease and ulcerative colitis.

In addition, the family of cretinidae, in particular the genus cretinina, can be used for the treatment of proliferative diseases selected from: lymphoma, glioblastoma, myeloma, leukemia, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, uterine cancer, pancreatic cancer, lung cancer, liver cancer, gallbladder cancer, and kidney cancer.

One or more bacteria useful according to the invention are bacteria of the genus creistigma. The bacteria of the genus Klebsiella may in particular be Marek's disease bacteria, Perkinsonia species, human enterocricetosis species and/or Microripisia species. According to a particularly suitable variant, the bacterium of the genus Klebsiella is a bacterium of the genus Klebsiella.

These bacteria can be Isolated from human feces, for example, according to the Description OF Morotomi et al in 2012 (Morotomi, M., Nagai, F., and Watanabe, Y., for the New species OF Klistesmann-Straussler, which forms a unique branch in the order Clostridiales, Isolated from human feces, and the proposal OF the family Neobrassinomycetes (Description OF Christenella minuta. nov., Sp. nov., Isolated from human feces, microorganisms OF the family Isatella from human fareces, where human for a protein bacterium is in the order Clostridiales, and Proposal OF Christenceaceae NAnov. nov.) (International JOURNAL OF phylogeny and EVOLUTIONARY microorganisms OF Escherichia coli SYSTEMATIC AND Enterobacteria, Microbacterium OF 62,144, Nanobacter, and microorganisms OF the family OF New strains, microorganism OF Escherichia, microorganism OF the family Legionnella, Escherichia, strain, Escherichia, strain, 32-33 (2016). These documents also describe methods for the cultivation of bacteria useful according to the invention.

Optionally, the bacteria are available in the collection of microorganisms and cell cultures and available [ possible missing text ] German corporation (German GmbH), in particular under application numbers DSM22607, DSM 102344, DSM 102800.

Compositions according to the invention

The bacterium or bacteria useful according to the invention are preferably administered in a composition comprising at least the useful bacterium or bacteria and/or a supernatant thereof in a physiologically acceptable medium, for the uses described hereinabove.

The invention therefore also relates to a composition comprising at least one bacterium of the genus cretinismus for use in the prophylaxis and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer in a human or animal, preferably in a human or animal which exhibits a overproduction of interleukin 6.

In particular, the invention makes it possible to provide an effective amount of a bacterium of the family of the Klebsiella family, such as Marek's disease, Drechslera, human enteroclepias and/or Microclepias, for the treatment of:

-diseases associated with increased interleukin 6, such as inflammatory disorders, allergic disorders, autoimmune disorders, graft rejection disorders, atherosclerosis, osteoporosis, neuropathic pain, sepsis, Castleman's disease, fibrotic conditions, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis or ankylosing spondylitis,

diseases associated with an increase in interleukin 8, such as atopic dermatitis, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, stroke, septic shock, endotoxic shock, psoriasis, gram negative sepsis, toxic shock syndrome, reperfusion kidney injury, glomerulonephritis, thrombosis, graft versus host reaction, allograft rejection, malaria, restenosis, angiogenesis, atherosclerosis, osteoporosis, gingivitis and unwanted release of hematopoietic stem cells, diseases caused by respiratory viruses, herpes viruses and hepatitis viruses, meningitis, herpetic encephalitis, CNS vasculitis, traumatic brain injury, CNS tumors, subarachnoid hemorrhage, post-operative trauma, cystic fibrosis, pruritus, interstitial pneumonia, hypersensitivity reactions, Crystalline arthritis, Lyme arthritis, progressive ossified fibrodysplasia, acute and chronic pancreatitis, necrotizing enterocolitis, chronic sinusitis, uveitis, polymyositis, vasculitis, acne, gastric and duodenal ulcers, celiac disease, lupus, and/or

Diseases associated with a decrease in interleukin 10 or which can be treated by increasing interleukin 10, such as autoimmune diseases, organ and bone marrow transplant rejection, graft-versus-host disease, parasitic infections, granulomatous tumors, crohn's disease, colitis, pancreatitis, inflammatory lung disease, inflammatory eye disease, atopic dermatitis and rhinitis.

According to one embodiment, the invention relates to a composition for the treatment and/or prophylaxis of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer in humans or animals, comprising (i) at least one bacterium of the genus cretinismus and/or (ii) a culture supernatant of at least one bacterium of the genus cretinismus in a physiologically acceptable medium.

The invention also relates to a composition for treating and/or preventing inflammatory gastrointestinal diseases in an individual, said composition comprising, in a physiologically acceptable medium

(i) At least one bacterial strain selected from the group consisting of tomorrow's bacilli, cremopsis parvum and/or mixtures thereof, and/or

(ii) A culture supernatant of at least any one of these bacterial strains and/or a mixture thereof.

According to any of the preceding embodiments, the composition of the invention may be administered to a human having a chronic inflammatory disease and/or an inflammatory gastrointestinal disease and/or cancer, said human having a Body Mass Index (BMI) of less than 25, in particular said composition is administered to a human having an inflammatory gastrointestinal disease.

The term "BMI less than 25" means a BMI strictly less than 25, more specifically a BMI less than or equal to 24.9. According to any of these embodiments, the human BMI may specifically be less than 25 and greater than or equal to 18.5.

Preferably, the composition according to the invention is characterized in that the bacteria of the genus cretinism are selected from the group consisting of Maritensinsecticeps, Thymodensiceps and/or Microcretiniceps and/or human Enterobacter and/or mixtures thereof. The bacteria of the genus creistens may in particular be a mixture of: maricitescens and Pemeridus or Maricitescens and Pemeridus hominis or Pemeridus hominis and Pemeridus hominis or Maricidus, bimicristesynia and small christmas, or Maricitesia, bimicrynia and human enterotesia, or bimicrynia and small christmas and human enterotesia, or Maricitesia, Maricitesia and small christmas and human enterotesia.

According to any of the preceding examples, the composition may be used in individuals whose gut microbiota is not deficient in cretinis, in particular humans or animals whose cretinis account for at least 0.01% of the total number of bacterial genera of their gut microbiota.

One or more bacteria are present in the composition in an effective amount to provide an effect on chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer affecting the treated human or animal. The term "human" is understood to mean an individual, in particular a human or an animal.

Preferably, a composition useful according to any of the preceding embodiments comprises a daily dose of said composition 10 to be administered⁶To 10¹²Colony Forming Units (CFU) of a bacterium of the genus Klitestonia. Preferably, this corresponds to the daily dose of bacteria to be administered, regardless of the body weight of the human or animal. Preferably, this dose is administered once daily. In particular, useful compositions include a daily dose 10 to be administered⁷To 10¹¹Bacteria of the genus CFU Cristermann, preferably 10⁹And (4) CFU. In particular, the bacteria present in the composition may be of the same species, a mixture of species or a mixture of bacterial strains. The compositions useful according to the invention may be in liquid form. The composition may in particular comprise a bacterium of the family of Cristesyniaceae, in particular the genus Cristesyneanus and/or may be made availableTo preserve the bacteria in its culture medium, such as anaerobic Columbia agar medium preferably enriched in sheep blood or an equivalent medium free of any animal by-products. For the purposes of the present invention, the term "culture medium" means the culture supernatant or extracellular medium in which the bacteria or bacterial strains reside during their cultivation.

According to one variant, the compositions useful according to the invention may be in solid form. In this case, the bacteria may be present in lyophilized form and may further comprise excipients such as microcrystalline cellulose, lactose, sucrose, fructose, levulose, starch (starch), stachyose, raffinose, starch (amylum), calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin, galacto-oligosaccharides, fructo-oligosaccharides, pectin, beta-glucan, lactoglobulin, isomalto-oligosaccharides, polydextrose, sorbitol and/or glycerol.

Compositions useful according to the invention may be in the form of: powders, microencapsulated powders, gelatin capsules, tablets, lozenges, granules, emulsions, suspensions, suppositories, beverages, foods, pharmaceutical or nutraceutical products, food additives, dietary supplements or dairy products.

According to a particularly suitable embodiment, the composition may be in a gastro-resistant form, such as a coated tablet containing microencapsulated bacteria. Thus, the composition may be provided with a gastro-resistant coating to ensure that the bacteria of the invention contained in the composition can pass through the damaged stomach. The release of the one or more bacteria may thus take place for the first time in the upper intestinal tract.

More specifically, the composition according to the invention may be comprised in a food supplement. The food supplement for oral administration may be present in capsules, hard capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes, lozenges, gums, oral solutions or emulsions, syrups or gels.

