Parabacteroides distasonis strains isolated from human feces and their applications

Abstract

The present invention relates to the Parabacteroides distasonis strain deposited under accession number CNCM I-5828. This strain may be useful in several applications, in particular when incorporated into a composition for use as a dietary supplement or medicine, preferably in the prevention or treatment of visceral pain induced by inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, or induced by irritable bowel syndrome.

Description

[Technical field]

[0001] The present invention relates to the Parabacteroides distasonis strain deposited under accession number CNCM I-5828. This strain may be useful in several applications, in particular when incorporated into a composition for use as a dietary supplement or medicine, in particular in the prevention or treatment of visceral pain, as observed in particular in inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, or in irritable bowel syndrome.

[0002] The present invention is in the field of bacterial strains isolated from humans, particularly human feces, and compositions comprising said strains.In a preferred embodiment, the present invention is in the field of dietary supplement compositions and compositions for medicinal use comprising said strains.The compositions for medicinal use are particularly for preventing or treating visceral pain. [Background technology]

[0003] The microbiome includes several types of microorganisms (bacteria, archaea, viruses, parasites and fungi), their genomes and their surrounding environment, including the gastrointestinal tract, oral mucosa, genitourinary and respiratory systems, and the skin surface [1]. Bacteria are the most studied group of microorganisms that make up the microbiome [2]. Their number in the whole human body is estimated to be of the same order of magnitude as the number of human cells [3]. Many bacteria are present in the intestine [4]. Due to their sheer number and diversity, they can significantly affect normal physiological functions and modify the host's susceptibility to diseases [5]. Indeed, alterations in the microbiota composition have been demonstrated in inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).

[0004] Crohn's disease (CD) and ulcerative colitis (UC) are part of IBD and are characterized by inflammation of the intestinal epithelial barrier. [6] The pattern of dysbiosis primarily associated with IBD patients is a decrease in commensal bacterial diversity and a relative increase in bacterial species belonging to the family Enterobacteriaceae. [7] Although the data are heterogeneous, the gut microbiota appears to play a key role in IBD pathogenesis. A recently published study conducted on 132 IBD patients provided the most comprehensive description to date of host and microbial activity in IBD and demonstrated that the gut microbiome, molecular functional profile, and host immune factors are important components in IBD. [8] In addition to identifying microbial, transcriptomic, and metabolomic profiles, recent studies have highlighted metabolite signatures in patients suffering from IBD. [9] Similarly, another recently published study showed disruptions in taxa of the Lachnospiraceae and Ruminococcaceae families in IBD patients compared to controls, demonstrating that impairments in distinct taxon-associated networks are involved in CD and UC disease development [ 8 ].

[0005] Recognizing the positive effect that some bacterial strains may have on the intestines of animals, various strains have been proposed as new therapeutic strategies in various diseases.Some strains, mainly including Lactobacillus and Bifidobacterium strains, have also been proposed for the treatment of inflammatory and autoimmune diseases that are not directly related to the gastrointestinal tract.However, the relationship between different diseases and different bacterial strains, and the exact effect of bacterial strains on any kind of disease at the intestinal and systemic levels, has hardly been characterized.

[0006] IBS or spastic colon is a disorder characterized by abdominal pain and altered bowel habits, not associated with any abnormalities seen in routine clinical diagnostics. It is common, accounting for 20-50% of gastroenterologist visits. Chronic pain in the digestive tract is a major unmet medical problem. It accompanies common digestive disorders, is poorly understood, and is difficult to treat.

[0007] Lower abdominal pain and bloating associated with changes in bowel habits, as well as abdominal discomfort relieved by defecation, are the most frequent symptoms.For a good understanding of the present invention, it must be understood that IBS is a syndrome, and under this expression, several symptoms observed in patients suffering from gastrointestinal tract are collected.Therefore, therapeutic solutions aim to treat or improve some symptoms associated with IBS.