The food supplement according to the present invention may further comprise sweeteners, stabilizers, antioxidants, additives, flavouring agents and/or colouring agents. The formulation thereof is carried out by means of the usual methods for producing sugar-coated tablets, capsules, gels, controlled-release hydrogels, emulsions, tablets or capsules.

The composition according to the invention may also be in the form of a nutritional composition. Such nutritional compositions according to the invention are in the form of: yogurt, cereal bars, breakfast cereals, desserts, frozen foods, soups, pet foods, liquid suspensions, powders, tablets, gums or candy pieces.

The composition according to the invention may further comprise at least one ingredient selected from the group consisting of: antioxidants, fish oil, DHA, EPA, vitamins, minerals, phytonutrients, proteins, lipids, probiotics, and combinations thereof. The bacteria according to the invention, alone or comprised in a composition according to any of the preceding embodiments, may be used in live, semi-active, inactivated or dead form, preferably in live form. The semi-active, inactivated or dead form of the bacteria may be achieved, for example, by irradiation, heat inactivation or lyophilization, in particular by heating, exposure to a suitable pH, UV, gamma rays, X-rays or high pressure. Thus, the term "semi-active" designates a bacterium of low physiological activity whose proliferative capacity is temporarily or permanently reduced. The term "inactivation" designates a bacterium that is no longer capable of proliferation, either temporarily or permanently. The term "dead" designates bacteria which are clearly no longer capable of proliferation. Dead or inactivated bacteria may have intact or ruptured cell membranes. Thus, the term "inactivation" also designates extracts and lysates of the resulting bacteria.

The bacteria according to the invention, alone or in the composition according to the invention, can be used in intact form, i.e. essentially in their natural form or in the form of extracts or lysates comprising various parts and/or metabolites of this microorganism. Such lysate may represent all or part of the lysate obtained by lysis and may be prepared according to one of the conventional methods well known to the person skilled in the art, such as thermal shock, ultrasound, osmotic shock or under mechanical stress, such as by centrifugation. The bacteria may be living, semi-active, inactivated or dead. In particular, the bacteria may be a mixture of live, semi-live, inactivated and dead bacteria.

Preferably, at least some of the bacteria are viable, in particular at least 50% (by number), even more preferably at least 90% (by number).

Thus, according to a suitable embodiment, at least 50% (by number) of the bacteria present in a useful composition according to the invention are live bacteria, preferably at least 90% (by number) are live bacteria, even more preferably all are live bacteria.

The storage conditions of the liquid formulation according to the invention are in the form of a frozen product maintained at-20 ℃ in a sealed package. For solid formulations, the storage conditions according to the invention include a light and oxygen barrier capsule or coating maintained at ambient temperature between 15 ℃ and 40 ℃ and humidity content between 3% and 70%.

The composition according to any one of the preceding embodiments is provided for use in the digestive tract, in particular the intestinal tract. As a result, the useful bacteria according to the invention and in particular the compositions comprising said bacteria can be administered by the oral, topical, inhalation or rectal route, preferably the rectal or intrarectal route.

Specifically, rectal or intrarectal administration is in the form of suppositories, enemas, or foams.

Surprisingly, the inventors have also determined that a klebsiella strain is capable of enhancing the anti-inflammatory properties of an akkermansia strain and in particular of an akkermansia muciniphila strain. Thus, the inventors have demonstrated that the combination of a klebsiella strain (or culture supernatant of such a strain) and an akkermansia strain (or culture supernatant of such a strain) is synergistic in the prevention and/or treatment of gastrointestinal inflammation, and in particular a klebsiella and/or delmopsis strain (or culture supernatant of such a strain) and a mucinous akkermansia strain (or supernatant of such a strain) is synergistic in the prevention and/or treatment of gastrointestinal inflammation.

Thus, according to any of the preceding embodiments, the composition may further comprise:

(i) at least one additional bacterium of the family verrucomicrobiaceae; and/or

(ii) A culture supernatant of the additional bacteria.

Preferably, the further bacterium of the family of verrucomicrobiaceae is a bacterium of the genus akkermansia, more particularly akkermansia muciniphila. Within the meaning of the present invention, a bacterium of the species Ackermansia muciniphila is also understood to mean a bacterium known as "Ackermansia muciniphila".

According to one embodiment, the bacterium as mentioned above and the further bacterium are in a live, semi-active, inactivated and/or dead form, and in particular in a live form, independently of each other.

In certain embodiments, the daily dose of a bacterium of the genus akkermansia, more particularly akkermansia muciniphila, to which a composition according to the invention is suitable for administration represents 10⁷To 10¹¹Colony Forming Units (CFU), preferably in a daily dose equivalent to 10⁹CFU。

In case a culture supernatant of any of the above mentioned bacteria is used, the composition according to the invention may specifically be comprised in an amount comprised between 0.1 and 99 wt. -%, specifically 10 to 90 wt. -%, specifically 25 to 70 wt. -% and more specifically 30 to 50 wt. -%, based on the total weight of the composition.

Thus, a composition according to any of the above embodiments may comprise a supernatant in an amount of between 0.1 wt% and 99 wt%, specifically 10 wt% to 90 wt%, specifically 25 wt% to 70 wt%, more specifically 30 wt% to 50 wt%, based on the total weight of the composition.

In addition to the useful bacteria according to the invention, the useful compositions according to the invention may also comprise at least one probiotic and/or at least one prebiotic.

In addition to the useful bacteria according to the invention, the useful compositions according to the invention may also comprise other compounds, such as:

at least one probiotic, and/or

-at least one lactic acid producing bacterium that makes it possible to create an anaerobic environment that is favorable to the family of crewsoniaceae, such as at least one bacterium chosen from the group consisting of Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), Streptococcus (Streptococcus), and/or at least one other organism that promotes the anaerobic conditions necessary for the survival of the family of crewsoniaceae, such as a microorganism chosen from at least one yeast of the genus saccharomyces (Lactobacillus) or Methanobacteriaceae (Methanobacteriaceae), and/or

At least one bacterium associated with the ecosystem of the family of the Cladosteinaceae, since it promotes its own survival in the intestinal tract, such as at least one bacterium selected from the group consisting of Firmicutes, Bacteroidetes, actinomycetes, Tenericutes and Verrucomicrobia, and/or

-at least one bacterium selected from the group consisting of: clostridiales, Verrucomicrobiales (Verrucomicrobiales), aeromonales (Aeromonadales), alteromonales (Alteromonadales), ML615J-28, RF32, YS2, clostridiaceae, Lachnospiraceae (Lachnospiraceae), Ruminococcaceae (Ruminococcaceae), Bacteroidaceae (bacteroides), Enterococcaceae (Enterococcaceae), riellaceae (rikennellaceae), desaliniceae (dehalogenaceae), Veillonellaceae (veillonellacee), and/or

-at least one bacterium selected from: bacteria of the genera Faecalibacterium, akkermansia, Eubacterium and oscillatoria, such as Faecalibacterium prausnitzii, akkermansia muciniphila, Eubacterium halodurans, oscillatoria giganteum, and/or oscillatoria

-at least one prebiotic, such as at least one prebiotic selected from the group consisting of: galacto-oligosaccharides, fructo-oligosaccharides, inulin, arabinoxylans, beta-glucans, lactoglobulins and/or beta-casein, and/or

-at least one polyhydric phenol, such as at least one polyhydric phenol selected from: quercetin, kaempferol, resveratrol, flavone (such as luteolin), flavan-3-ol (such as catechin), flavanone (such as naringenin), isoflavone, anthocyanin, procyanidin, and/or

-at least one mineral and/or at least one vitamin and/or at least one nutritional agent, and/or

-at least one active pharmaceutical ingredient, preferably selected from: non-steroidal anti-inflammatory drugs, antibodies to pro-inflammatory targets (anti-TNF α), anti-rheumatic drugs, analgesics, antibacterial agents, corticosteroids, anabolic steroids, antidiabetic agents, thyroid agents, antidiarrheal agents, cough suppressants, antiemetics, antiulcer agents, laxatives, anticoagulant agents, erythropoietin, immunoglobulins, immunosuppressive agents, growth hormones, hormonal agents, estrogen receptor modulators, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma agents, β -agonists, inhaled steroids, leukotriene inhibitors, cromolyn salts or cromolyn, epinephrine, alpha chain enzymes, cytokines, cytokine antagonists.

Finally, the invention relates to a composition comprising, in a physiologically acceptable medium:

(I) (a) at least one bacterium selected from the group consisting of: dimodermacetrin, cremopsis parvum, Maricillethrix parvum, human enterocremopsis parvum and/or mixtures thereof;

and/or

(b) At least one culture supernatant of at least one bacterium selected from the group consisting of: dimodermacetrin, cremopsis parvum, Maricillethrix parvum, human enterocremopsis parvum and/or mixtures thereof;

and

(II) (a) at least one further bacterium of the genus akkermansia, more particularly akkermansia muciniphila;

and/or

(b) At least one culture supernatant of at least one further bacterium of the genus akkermansia, more particularly akkermansia muciniphila.

Method of prevention and/or treatment

According to another aspect, the present invention relates to a method for reducing and/or preventing inflammation in an individual in need thereof, the method comprising administering to the individual an effective amount of a bacterium of the family cremastaceae, in particular a bacterium of the family cremastesynellaceae.

The invention also relates to a method for reducing IL-6 levels and/or stabilizing IL-6 levels and/or preventing IL-6 levels from increasing in a subject in need thereof, which method comprises administering to the subject an effective amount of a bacterium of the family Clostridiaceae, in particular a bacterium of the family Clostridiaceae, preferably the subject exhibits an elevated IL-6 serum level prior to treatment. Specifically, the elevated serum IL-6 level is greater than 5pg/ml, greater than 10pg/ml, greater than 20pg/ml, greater than 30pg/ml, greater than 40pg/ml, greater than 50pg/ml, greater than 70pg/ml, greater than 90pg/ml, or greater than 100 pg/ml.

The invention also relates to a method for reducing IL-8 levels and/or stabilizing IL-8 levels and/or preventing increased IL-8 levels in a subject in need thereof, which method comprises administering to the subject an effective amount of a bacterium of the family Clostridiaceae, in particular a bacterium of the family Clostridiaceae, preferably the subject exhibits an elevated serum IL-8 level prior to treatment. Specifically, the elevated serum IL-8 level is greater than 5pg/ml, greater than 10pg/ml, greater than 20pg/ml, greater than 30pg/ml, greater than 40pg/ml, greater than 50pg/ml, greater than 70pg/ml, greater than 90pg/ml, or greater than 100 pg/ml.

The invention also relates to a method for increasing IL-10 levels and/or stabilizing IL-10 levels and/or preventing a decrease in IL-10 levels in a subject in need thereof, which method comprises administering to the subject an effective amount of a bacterium of the family Clostridiaceae, in particular a bacterium of the family Clostridiaceae, preferably the subject exhibits a low serum IL-10 level prior to treatment. Specifically, the low serum IL-10 level is less than 15pg/ml, less than 10pg/ml, or less than 5 pg/ml.

In addition, the present invention relates to a method for reducing and/or stabilizing and/or preventing an increase in kynurenine levels in a subject in need thereof, which method comprises administering to the subject an effective amount of a bacterium of the family cretinidae, in particular cretinia bacteria, preferably the subject exhibits a high fecal kynurenine level prior to treatment.

The invention also relates to a method of treating and/or preventing cancer associated with inflammation in an individual in need thereof, the method comprising administering to the individual an effective amount of a bacterium of the family Klebsiella.

The method according to the invention makes it possible to reduce the level of at least one of the above-mentioned markers of inflammation in said individual, said marker preferably being reduced in serum, blood or faeces.

Preferably, the method of the invention is carried out in a subject, preferably a human, suffering from one or more of the diseases described herein above.

Preferably, the 16S ribosomal RNA gene comprised by a bacterium of the genus cremastra exhibits at least 50%, at least 60%, at least 70%, at least 80% or preferably at least 90% sequence identity with the 16S ribosomal RNA gene from cremastesma minutissima, Marithelinus or Tellimelinus, more preferably at least 97% sequence identity with a sequence selected from the group consisting of SEQ ID No. 1, 2, 3, 4,5 or 6.

SEQ ID NO 1 is the sequence of the 16S ribosomal RNA gene of the bacterium Klebsiella parvula DSM22607, isolated from human fecal samples. The 16S rRNA sequence of Klitestonia parvum is identified by GenBank accession number AB 490809. SEQ ID NO 2 is part of the 16S ribosomal RNA gene sequence from the bacterium Klebsiella parvula DSM 22607. Additional 16S sequences of bacteria of the genus Kleinstein-Barr are provided, as SEQ ID NOs:3-6 (OTU numbering in the Greenenges database is 701845, 177179, 1146771 and 361793, respectively). The identity of each sequence relative to SEQ ID NO 1 is as follows: 3, 93% identity to SEQ ID No. 1; 4, 95% identity to SEQ ID No. 1; 5, 97% identity to SEQ ID NO 1; SEQ ID NO 6, 95% identical to SEQ ID NO 1.

In particular, the method comprises the administration of at least one bacterium or composition according to the invention, characterized by any one of the above examples, such as a mixture or non-mixture of bacteria, an amount administered, an amount of living cells, additional components, such as active pharmaceutical ingredients, prebiotics or probiotics, vitamins, minerals, nutrients, other microorganisms, specific formulations of the composition, such as powders, capsules, etc.

Understandably, the methods of the invention comprise a sufficient amount of oral, inhalation, topical, rectal or intrarectal administration.

The invention is now illustrated by the following items: examples of useful bacteria according to the invention, methods of culturing these bacteria, examples of compositions containing the bacteria, and test results demonstrating the effectiveness of the invention, are shown for illustrative purposes only.

Examples of the invention

Example 1: klisteinson bacteriaceae

The Klebsiella parvula can be cultured according to the protocol described below.

1/dissolving dehydrated RCM (enhanced Clostridium culture Medium) in distilled water

2/Add 0.5ml/L Resazurin-Na solution (0.1% w/v)

3/boil to boil and cool to room temperature while injecting 80% N₂And 20% CO₂Of a gas mixture

4/diffusion of the culture medium in anoxic Hungate tubes or serum vials under the same gaseous atmosphere followed by autoclaving

5/before use, 80% N₂And 20% CO₂Prepared from a gaseous mixture to which 1.0g of sodium carbonate per litre was added

The pH of the medium was checked after 6/autoclaving and used at 80% N₂And 20% CO₂Prepared in gaseous atmosphere ofStock solution of bacterial hypoxic sodium bicarbonate (5% w/v) adjusted the pH to between 7.3 and 7.5.

Example 2: maricitescens sp

Marasmius mosaic may be cultured according to the protocol described below.

1/preparation of carboxymethyl cellulose (N) according to the following description of DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen)₂/CO₂) And (4) a culture medium.

[ Table 1]

Tyrose peptone	30.0g
		Yeast extract	5.0g
K2HPO4	5.0g
		Na-Resazurin solution (0.1% w/v)	0.5ml
L-cysteine-HCl xH2O	0.5g
		D-glucose	4.0g
Cellobiose	1.0g
		Maltose	1.0g
Minced meat (without fat)	500.0g
		NaOH 1N	25.0ml
Distilled water	1000ml
		Starch (soluble)	1.0g
Na2CO3	1.5g
		Meat filtrate (see Table 2)	1000ml

2/dissolve the various ingredients listed in the table above, except cysteine, carbohydrate and carbonate.

3/boiling the medium for 1 min, then bringing it to 80% N₂And 20% CO₂Is cooled to room temperature.

4/adding 0.5g/L of L-cysteine-HCl xH₂O and poured into a Hungate type tube under the same gaseous atmosphere (for strains requiring meat particles, these are first introduced into the tube, using 1 part meat particles for 4 or 5 parts liquid).

5/autoclaving at 121 ℃ for 20 minutes.

6/after autoclaving, from 100% N₂Adding glucose, cellobiose, maltose and starch into sterile anoxia stock solution prepared from gas at a ratio of 80%N₂And 20% CO₂The sterile anoxic stock solution prepared under the gas mixture of (a) is added with carbonate.

7/if necessary, the pH of the medium is adjusted to 7.

Preparation of meat filtrate:

[ Table 2]

Lean beef or horse meat is used.

Fat and connective tissue are removed prior to mincing.

Meat, water and NaOH were mixed and then boiled for 15 minutes while stirring.

Cooled to room temperature, fat was removed from the surface and filtered off, thereby retaining the meat particles and filtrate.

To the filtrate was added water to a final volume of 1000.0 ml.

The bacteria should be cultured under anaerobic conditions at 37 ℃.