[0008] Visceral pain is a common symptom observed in inflammatory bowel disease (IBD: Crohn's disease, ulcerative colitis). Pain is an important sign of inflammation, as inflammatory cytokines and mediators sensitize primary afferent neurons. It is therefore not surprising that pain is one of the symptoms presented in approximately 50%-70% of patients experiencing an initial or exacerbation of IBD

[10] . Current treatment options are mainly focused on reducing inflammation in IBD or intestinal motility disorders in IBS. There are only a few therapies that directly aim at reducing abdominal pain

[11] . Today, only symptomatic treatments are proposed, and alternative solutions are currently being explored to reduce visceral and abdominal pain in patients suffering from IBD or IBS. Among them, adapted diets, fecal microbiota transplants, or the use of "biotic" products (including probiotics, prebiotics, synbiotics, metabiotics, or parabiotics) are explored strategies to restore microbiota homeostasis and limit inflammation.

[0009] At the gut level, pain stimuli can be altered both mechanically (intestinal peristalsis, distension, stretching) or chemically (e.g. neurotransmitters or inflammatory mediators) and can originate from various sources: enterocytes, immune cells or gut microbiota. There are many receptors involved in pain signaling. Nociceptive stimuli, whether mechanical, chemical or thermal, are picked up by nociceptors, called primary afferent neurons, which carry the information to the dorsal horn of the spinal cord. In the pain perception process, the transmission of sensory information from the gut to the brain is ensured by the extrinsic nervous system, whose cell bodies localized in the dorsal root ganglion (DRG) play an important function, relaying the nociceptive information to the brain. Spinal afferents play an important role in the transmission of information about physiological disorders, causing distension, discomfort or visceral pain. As a true sentinel system, the extrinsic innervation ensures communication between the gut and the brain, transmitting digestive and physiological information to the central nervous system to be treated. It is the pathway that nerve impulses use to reach the central nervous system. Pain perception involves complex cellular and molecular mechanisms in several stages. In the first step, the gut information is encoded and relayed to the dorsal horn of the spinal cord. This is the transduction step. During transduction, the nociceptive stimulus depolarizes the free end of the nociceptor, thereby activating the opening of voltage-gated sodium channels and triggering the generation of an action potential (PA). This nerve impulse induces a massive influx of positively charged ions at the central end of the dorsal horn of the spinal cord and is transmitted to second-order neurons up to the thalamus, where it synapses with third-order neurons. This is the transduction step in the brain. The latter then directs the nociceptive information to different areas of the somatosensory cortex, where sensory information can be differentiated from affective or emotional information.

[0010] Animal models of colitis have been studied to develop alternative treatment options for patients with visceral pain by measuring colonic hypersensitivity. In addition to showing a response to inflammation similar to that seen in humans, rodent models have allowed the identification of unique characteristics of the population of sensory neurons that innervate the gut. Interestingly, there are common features of sensory neurons that innervate different organs, including the colon, stomach, and pancreas. All of these structures are densely innervated by sensory fibers that express calcitonin gene-related peptide (CGRP). This peptide, a potent vasodilator, is released by peripheral sensory nerve endings in response to noxious and non-noxious stimuli and can affect the vasculature and other structures. This property highlights one of the less understood aspects of visceral (and most somatic) sensory neurons. That is, in addition to detecting internal (visceral) or external (environmental) stimuli, sensory neurons release several compounds, including CGRP and substance P (SP), that can have peripheral motor and / or autocrine / paracrine effects on other cells or sensory neurons. Under normal conditions, these compounds contribute to homeostatic regulation, but in disease states, including those with an inflammatory component, they can exacerbate symptoms ("neurogenic inflammation"). Most TRPV1-expressing neurons release SP and / or CGRP, and it is believed that the release of these compounds can drive the inflammatory response.