Example 3: thymus delbrueckii

The tessercenella marcescens can be cultured according to the same operating protocol as described for the tessercenella marcescens in example 2.

Example 4: specific strains according to the invention

All strains described below were obtained from the DSMZ Collection in Germany (Leibnitz DSMZ Institute). Is full of N₂＝85％、CO ₂10% and H₂YBHI medium [ brain-heart perfusion medium supplemented with 0.5% yeast extract (Difco) ] in a 5% anaerobic chamber supplemented with cellobiose (1 mg/ml; Sigma)), maltose (1 mg/ml; Sigma), cysteine (0.5 mg/ml; Sigma) and mucin (1 mg/ml; Sigma)]Culturing of Klebsiella parvum DSM22607, of Klebsiella delbrueckii DSM 102800 and of Ackermanella muciniphila DSM 22959 at 37 ℃.

Example 5: supernatant-based useful compositions

Examples of compositions useful according to the invention are compositions comprising between 0.1% and 95% of the culture supernatant of the strains cretinisson DSM22607, dermocritesin DSM 102800 and akkermansia muciniphila DSM 22959.

Example 6: compositions in liquid form useful according to the invention

An example of a liquid form composition useful according to the invention is the inclusion of Klebsiella parvula 10 in the anaerobic RCM medium described above⁹CFU/ml, said medium modified to be free of animal by-products and enriched in 5% glycerol.

The composition of example 6 obtained from RCB (Research Cell Bank) was 10¹⁰CFU/ml Microcretini was prepared and then stored frozen at-20 ℃ in oxygen-tight packaging.

The frozen composition must be warmed to room temperature until it returns to a liquid form before use.

Example 7: solid form compositions useful according to the invention

Examples of compositions in lyophilized form useful according to the present invention can be obtained by lyophilizing the composition of example 6 in a frozen state.

Test results demonstrating the effects of the present invention

1. Demonstration of an in vitro anti-inflammatory action of a bacterium of the genus Klitenststosenia (kynurenine)

The aim of the study was to demonstrate the in vitro anti-inflammatory action of the bacteria of the genus crewsonia according to the invention. This effect was demonstrated for the known inflammation marker kynurenine. Human feces were collected and cultured in a fermenter.Kries The abundance of Tensenna is related to the abundance of kynurenine present in the fermenter。

The following describes the protocol of the study.

Fermentation protocol for feces from human origin containing Klitestonia sp：

Donors should not take antibiotics within six months prior to the experiment and have no history of gastroenteropathy. Donors are between 18 and 60 years of age.

-collecting a fresh sample of donor feces in a sterile plastic container, stored in an anaerobic vial containing a sachet of oxoid tmananoerogen 2.5l (O)₂<0.1％；CO₂: 7-15%). These samples were taken to the laboratory within two hours after production.

-stool samples were diluted 1/5 (weight/volume) in pH 7.4 phosphate buffered saline (1M) (PBS). The suspension was homogenized in a homogenizer (stomacher) for 120 seconds.

-basic nutrient medium: the basal nutrient medium was prepared from: 2g/L tryptone soy broth, 2g/L yeast extract, 0.1g/L NaCl, 0.04g/L K₂HPO₄、0.01g/L MgSO₃.7H₂O、0.01g/L CaCl₂.6H₂O、2g/L NaHCO₃0.5g/L L-cystine HCl, 2ml/L Tween 80, 10 μ L/L vitamin K1, 0.05g/L heme, 0.05g/L bile salt, 4ml/L resazurin (pH 7).

-fermentation in a fermenter: a20 ml fermenter containing 18ml of the basal nutrient medium was autoclaved (15 minutes at 121 ℃) and aseptically poured into a sterile fermenter. The system was allowed to stand overnight with anaerobic nitrogen bubbling through the medium at a rate of 2 ml/min. The pH was maintained between 6.7 and 6.9 using HCl or NaOH (0.5M). The temperature of each of the fermentors was controlled at 37 ℃, and the contents of the vessels were homogenized with a magnetic mixer.

-adding a mixture of pre-digested proteins (0.35g) to the container before inoculating 2ml of faecal inoculum at T0. The pre-digested protein was obtained according to a gastrointestinal digestion protocol adapted from Versantvorort et al (2005).

Samples were collected before fermentation (T0) and after 48 hours of fermentation (T48) and frozen at-80 ℃ until analysis.

2/Quantification of kynurenine

-mixing 50 μ L of the sample collected and stored at-80 ℃ with 20 μ L of Milli-Q water containing an internal standard.

-mixing and filtering the mixture through a 5-kDa cut-off filter to remove macromolecules.

-detecting the metabolites by capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS) analysis. The peak detection limit is determined based on the signal-to-noise ratio S/N-3.

Relative peak area (peak area of metabolite … …) (peak area of internal standard … … x sample amount).

3/Quantification of Kliteston bacterium

Use of a catalyst from Macherey-Nagel

The 96Soil kit extracts the DNA contained in the sample according to the manufacturer's instructions.

The total extracted DNA was then randomly fragmented into 350bp fragments, which were then used to construct libraries using the NEBNext Ultra II kit from New England biologies laboratories (New England Biolabs) according to the manufacturer's instructions.

The library was then sequenced on the Illumina HiSeq platform using 2x150bp paired-end sequencing.

-measuring the abundance of bacteria by creating a catalogue of Metagenomic (MGS) species from a reference catalogue containing 22M genes. These MGS are then associated at the appropriate classification level. In the case of the genus crewsonia, these MGS were detected at the genus level and were therefore cited in this experiment by crewsonia.

The relative amounts of kynurenine (relative to the total amount measured) and the relative abundance of klebsiella (relative to the total amount measured) were analyzed and correlated to obtain a linear regression R-0.44 (n-18). The results are shown in table 3.

[ Table 3]

2. Demonstration of an in vitro anti-inflammatory action of a bacterium of the genus Klitenststosenia (IL-8)

The aim of this study was to demonstrate the in vitro anti-inflammatory effect of the bacteria according to example 4. The demonstration was performed on one of the inflammatory markers: interleukin 8 (IL-8).

The study was carried out on HT-29 cells obtained as follows:

HT-29(ATCC HTB-38) human colonic epithelial cells (LGC Standards) were cultured in Dulbecco's Modified Eagle's minimal essential medium (DMEM) (Sigma-Aldrich) supplemented with 10% (w/v) heat-activated Fetal Bovine Serum (FBS) (GibcoBRL, Eragny, France) of Erhagoni France and penicillin G/streptomycin (5000IU/ml, 5000. mu.g/ml) (Sigma-Aldrich)). Cell cultures were grown at 25-cm²Tissue culture flasks (Nunc, Roskilde, Denmark) from Rosschel, Denmark) at 37 ℃ in 5% (v/v) CO₂Incubation in atmosphere until reaching confluent state.

The following describes the protocol of the study.

Single and co-cultures consisting of combinations between the listed strains were prepared at time 0. At 24 hours, the bacterial samples were centrifuged at 6000rpm for 10 minutes. The supernatant thus obtained was stored at-80 ℃ to continue the analysis of the in vitro immunomodulatory properties.

Anti-inflammatory tests were performed according to the procedure described in Kechaou et al, 2012 (Kechaou N et al, applied to environmental microbiology (apple Environ Microbiol), 79: 1491-. Specifically, 50,000 Hela (HeLa) or HT-29 cells were seeded per well in 24-well culture plates (Nunc Corp.). Twenty-four hours prior to bacterial contact, the medium was replaced with 5% FBS-containing medium. Experiments were performed on day 7 post-inoculation when cells were in confluent state (1.83x 10)⁶Individual cells/well). Twenty-four hours before the bacterial co-culture (day 6), the medium was replaced with a medium containing 5% heat-inactivated FBS and 1% glutamine. On the day of co-cultivation, 10% bacterial supernatant was added to DMEM in a total volume of 500 μ l. At 37 ℃ in 10% CO₂While using human TNF-alpha (5ng/ml, Peprotech, NJ) in Western PaptakeCells were stimulated for 6 hours. All samples were analyzed in triplicate. After co-incubation, cell supernatants were collected and stored at-80 ℃ until further analysis of interleukin-8 (IL-8) by ELISA (Biolegend, San Diego, Calif.). Experiments were performed in at least triplicate.

FIG. 1 shows the production of IL-8 in HT-29 cells stimulated by TNF- α in the presence of Klebsiella parvum, Klebsiella delbrueckii, Ackermanella mucovora, Klebsiella parvum + Ackermanella mucovora, and Klebsiella delbrueckii + Ackermanella mucovora. The results are expressed as IL-8(pg/mL) and normalized using IL-8 produced after co-incubation with PBS as a negative control as a reference. The results were analyzed using a Kruskal-Wallis test (Kruskal Wallis test) followed by dunn multiple comparison test (p < 0.05;. p < 0.01).

Statistical analysis was performed using GraphPad Software (GraphPad Software, La Jolla, CA, USA, ralasia, CA). The results are presented in the form of a box plot (min to max). Comparisons were made with a kruskal-voriss nonparametric test and subsequent dunne multiple comparisons. p values less than 0.05 were considered significant.

The results are described below:

the reference strain Ackermansia muciniphila DSM 22959 is well known for its immunomodulatory properties and more specifically for its anti-inflammatory effects (Derrien M, Belzer C, de Vos WM., "microbial pathogenesis (Microb disease.)" in 2017, 5 months; 106:171-181.doi:10.1016/j. micropath.2016.02.005.2016, 11. d. 2 months). To determine whether the strains of the small critique-stevenson strain DSM22607 and the timothrix-stevenson strain DSM 102800 were able to modulate the immune response, the inventors tested the immunomodulatory properties of the supernatant of the strains in vitro in a model based on the ability to block the production of IL-8 (pro-inflammatory cytokine) induced by TNF- α stimulation in HT-29 colonic epithelial cells.

Thus, the inventors observed that the two strains were able to block the production of IL-8 in the same way as the positive control (akkermansia muciniphila DSMZ 22959) (fig. 1).

The Klebsiella parvula DSM22607 and the Klebsiella timoni DSM 102800 increase the anti-inflammatory properties of the Ackermanella muciniphila DSMZ 22959 in vitro.

To test whether members of the genus klebsiella can increase the anti-inflammatory properties of other members of the gut microbiota, co-culture with akkermansia muciniphila was performed. As also shown in fig. 1, cretinism parfait DSM22607 and dermasepsis deltoid DSM 102800 were able to increase the anti-inflammatory properties of akkermansia muciniphila DSMZ 22959 in a statistically significant manner.

3. Demonstration of an in vitro anti-inflammatory action of a bacterium of the genus Klitenststosenia (IL-8)

The aim of this study was to demonstrate the in vitro anti-inflammatory effect of the bacteria according to the invention. The demonstration was performed on one of the inflammatory markers: interleukin-8.

The following describes the protocol of the study.

When HT-29 cells were in confluent state (approximately 7 days after seeding), complete medium (DMEM Glutamax + 10% FCS) was removed and replaced with 5% FCS-containing medium (D0). On day D +1, the medium was removed. Two media were prepared (DMEM Glutamax + 5% FCS +/-TNF-. alpha.). 450 microliters/well of one of the two media was then added, followed by 50 μ L of the supernatant to be tested. After 6 hours of co-incubation, the supernatant was recovered and stored at-80 ℃. Then, an IL-8 chemokine ELISA assay (ELISA MAX luxury version of IL-8(ELISA MAX Deluxe set Human IL-8) from Biolegend) was performed.

FIG. 2 shows the determination of the chemokine IL-8 secreted by HT-29 cells (colon adenocarcinoma) in an inflammatory state induced by TNF-alpha. Different bacterial supernatants of bacteria belonging to the species cretinissella (cretini 1, cretini 2, cretini 3, cretini 4 and DSMZ, corresponding to strain DSM22607) were tested in order to evaluate their immunomodulatory capacity. The bacterial culture medium (GAM) represents a control, so that the effect of the supernatant can be assessed. Experiments were performed in triplicate four times.

All the small klebsiella strains tested had an anti-inflammatory immunomodulating effect (the level of secreted IL-8 was reduced by more than 50%), which makes it possible to demonstrate the anti-inflammatory potential of the klebsiella.

4. Demonstration of the Permeability of bacteria of the genus Klitenshensis to the epithelial lining of the intestinal tract

FIG. 3 shows transepithelial electrical resistance measurements performed on Caco-2 epithelial cells. The latter, by differentiating, establish a link (called a tight link) between them, thus maintaining the integrity of this barrier produced by the cells, which mimics the intestinal barrier present in the digestive tract.

The addition of the cytokine TNF-alpha (tumor necrosis factor) destroys these tight junctions and then increases the membrane permeability, thus promoting, for example, inflammatory or infectious diseases and thus reducing the TEER ratio (DMEM + TNF-alpha control). "DMEM" control group refers to wells in which only cell culture medium is present and no TNF-. alpha.or bacteria are present.

Transepithelial electrical resistance (TEER) is a method for quantifying the integrity of the epithelial tissue barrier by measuring the electrical resistance across the epithelial tissue barrier. TEER measurements are performed by placing electrodes at both poles of a layer of epithelial cells that have been cultured on a semi-permeable membrane. The electrodes apply an alternating current, allowing the resistance through the layer of epithelial cells to be measured.

The TEER ratio is obtained by the following calculation:

TEER was measured 7 days after inoculation of Caco-2 cells (intestinal epithelial cells) in Transwell plates using an automated machine (REMS). The value is recorded and represents a start value (T0). The cells were then co-incubated at MOI 1/40 for 3 hours in the presence of bacteria, which was the time for the latter to adhere to the cells. The cells were then challenged with TNF- α, inducing destruction of the epithelial cell layer, specifically at the tight junctions. TEER was measured again 24 hours after addition of bacteria (T24).

This figure demonstrates that cretini bacteria affect the integrity of the intestinal barrier by increasing transepithelial resistance, indicating a potential effect on claudin and decreased intestinal permeability. Thus, the bacteria of the species cretini make it possible to restore the electrical resistance of the epithelial cell membrane.

5. Demonstration of in vivo anti-inflammatory action of bacteria of the genus Klitestonia

The aim of this study was to demonstrate the in vivo anti-inflammatory effect of the bacteria according to the invention on mice. The demonstration was performed on the main inflammatory markers, namely the following two cytokines: IL-6 (Interleukin 6) and TNF- α (tumor necrosis factor- α). This study was performed as a blind study: the experimenter is unaware of the treatment modality, so that his prior knowledge does not affect the results of the study in any way.

The operating scheme is described below.

Four week old C57BL/6 male mice were purchased from Charles River (Charles River, St german sur I' arblesle, France).

Mice underwent a one week adaptation to a standard diet based diet ("feed (SafeA 04)") before starting the study.

On the first day (D0), animals were subjected to a fat-rich Diet (45% kcal "Research Diet D12451") and randomized into 2 groups (n ═ 5 per group), each group receiving 150 μ L of cretinism parva (10 μ L)⁹CFU/mL) solution for 12 weeks, i.e. a control solution consisting of the medium used to grow the bacteria. Animals were provided food and water ad libitum throughout the duration of the study and were housed in a room at a controlled temperature (22.0 ± 2.0 ℃) and humidity (40-50%) with a 12 hour light (8 am to 8 pm)/12 hour dark cycle.

At the end of the experiment (D85), a final blood sample was taken from the anesthetized animal (with a mixture of ketamine/xylazine 80/10 mg/kg). Blood samples were collected in centrifuge tubes (200IU/ml blood) pre-filled with heparin sulfate. Plasma was separated by centrifugation (3500rpm, 15 min, 4 ℃), collected and stored at-80 ℃ until analysis.

Use "

Cytokine Mouse Magnetic 10 plexus (Cytokine Mouse Magnetic 10-Plex) "ELISA assay kit and plasma levels of cytokines IL-6 and TNF-alpha were determined according to the manufacturer's instructions. The measurements were carried out by the company VEBIO (Archie, France) in France.

The results of the assays for the proinflammatory cytokines TNF-. alpha.and IL-6 (presented in Table 4) after 85 days on a 45% (calorie) high fat diet show that bacteria of the family Klitenstaceae strongly limit inflammation.