[0011] IBD and IBS are characterized, among others, by the development of visceral hypersensitivity. Several mediators, such as serotonin, histamine, bradykinin, substance P, ATP, prostaglandins, as well as all mediators of inflammation, such as cytokines and chemokines, may originate. These molecules interact with neuronal receptors involved in the signal transduction of visceral pain. The activation of these receptors can result in an anti-nociceptive effect, or, conversely, a nociceptive effect, depending on whether they are activators or inhibitors, or are involved in pain modulation. Some receptors, such as serotonergic, histaminergic, bradykinin receptors, or receptors belonging to the TRP (Transient Receptor Potential) family, such as TRPV1, TRPV4, Transient Receptor Potential Ankyrin 1 (TRPA1), but also purinergic or tachykinin receptors, involved in the regulation of visceral pain processes, have been studied and have rapidly become therapeutic targets. One of them, TRPV1, may play a central role in setting the overall sensitivity of colonic afferents. Indeed, colonic hypersensitivity that occurs in response to inflammatory mediators in wild-type mice is absent in TRPV1-deficient mice. Moreover, TRPV1 is overexpressed and correlates with pain severity in IBD

[13] and IBS

[14] . Moreover, pharmacological inhibition of TRPV1 blocks the development of hyperalgesia during colitis

[15] . Although TRPV1 has received considerable attention recently, no single channel appears to be responsible for colonic hypersensitivity. Deletion of a member of the acid-sensing ion channel (ASIC3) or another member of the TRP (transient receptor potential) family (TRPV4) blunts or completely blocks the development of hyperalgesia in response to colonic inflammation

[15] . TRPA1 has also been reported to contribute to visceral pain-like behavior in dextran sulfate sodium (DSS)-induced colitis. Pain processes involve complex molecular pathways and various neuronal receptors that may together contribute to pain generation. This is the case for G-coupled protein receptors (GPCRs), whose activation can sensitize nociceptors belonging to the TRP family. This is why GPCRs have become therapeutic targets for the treatment of chronic pain in the digestive system

[16] .

[0012] Parabacteroides distasonis is a bacterium belonging to the Porphyromonas family and the genus Parabacteroides. Initially classified in the genus Bacteroides based on its phenotypic and morphological characteristics, this bacterial species has recently been reclassified and belongs to the family Tannerellaceae, which includes the genera Parabacteroides and Tannerella. In fact, beyond its phenotypic characteristics, it has been demonstrated that this species is phylogenetically closer to the species Tannerella phositensis. The genus Parabacteroides currently counts 15 species: Parabacteroides acidifaciens, Parabacteroides bouchesdarhonensis, Parabacteroides chartae, Parabacteroides chinchillae, Parabacteroides chongii, Parabacteroides distasonis, Parabacteroides faeces, Parabacteroides goldsteinii, Parabacteroides goldsteinii, Parabacteroides johnsonii, Parabacteroides masiliensis, Parabacteroides merdae, Parabacteroides spacaensis, Parabacteroides provencensis, and Parabacteroides timonensis. Parabacteroides distasonis represents the reference species, and strain Parabacteroides distasonis ATCC 8503 (DSM20701) is the reference strain for the reclassified genus Parabacteroides

[17] .

[0013] Parabacteroides distasonis is a type of strain of the genus Parabacteroides, a group of Gram-negative anaerobic bacteria that commonly colonize the gastrointestinal tract of many species. The anti-inflammatory properties of Parabacteroides distasonis have been demonstrated

[18] . In addition to exerting anti-inflammatory properties, some Parabacteroides distasonis strains have shown beneficial effects on strengthening the epithelial barrier.

[0014] For the above reasons, there is a need for new methods to treat visceral pain, especially induced by IBS or IBD.Furthermore, there is a constant need in many technical fields to identify new bacterial strains that may be useful, especially in dietary supplements and as medicines.

[0015] Surprisingly, the Applicant has identified a new strain of the genus Parabacteroides from the species Parabacteroides distasonis in human faeces, which shows unexpectedly strong efficacy for preventing or treating visceral pain. Summary of the Invention

[0016] Thus, in a first aspect, the present invention relates to the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0017] The Parabacteroides distasonis strain according to the invention has a 16s rRNA gene sequence as shown in SEQ ID NO:1.

[0018] The Parabacteroides distasonis strain according to the invention is isolated from the faeces of healthy humans.

[0019] The bacterial strain according to the invention has been designated "Parabacteroides distasonis F1-2", a name which is used in particular in the examples to describe the strain according to the invention, deposited under accession number CNCM I-5828 and having the 16s rRNA gene sequence shown in SEQ ID NO:1.