[ Table 4]

6. Demonstration of in vivo anti-inflammatory action of bacteria of the genus Klitestonia

Once the animals arrived, they were placed in cages (n-5 per cage) and an acclimation period was initiated to recover the mice from transport and to relieve stress by habituation to this new environment. One week later, gavage feeding (except for the 5-ASA control group, i.e. the pharmacological anti-inflammatory control) and body weight monitoring of all groups were started. Control mice (control-vehicle and DNBS-vehicle) were gavaged with 150 μ L of PBS/16% glycerol. DNBS-Christenscenella minuta or C.minuta mice were gavaged with 150 μ L of 10 resuspended in PBS/16% glycerol¹⁰CFU/mL of Microcretinis DSM 22607. After two weeks, inflammation is induced by a chemical called DNBS (dinitrobenzene sulfate). The latter was injected directly into the colon of the mice by a probe introduced into the rectum. DNBS is dissolved in 30% ethanol, thereby making it possible to weaken the intestinal barrier to facilitate DNBS entry into the tissue, thus triggering the immune system and hence inflammation. Gavage continued during this period, and gavage of the 5-ASA group began with 150 μ L on the day of injection (5-ASA prepared daily in the morningSolution, 2 mg/mouse, resuspended in PBS). Euthanasia was performed 3 days after injection. Therefore, the so-called "recovery" period was short, but was necessary to be able to observe the difference from the DNBS control group.

Fig. 4 shows various parameters evaluated during the testing of DNBS-induced inflammatory models.

In fig. 4A, monitoring of weight loss was observed from the day of injection and thus during the subsequent recovery period (D1, D2, and D3). The inventors have thus observed an effect on the recovery that seems to be faster after the application treatment.

In fig. 4B, the macroscopic scores show the severity level of the disease, indicating that a significant decrease in score was observed after treatment with DSM22607, as well as after treatment with the pharmacological anti-inflammatory agent 5-ASA (5-aminosalicylic acid, used as a comparative positive control).

Figures 4C and 4D show that DSM22607 has immunomodulatory potential, evaluated by monitoring myeloperoxidase activity, specifically by reducing the presence of neutrophils in the colon. In addition, an increase in interleukin-10 in the spleen (FIG. 4D) showed an anti-inflammatory effect associated with Microchristian bacteria.

7. Demonstration of in vivo anti-inflammatory action of bacteria of the genus Klitestonia

Three days after injection, animals were euthanized. The body weight change of the mice was measured by taking the body weight of the mice daily. During sacrifice, the colon was opened and cleaned for macroscopic scoring. The latter is based on the presence of blood in the tissue (engorgement), the presence of ulcers, intestinal transit (e.g. if the mouse is constipation) and DNBS-induced colon thickening.

A colon was also recovered to determine Myeloperoxidase (MPO) activity. This enzyme is present in neutrophils, an inflammation marker. It is also involved in the production of Reactive Oxygen Species (ROS). During this test, after weighing the colon to normalize the results according to sample size, a calorimetric reaction was performed to measure the activity of this enzyme.

Spleens (peripheral lymphoid organs) were also recovered during sacrifice and cells stimulated for innate and adaptive immune responses to measure secreted cytokines such as IL-10, anti-inflammatory cytokines.

Figure 5 shows the effect of other strains of cretinis during the repetition of the experiment presented in figure 4. The experimental design and procedure remained similar. This experience teaches that all small klebsiella strains tested had the same efficacy in reducing macroscopic scores after intrarectal injection of DNBS. Also, all strains tested showed an anti-inflammatory effect, which resulted in a reduction of MPO activity.

8. Demonstration of the antiproliferative Effect of bacteria of the genus Klitenstein

HT-29 cells were seeded in 96-well plates at a density of 10,000 cells/well in a total volume of 100. mu.l. After 24 hours of incubation, the medium was removed from the adherent cells and new medium supplemented with 10% cretinissella parvum stationary phase supernatant (DSMZ, cretinissella 1 and cretinilla 2 and control GAM (bacterial culture) was added. Each condition was repeated in 4 replicates. The cells were incubated for an additional 48 hours or 72 hours.

Cell proliferation was determined by the CellTiter-Glo 2.0 assay kit (Promega). Measurements were made according to the manufacturer's instructions. Briefly, plates were removed from the incubator and equilibrated at room temperature for 30 minutes, and an equal volume of CellTiter-Glo 2.0 reagent was added directly to wells (100. mu.l). The plates were stirred for 2 minutes at 300rpm using a rotary shaker, and then incubated for 10 minutes at room temperature. The reaction mixture was then transferred to a white-walled 96-well plate and the luminescence signal was measured using a microplate reader (FLUOstar Omega, BMG Labtech).

FIG. 6 shows the effect of Klebsiella parvula on tumor cell proliferation.

The inventors observed the effect of the microcystis tenella strains on tumor cell proliferation. The effect of these supernatants was analyzed by treating human colon adenocarcinoma cell line HT-29 line. After 48 and 72 hours, the effect of the supernatant of Klebsiella parvula on the proliferation of HT-29 cells was evaluated. Strains DSM22607(DSMZ), cretinissella parvula 1 and cretinica parvula 2 significantly reduced proliferation of cells treated for 48 and 72 hours compared to the "GAM" control corresponding to cells treated with bacterial culture medium (statistical test ═ dannett, where the control group was GAM;. GAM to DSMZ: p <0.0001,. GAM to cretinica parvula 1: p ═ 0.0140,. GAM to cretinica parvula 2: p ═ 0.0027, 48 hours and: <0.0001, 72 hours). Akt inhibitor VIII was used as a control for blocking cell proliferation.

This experiment makes it possible to draw the following conclusions: supernatants of different strains of the tested klebsiella minutissima induced a reduction in proliferation of the HT-29 tumor line and therefore these strains play a role in inhibiting tumor development.

Sequence listing

<110> Isobia Biotech (YSOPIA BIOSCIENCES)

French NATIONAL institute for agricultural food and environmental research (INSTITUT NATIONAL DE RECHERCHE)

POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT）

Bolonia university (ALMA MATER STUDIORUM-UNIVERSIT À DI BOLOGNA)

<120> for preventing and/or treating chronic inflammatory diseases and/or inflammatory gastrointestinal diseases

And/or cancer bacteria of the family of the Klysteinaceae

<130> PPI21171963US

<150> US62/816,539

<151> 2019-03-11

<160> 6

<170> BiSSAP 1.3.6

<210> 1

<211> 1495

<212> DNA

<213> Microcretinis

<400> 1

ggctcaggac gaacgctggc ggcgtgctta acacatgcaa gtcgaacgag gttgcccttt 60

gtgaatcctt cgggaggaac tgtgggtata ccgagtggcg gacgggtgag taacgcgtga 120

gcaacctgcc ctgcaacggg ggacaacagt tggaaacgac tgctaatacc gcatgagacc 180

acgaaaccgc atggttttga ggtaaaagga tttattcgat gcaggatggg ctcgcgtccc 240

attagatagt tggtgaggta acggcccacc aagtcaacga tgggtagccg acctgagagg 300

gtgatcggcc acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg 360

aatattgggc aatgggggaa accctgaccc agcaacgccg cgtgagggaa gaaggtcttc 420

ggattgtaaa cctttgtcct atgggacgaa agaaatgacg gtaccatagg aggaagctcc 480

ggctaactac gtgccagcag ccgcggtaat acgtagggag caagcgttgt ccggaattac 540

tgggcgtaaa gggtgcgtag gtggtcatgt aagtcagatg tgaaagaccg gggcttaacc 600

ccggggttgc atttgaaact gtgtgacttg agtacaggag agggaagtgg aattcctagt 660

gtagcggtga aatgcgtaga tattaggagg aacaccagtg gcgaaggcga ctttctggac 720

tgtaactgac actgaggcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780

ccacgccgta aacgatggat actaggtgtg gggcccgata gggttccgtg ccgaagcaaa 840

cgcattaagt atcccgcctg gggagtacga tcgcaaggtt gaaactcaaa ggaattgacg 900

ggggcccgca caagcagcgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960

aaggcttgac atcctctgac gactgtagag atacagtttc ccttcggggc agagagacag 1020

gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080

gcaaccctta ttgctagttg ccagcgcgta aaggcgggaa ctctagtgag actgccgggg 1140

acaactcgga ggaaggtggg gacgacgtca aatcatcatg ccccttatgt cttgggctac 1200

acacgtgcta caatggccgg tacaaagggc agcgaacccg taaggggaag cgaatctcaa 1260

aaagccggtc ccagttcgga ttgtgggctg caacccgccc acatgaagtc ggagttgcta 1320

gtaatcgcga atcagcatgt cgcggtgaat gcgttcccgg gccttgtaca caccgcccgt 1380

cacaccacgg aagttgggag cacccgaagc cagtggctta accgtaagga gagagctgtc 1440

gaaggtgaga tcaatgactg gggtgaagtc gtaacaaggt agccgtatcg gaagg 1495

<210> 2

<211> 292

<212> DNA

<213> Microcretinis

<400> 2

gtgccagcag ccgcggtaat acgtagggag caagcgttgt ccggaattac tgggcgtaaa 60

gggtgcgtag gtggtcatgt aagtcagatg tgaaagaccg gggcttaacc ccggggttgc 120

atttgaaact gtgtgacttg agtacaggag agggaagtgg aattcctagt gtagcggtga 180

aatgcgtaga tattaggagg aacaccagtg gcgaaggcga ctttctggac tgtaactgac 240

actgaggcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt cc 292

<210> 3

<211> 1357

<212> DNA

<213> Microcretinis

<220>

<221> misc_feature

<222> 550..554

<223 >/Note = "n is a, c, g, or t"