[0020] The bacterial strain used in the present invention has been characterized, in particular by sequencing of the 16s rRNA gene. The 16s rRNA gene sequence of this bacterial strain (SEQ ID NO: 1) is at least 99.61% identical to the 16s RNA sequence of the species Parabacteroides distasonis (Parabacteroides distasonis ATCC 8503). 16s rRNA gene sequencing is commonly used as a tool to identify bacteria at the species level and to aid in the differentiation between closely related bacterial species. The sequence of the complete genome of Parabacteroides distasonis ATCC 8503 is known in the art and can be found on the following link: https: / / www.ebi.ac.uk / ena / browser / api / fasta / CP000140.1?lineLimit=1000

[0021] The bacterial strain of the present invention has been isolated from a healthy male human.

[0022] In a second aspect, the present invention relates to a composition comprising the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0023] In one embodiment, the composition may further comprise any type of other compound that may be suitable for the purpose of the composition.

[0024] In a third aspect, the present invention relates to a dietary supplement composition comprising a bacterial strain according to the present invention.

[0025] "Dietary supplement", "food", "food supplement", "nutraceutical composition" or "dietary supplement" are intended according to the present invention to be identical to a product that may provide a nutritional benefit. The supplement may be taken alone or may be formulated with other compounds to make the composition more attractive to consume by being more similar to common foods. The supplement may be a contributing factor to prevent or reduce any superficial visceral pain problem that does not require therapeutic treatment.

[0026] In one embodiment, the dietary supplement composition further comprises at least one acceptable nutritional ingredient, preferably selected from probiotics, prebiotics, synbiotics, parabiotics, metabiotics, vitamins, minerals, herbs, amino acids and enzymes.

[0027] Probiotics, according to the present invention, are intended to be viable microorganisms that, when administered in appropriate amounts, confer a health benefit to the host.

[0028] More preferably, the probiotic is selected from among bacteria belonging to the genera Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus, or next-generation probiotics such as bacterial strains identified as Faecalis prausnitzii, Akermansia muciniphila, Bacteroides fragilis, Bacteroides taiotaomicron, Clostridium butyricum, Roseburia hominis, Roseburia intelligentis, Hafnia alvei, or Christensenella minuta. It also includes Saccharomyces fungal strains.

[0029] By prebiotics is intended, according to the present invention, selectively fermented ingredients that bring about specific changes in the composition and / or activity of the gastrointestinal microbiota and thus benefit the health of the host.

[0030] More preferably, the prebiotics are selected from among galactooligosaccharides, fructooligosaccharides or polysaccharides.

[0031] By synbiotics, according to the present invention, is intended a mixture of probiotics and prebiotics that beneficially affect the host by improving the survival and activity of beneficial microorganisms in the intestine.

[0032] More preferably, the symbiotic is selected from the next generation probiotics, such as bacteria belonging to the genera Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus, or bacterial strains identified as Faecalibacterium prausnitzii, Akermansia muciniphila, Bacteroides fragilis, Bacteroides taiotaomicron, Clostridium butyricum, Roseburia hominis, Roseburia integinalis, Hafnia alvei, or Christensenella minuta. It also includes Saccharomyces fungal strains. All of these bacteria or fungi should be associated with prebiotics, such as galactooligosaccharides, fructooligosaccharides, or polysaccharides, in the symbiotic formulation.

[0033] By parabiotics, paraprobiotics or inactivated probiotics is intended, according to the present invention, non-viable microbial cells (either intact or disrupted) or crude cell extracts that, when administered in appropriate amounts (either orally or topically), confer a benefit to the human or animal consumer.

[0034] More preferably, the parabiotic is selected from among inactivated microbial cells belonging to the Lactobacillus, Lactococcus, Streptococcus or Bifidobacterium genera or based on Faecalibacterium prausnitzii, Akermansia muciniphila, Bacteroides fragilis, Bacteroides taiotaomicron, Clostridium butyricum, Roseburia hominis, Roseburia intelligentis, Hafnia alvei or Christensenella minuta bacterial species or Saccharomyces fungal strains.

[0035] Metabiotics or "postbiotics" are bioactive molecules produced by probiotics that provide a physiological benefit to the host by modulating the interactions between the host and the gut microbiome, for example through epigenetic regulation and cellular communication. They can be found in any product fermented by live bacteria.

[0036] Preferably, the metabiotics are selected from among products fermented with live bacteria.

[0037] In one embodiment, the dietary supplement composition according to the present invention comprises at least 10 9 , preferably at least 10 10 colony forming units / mL of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0038] In one embodiment, administration of the dietary supplement is by oral ingestion.

[0039] In one embodiment, the dietary supplement is taken in one or several successive doses.

[0040] In one embodiment, consecutive doses are separated by at least 12 hours, preferably at least 1 day, more preferably at least 2 days, even more preferably at least 3 days, even more preferably at least 4 days, even more preferably at least 5 days, and even more preferably at least 6 days.

[0041] In an even more preferred embodiment, consecutive doses are separated by at least one week.

[0042] In one embodiment, oral intake of the dietary supplement is via gelatin capsules, capsules, tablets, powders, granules, oral solutions or suspensions.

[0043] In one embodiment, oral ingestion of the dietary supplement occurs via a food, beverage, food additive, or dairy product that contains the dietary supplement.

[0044] In one embodiment, a dietary supplement composition according to the present invention comprises only live bacteria.

[0045] In one embodiment, the dietary supplement composition according to the invention comprises only killed bacteria and / or fractions of bacteria obtained by thermal, chemical or mechanical inactivation or UV inactivation, which may be fractionated or purified.

[0046] According to the present invention, "dead bacteria" are bacterial strains that have been inactivated and are no longer capable of replicating.

[0047] According to the present invention, killed bacteria can be obtained by heat treatment at 121° C. for 15 to 30 minutes.

[0048] In one embodiment, the dietary supplement composition according to the present invention comprises live and / or dead bacteria and / or bacterial fractions.

[0049] In a fourth aspect, the present invention relates to a composition comprising a bacterial strain according to the present invention for use as a medicament.

[0050] In one embodiment, the composition is for use in the treatment and / or prevention of visceral pain.

[0051] In preferred embodiments, the visceral pain is induced by inflammatory bowel disease, including Crohn's disease and ulcerative colitis, or by irritable bowel syndrome.

[0052] "Visceral pain" according to the present invention includes chronic abdominal pain, chest pain, pelvic pain, bloating associated with altered bowel habits, abdominal discomfort relieved by defecation, chronic pain in the digestive tract. According to the present invention, this term includes pain associated with the stomach, pancreas, liver, gallbladder, colon, and small and large intestines.

[0053] According to the present invention, the term "prevent" refers to a reduction in the risk of developing a pathology.

[0054] According to the present invention, the term "treating" means the alleviation of symptoms and / or elimination of symptoms associated with a particular disorder or condition.

[0055] As shown in the examples of the present application, this bacterial strain exhibits unique antinociceptive properties by reducing visceral hypersensitivity in inflammatory and non-inflammatory mouse models that was not observed with reference bacterial strains belonging to the same species.Furthermore, Parabacteroides distasonis F1-2 strain exerts unique neuromodulatory properties that contribute to the reduction of pain processing and reduce the severity of visceral pain, including analgesic effects.Furthermore, this composition reduces the pain observed in IBS.Finally, this strain of Parabacteroides distasonis has neuroinhibitory properties on nociceptors expressing TRPV1 channels and G-coupled protein receptors.

[0056] In one embodiment, the composition for use according to the invention comprises at least 10 9 , preferably at least 10 10colony forming units / mL of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0057] In one embodiment, a composition for use according to the invention comprises only live bacteria.

[0058] In one embodiment, a composition for use according to the invention comprises only killed bacteria and / or a fraction of bacteria.

[0059] In one embodiment the composition for use according to the invention comprises live and / or dead bacteria and / or a fraction of bacteria.

[0060] In one embodiment, the composition for use according to the invention further comprises at least one other acceptable active pharmaceutical ingredient, and / or at least one acceptable pharmaceutical excipient, and / or at least one acceptable pharmaceutical carrier.