<220>

<221> misc_feature

<222> 568..569

<223 >/Note = "n is a, c, g, or t"

<220>

<221> misc_feature

<222> 605..605

<223 >/Note = "n is a, c, g, or t"

<220>

<221> misc_feature

<222> 612..612

<223 >/Note = "n is a, c, g, or t"

<220>

<221> misc_feature

<222> 617..619

<223 >/Note = "n is a, c, g, or t"

<400> 3

ggacgaacgc tggcggcgtg cttaacacat gcaagtcgaa cgaggtgcag cgagcggacc 60

ccttcgggga gaagcatgct gtatcctagt ggcggacggg tgagtaacgc gtgagcaacc 120

tacccttgaa tgggggacaa cagctggaaa cggctgctaa taccgcataa gaccacgtca 180

tcgcatggtg aagaggtaaa aggattaatt cgatcaagga tgggctcgcg tcccattaga 240

tagttggtga gatagcagct caccaagccg acgatgggta gccgacctga gagggtgaac 300

ggccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt ggggaatatt 360

gggcaatggg ggaaaccctg acccagcaac gccgcgtgaa ggaagaaggc cttcgggttg 420

taaacttttg tcctatggga cgaagaagtg acggtaccat aggaggaagc tccggctaac 480

tacgtgccag cagccgcggt aatacgtagg gagcgagcgt tgtccggaat tactgggcgt 540

aaagggtgcn nnnncggaga tgtaagtnng gtgtgaaagc ccggggctca accccgggat 600

tgcanttgaa antacannnc ttgagtacag gagaggcaag tggaattcct agtgtagcgg 660

tgaaatgcgt agatattagg aggaacacca gtggcgaagg cgacttgctg gactgtaact 720

gacgctgagg cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780

gtaaacgatg gatactaggt gtaggggtcg ataggcctct gtgccgaagc aaacgcatta 840

agtatcccgc ctggggagta cgatcgcaag gttgaaactc aaaggaattg acgggggccc 900

gcacaagcag cggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaaggctt 960

gacatcctct gacggctata gagatatagc ttcccttcgg ggcagagaga caggtggtgc 1020

atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080

ttattgctag ttgccagcac gtaaaggtgg gaactctagt gagaccgccg gggacaactc 1140

ggaggaaggt ggggacgacg tcaaatcatc atgcccctta tgtcttgggc tacacacgtg 1200

ctacaatggc cggtacaaag ggtagcgaag tcgtaagatg aagcgaatct caaaaagccg 1260

gtcccagttc ggattgaggg ctgcaacccg ccctcatgaa gtcggagttg ctagtaatcg 1320

cgaatcagca tgtcgcggtg aatgcgttcc cgggcct 1357

<210> 4

<211> 1402

<212> DNA

<213> Microcretinis

<400> 4

ttcgccctta gagtttgatc ctggctcagg acgaacgctg gcggcgtgct taacacatgc 60

aagtcgaacg aagttgctct ttgggaagcc ctcgggtgga actgtgagta tacttagtgg 120

cggacgggtg agtaacgcgt gagcaatctg ccctgcaatg ggggacaaca gttggaaacg 180

actgctaatg ccgcataaga ccacgaaacc gcatggttta gaggtaaaag gattcattcg 240

atgcaggatg agctcgcgtc ccattagata gttggtgagg taacggccca ccaagtcaac 300

gatcggtagc cggactgaga ggttgaacgg ccacattggg actgagacac ggcccagact 360

cctacgggag gcagcagtgg ggaatattgc acaatggggg aaaccctgat gcagcgacgc 420

cgcgtggagg aagaaggtct tcggattgta aactcctgtt gttggggaag ataatgacgg 480

tgcccaacaa ggaagtgacg gctaactacg tgccagcagc cgcggtaata cgtagggagc 540

aagcgttgtc cggaattact gggcgtaaag ggtgcgtagg tggccatgta agtcaggtgt 600

gaaagaccgg ggctcaaccc cggggttgca cttgaaactg tgtggcttga gtacaggaga 660

gggaagtgga attcctagtg tagcggtgaa atgcgtagat attaggagga acaccagtgg 720

cgaaggcgac tttctggact gtaactgaca ctgaggcacg aaagcgtggg gagcaaacag 780

gattagatac cctggtagtc cacgccgtaa acgatggata ctaggtgtgg gggggcgata 840

gtcctccgtg ccgaagctaa cgcattaagt atcccgcctg gggagtacga tcgcaaggtt 900

gaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggt ttaattcgaa 960

gcaacgcgaa gaaccttacc aaggcttgac atcctctgac gcatatagag atatatgttc 1020

ccttcggggc agagagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080

ggttaagtcc cgcaacgagc gcaacccttg ttgctagttg ccagcacgta aaggtgggaa 1140

ctctagtgag actgccgggg acaactcgga ggaaggtggg gacgacgtca aatcatcatg 1200

ccccttatgt cttgggctac acacgtgcta caatggccgg tacaaagggc agcgacctcg 1260

taagagtaag cgaatctcaa aaagccggcc ccagttcgga ttgtgggctg caacccgccc 1320

acatgaagtc ggagttgcta gtaatcgcga atcagcatgt cgcggtgaat gcgttcccgg 1380

gccttgcaca caccgcccgt ca 1402

<210> 5

<211> 1497

<212> DNA

<213> Microcretinis

<400> 5

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60

gaagttgctc tttgggaagc cctcgggtgg aactgtgagt atacttagtg gcggacgggt 120

gagtaacgcg tgagcaatct gccctgcaat gggggacaac agttggaaac gactgctaat 180

accgcataag accacgaaac cgcatggttt agaggtaaaa ggattcattc gatgcaggat 240

gagctcgcgt cccattagat agttggtgag gtaacggccc accaagtcaa cgatgggtag 300

ccgacctgag agggtgatcg gccacattgg aactgagaaa cggtccaaac tcctacggga 360

ggcagcagtg gggaatattg ggcaatgggg gaaaccctga cccagcaacg ccgcgtgaag 420

gaagaaggcc ttcgggttgt aaacttttgt cctatgggac gaaaaaaatg acggtaccat 480

aggaggaagc tccggctaac tacgtgccag cagccgcggt aatacgtagg gagcaagcgt 540

tgtccggaat tactgggcgt aaagggtgcg taggtggcca tgtaagtcag gtgtgaaaga 600

ccggggctca accccggggt tgcacttgaa actgtgtggc ttgagtacag gagagggaag 660

tggaattcct agtgtagcgg tgaaatgcgt agatattagg aggaacacca gtggcgaagg 720

cgactttctg gactgtaact gacactgagg cacgaaagcg tggggagcaa acaggattag 780

ataccctggt agtccacgcc gtaaacgatg gatactaggt gtggggggcg atagtcctcc 840

gtgccgaagc taacgcatta agtatcccgc ctggggagta cgatcgcaag gttgaaactc 900

aaaggaattg acgggggccc gcacaagcag cggagcatgt ggtttaattc gaagcaacgc 960

gaagaacctt accaaggctt gacatcctct gacgcatata gagatatatg ttcccttcgg 1020

ggcagagaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080

tcccgcaacg agcgcaaccc ttgttgctag ttgccagcac gtaaaggtgg gaactctagt 1140

gagactgccg gggacaactc ggaggaaggt ggggacgacg tcaaatcatc atgcccctta 1200

tgtcttgggc tacacacgtg ctacaatggc cggtacaaag ggcagcgacc tcgtaagagt 1260

aagcgaatct caaaaagccg gccccagttc ggattgtggg ctgcaacccg cccacatgaa 1320

gtcggagttg ctagtaatcg cgaatcagca tgtcgcggtg aatgcgttcc cgggccttgt 1380

acacaccgcc cgtcacacca cggaagttgg gagcacccga agccagtggc ttaaccgtaa 1440

ggagagagct gtcgaaggtg agatcaatga ctggggtgaa gtcgtaacaa ggtaacc 1497

<210> 6

<211> 1495

<212> DNA

<213> Microcretinis

<400> 6

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60

gaagatgctc tttgtgaagc cttcgggtgg aactgtgagt agactgagtg gcggacgggt 120

gagtaacgcg tgagcaacct gccctgcaac gggggacaac agctggaaac ggctgctaat 180

accgcataag accacggaat cgcatggttc tgaggtaaaa ggatttattc gatgcaggat 240

gggctcgcgt cccattagat agttggtgag gtaacggctc accaagtcaa cgatgggtag 300

ccgacctgag agggtgatcg gccacactgg aactgagaca cggtccagac tcctacggga 360

ggcagcagtg gggaatattg ggcaakgggc gagcctgacc agcacgccgc gtgaaggaag 420

aaggccttcg ggttgtaaac ttttgtccta tgggaagaag aagtgacggt accataggag 480

gaagctccgg ctaactacgt gccagcagcc gcggtaatac gtagggagca agcgttgtcc 540

ggaattactg ggcgtaaagg gtgcgtaggc ggtttgacaa gtcagatgtg aaagcctggg 600

gcttaactcc aggattgcat ttgaaactgt taaacttgag tgcaggagag gcaagtggaa 660

ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa caccagtggc gaaggcgact 720

tgctggactg taactgacgc tgaagcacga aagcgtgggg agcaaacagg attagatacc 780

ctggtagtcc acgctgtaaa cgatggatac taggtgtagg gggcgatagt cttctgtgcc 840

gcagctaacg cattaagtat cccgcctggg gagtacgatc gcaaggttga aactcaaagg 900

aattgacggg ggcccgcaca agcagcggag catgtggttt aattcgaagc aacgcgaaga 960

accttaccaa ggcttgacat cctctgacgc atgtagagat acatgttccc ttcggggcag 1020

agagacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080

caacgagcgc aacccttatt gctagttgcc agcacgtaaa ggtgggaact ctagtgagac 1140

cgccggggac aactcggagg aaggtgggga cgacgtcaaa tcatcatgcc ccttatgtct 1200

tgggctacac acgtgctaca atggccggta caaaggacag cgaacccgca aggggaagcg 1260

aatctcaaaa agccggtccc agttcggatt gagggctgca acccgccctc atgaagtcgg 1320

agttgctagt aatcgcgaat cagcatgtcg cggtgaatgc gttcccgggc cttgtacaca 1380

ccgcccgtca caccacggaa gttgggagcg cccgaagcca gtggcttaac cgtaaggaga 1440

gagctgtcga aggtgagatc aatgactggg gtgaagtcgt aacaaggtaa ccgta 1495

Claims (22)

1. A bacterium of the genus Christensenella for use in the prevention and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancer in a human or animal.

2. A bacterium of the genus Klitenstason for use according to claim 1, for use in a patient or animal exhibiting overproduction of interleukin 6 and/or interleukin 8 and/or insufficient production of interleukin 10.

3. A crenstella bacterium for use according to one of the preceding claims for use in the treatment of at least one disease selected from: chronic inflammatory bowel disease, chronic inflammatory disease of the liver, chronic inflammatory disease of the pancreas, polyarthritis, atopic dermatitis, neuroinflammatory disease, chronic obstructive pulmonary disease, and inflammatory bowel disease.

4. A bacterium of the genus crenstein for use according to the preceding claim, for use in the treatment of at least one disease selected from: crohn's disease, ulcerative colitis, pouchitis, ulcerative colitis, celiac disease, autoimmune gastritis, hepatitis, non-alcoholic steatohepatitis, primary sclerosing cholangitis, pancreatitis, rheumatoid arthritis, psoriatic arthritis, psoriasis and eczema.

5. The bacterium of the genus klebsiella for use according to one of claims 1 or 2, for use in the treatment of lymphoma, glioblastoma, myeloma, leukemia, colorectal, breast, prostate, ovarian, uterine, pancreatic, lung, liver, gall bladder and kidney cancers.

6. The bacterium of the genus klebsiella according to one of the preceding claims, characterized in that it is selected from the group consisting of Marisis mosaic (Christensella massiensis), Christensella (Christensella timenensis), human intestinal klebsiella (Christensella internihonisis) and small klebsiella (Christensella minuta).

7. A composition comprising, in a physiologically acceptable medium, at least one crewsonia bacterium according to one of the preceding claims and/or a culture supernatant of at least one bacterial strain of the crewsonia genus, for use in the prophylaxis and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancers.

8. The composition for use according to claim 7, wherein the Cristermum bacteria and the culture supernatant may comprise or be derived from the same or different strains or species of Cristermum.

9. Composition for use in accordance with one of claims 7 or 8, which is administered to a human having a body mass index of less than 25.

10. Composition for use according to one of claims 7 or 9, characterized in that the bacteria are selected from Maritestonia mosaic, Tumorestonia and Micromerstonia and/or mixtures thereof.

11. Composition for use according to one of claims 7 to 10, characterized in that at least 50% of the bacteria present are live bacteria (by number).

12. Composition for use according to one of claims 7 to 11, characterized in that at least 90% of the bacteria present are live bacteria (by number).

13. The composition for use according to one of claims 7 to 12, further comprising:

(i) at least one additional bacterium of the family Verrucomicrobiaceae (Verrucomicrobiaceae); and/or

(ii) A culture supernatant of the additional bacteria.

14. The composition for use according to claim 13, wherein the further bacterial strain is a bacterium of the genus Akkermansia (Akkermansia), more particularly Akkermansia muciniphila (Akkermansia muciniphila).

15. Composition for use according to one of claims 7 to 14, characterized in that it is provided in liquid form or in solid form.

16. The composition for use according to claim 15, wherein said composition in solid form comprises said bacteria in lyophilized form.

17. The composition for use according to one of claims 7 to 16, wherein the composition is administered orally, rectally, by inhalation or topically.

18. Composition for use according to one of claims 7 to 17, characterized in that it is in the form of: powders, microencapsulated powders, gelatin capsules, tablets, lozenges, granules, emulsions, suspensions, suppositories, beverages, foods, pharmaceutical or nutraceutical products, food additives, dietary supplements or dairy products.

19. Composition for use in accordance with one of claims 7 to 18, characterized in that it is in a gastro-resistant form.

20. Composition for use in accordance with one of claims 7 to 19, characterized in that it comprises at least one probiotic and/or at least one prebiotic.

21. Composition for use according to one of claims 7 to 20, characterized in that it further comprises:

at least one probiotic, and/or

-at least one lactic acid producing bacterium and/or at least one other organism promoting anaerobic conditions necessary for the survival of Christensellaceae (Christensellaceae), and/or

-at least one bacterium associated with the ecosystem of the family of the Krystenbacid, and/or

-at least one bacterium selected from the group consisting of bacteria of the genera faecalis (Faecalibacterium), Ackermansia, Eubacterium (Eubacterium) and Oscillatoria (Oscillospira), and/or

At least one prebiotic, and/or

At least one polyhydric phenol, and/or

-at least one mineral and/or at least one vitamin, and/or at least one nutritional agent, and/or

-at least one active pharmaceutical ingredient, preferably selected from the group consisting of non-steroidal anti-inflammatory drugs, antibodies against pro-inflammatory targets (anti-TNF α, anti-IL-6, anti-IL-8), anti-rheumatic drugs, analgesics, antibacterial agents, corticosteroids, anabolic steroids, antidiabetics, thyroid agents, antidiarrheal drugs, cough suppressants, antiemetics, antiulcer drugs, 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 agents, beta agonists, inhaled steroids, leukotriene inhibitors, Cromoglycate or cromoglycate, epinephrine, alpha-chain enzymes, cytokines, cytokine antagonists, interleukin 10 agonists.

22. A composition comprising, in a physiologically acceptable medium:

(I) (a) at least one bacterial strain selected from the group consisting of: tomorrow, cremastesmus parvum and/or mixtures thereof;

and/or

(b) At least one culture supernatant of at least one bacterial strain selected from the group consisting of: tomorrow, cremastesmus parvum and/or mixtures thereof; and

(II) (a) at least one further bacterial strain of the genus akkermansia, more particularly akkermansia muciniphila;

and/or

(b) At least one culture supernatant of at least one further bacterial strain of the genus akkermansia, more particularly akkermansia muciniphila.

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