[0061] In accordance with the present invention, an acceptable active pharmaceutical ingredient is intended to be any compound, molecule, or the like that may be useful to add to a composition in order to achieve the objectives of the composition according to the present invention. Included within this definition, among others, are anti-inflammatory molecules such as naproxen, aspirin, or acetaminophen, as well as opioids such as codeine or morphine, or painkillers.

[0062] According to the present invention, acceptable pharmaceutical excipients are intended to be any substances other than the active substance that have been properly evaluated for safety and that are intentionally included in the formulation, which allow the medicament to be effectively administered to the patient and support its correct delivery into the body.

[0063] According to the present invention, as an acceptable pharmaceutical carrier, any substance that serves to improve the selectivity, efficacy, and / or safety of administration of the composition according to the invention is contemplated.

[0064] In one embodiment, the compositions for use according to the invention are formulated to be suitable for oral, intravenous, enteral or subcutaneous administration.

[0065] In a preferred embodiment, the compositions for use according to the invention are administered orally via gelatin capsules, capsules, tablets, powders, granules, oral solutions or suspensions.

[0066] In one embodiment, administration is by one or several consecutive doses.

[0067] In one embodiment, consecutive doses are separated by at least 12 hours, preferably at least 1 day, more preferably at least 2 days, even more preferably at least 3 days, even more preferably at least 4 days, even more preferably at least 5 days, and even more preferably at least 6 days.

[0068] Preferably, successive doses are separated by at least one week.

[0069] In one embodiment, the subject is a mammal, including a non-human mammal, and in particular a human.

[0070] In a preferred embodiment, the subject is an adult human.

[0071] In another embodiment, the present invention relates to cells of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0072] In another aspect, the present invention relates to cells of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828 for use as a medicament.

[0073] In one embodiment the cells according to the invention are for use in the treatment and / or prevention of visceral pain.

[0074] More preferably, the visceral pain is induced by inflammatory bowel disease, including Crohn's disease and ulcerative colitis, or by irritable bowel syndrome.

[0075] In another aspect, the present invention relates to a dietary supplement composition comprising cells of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0076] In another aspect, the present invention relates to a method for preventing and / or treating visceral pain comprising the administration of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0077] In a preferred embodiment, the method of prevention and / or treatment according to the invention is the prevention and / or treatment of visceral pain induced by inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, or by irritable bowel syndrome.

[0078] In one embodiment, the method according to the present invention comprises at least 10 9 , preferably at least 10 10 The doses included colony forming units / mL of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0079] In another aspect, the present invention relates to a strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to the 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0080] In a preferred embodiment, the strain has a 16s rRNA gene sequence that is at least 85.00%, more preferably at least 90.00%, and even more preferably at least 95.00% identical to the 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0081] In a preferred embodiment, the strain has a 16s rRNA gene sequence that is at least 96.00%, more preferably at least 97.00%, and even more preferably at least 98.00% identical to the 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0082] In a preferred embodiment, the strain has a 16s rRNA gene sequence that is at least 99.00%, more preferably at least 99.50% identical to the 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0083] In a preferred embodiment, the strain has a 16s rRNA gene sequence that is at least 99.61% identical to the 16s RNA sequence of the bacterial strain Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0084] In a preferred embodiment, the strain has the 16s rRNA gene sequence set forth in SEQ ID NO:1.

[0085] In another aspect, the present invention relates to a composition comprising a strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0086] In one embodiment, the composition according to the present invention comprises at least 10 9 , preferably at least 10 10 The strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1, in colony forming units / mL.

[0087] In one embodiment, the composition comprises a strain that is at least 85.00%, preferably at least 90.00%, more preferably at least 95.00%, more preferably at least 96.00%, more preferably at least 97.00%, more preferably at least 98.00%, more preferably at least 99.00%, more preferably at least 99.50%, more preferably at least 99.61% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0088] In another aspect, the present invention relates to a dietary supplement composition comprising a strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0089] In one embodiment, the dietary supplement composition according to the present invention comprises at least 10 9 , preferably at least 10 10 The strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1, in colony forming units / mL.

[0090] In one embodiment, the dietary supplement composition comprises a strain that is at least 85.00%, preferably at least 90.00%, more preferably at least 95.00%, more preferably at least 96.00%, more preferably at least 97.00%, more preferably at least 98.00%, more preferably at least 99.00%, more preferably at least 99.50%, more preferably at least 99.61% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0091] In another aspect, the present invention relates to a composition comprising a strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to the 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1, for use as a medicament.

[0092] In one embodiment, the composition for use according to the invention comprises at least 10 9 , preferably at least 10 10 The strain of Parabacteroides distasonis having a 16s rRNA gene sequence that is at least 80.00% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1, in colony forming units / mL.

[0093] In one embodiment, the composition for use comprises a strain that is at least 85.00%, preferably at least 90.00%, more preferably at least 95.00%, more preferably at least 96.00%, more preferably at least 97.00%, more preferably at least 98.00%, more preferably at least 99.00%, more preferably at least 99.50%, more preferably at least 99.61% identical to a 16s RNA sequence of a bacterial strain of the species Parabacteroides distasonis selected from Parabacteroides distasonis ATCC 8503 or SEQ ID NO:1.

[0094] In one embodiment, the composition is for use in the treatment and / or prevention of visceral pain, hi one embodiment, the visceral pain is induced by inflammatory bowel disease, including Crohn's disease and ulcerative colitis, or by irritable bowel syndrome.

[0095] References to a percentage of sequence identity between two nucleotide sequences mean that, when aligned, that percentage of nucleotides are the same comparing the two sequences.

[0096] In another aspect, the present invention relates to a cosmetic skin care composition comprising the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0097] In one embodiment, the composition comprises at least 10 9 , preferably at least 10 10 colony forming units / mL of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0098] In one embodiment, the composition is administered by topical application.

[0099] By "topical application" is meant, within the meaning of the present invention, application to the skin (including the scalp) and mucous membranes.

[0100] In one embodiment, the cosmetic composition also comprises at least one other cosmetically acceptable agent, preferably selected from soothing agents, restructuring agents, regenerating agents, revitalizing agents, sunscreens, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fats, organic solvents, solubilizers, thickening and gelling agents, smoothing agents, firmness enhancing agents, skin elasticity and / or barrier effect, antioxidants, opacifying agents, warming agents, matting agents, chemical or mineral filters, trace elements, stabilizing agents, foaming agents, fragrances, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, fragrances, filters, essential oils, colorants, pigments, hydrophilic or lipophilic active ingredients, lipid vesicles encapsulating one or more active ingredients, and / or preservatives.

[0101] "Cosmetically acceptable" means, according to the present invention, compounds that are useful in the preparation of cosmetic compositions and that are generally safe, non-toxic, not biologically or otherwise undesirable, and acceptable for cosmetic use, in particular by topical application to the skin.

[0102] Preferably, the cosmetic composition is formulated in the form of a cream, an ointment, a balm, a mask, a milk, a lotion, a serum, a spray, a paste, a foam, an aerosol, a stick, a shampoo, a conditioner, a patch, an aqueous alcoholic or oily solution, an oil-in-water or water-in-oil or a multiphase emulsion, an aqueous or oily gel, a liquid, a pasty or a solid anhydrous product, and / or a dispersion of oil in an aqueous phase using spheroids, which can be polymeric nanoparticles such as nanospheres and nanocapsules, or lipid vesicles of ionic and / or non-ionic type.

[0103] In one embodiment, the cosmetic composition is applied to the skin area in an amount of 0.2-3 mg / cm2, preferably 2 mg / cm2.

[0104] In one embodiment, the cosmetic composition according to the present invention is applied once to twice daily for at least 7 days, preferably at least 15 days, even more preferably at least 1 month, and most preferably at least 2 months. Most preferably, the cosmetic composition is applied twice daily for at least 2 months in the method according to the present invention.

[0105] In another aspect, the present invention relates to a dermocosmetic composition for use in the treatment of atopic dermatitis and / or psoriasis.

[0106] In one embodiment, the composition comprises at least 10 9 , preferably at least 10 10 colony forming units / mL of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

[0107] According to one embodiment, the composition further comprises at least one other dermatologically active ingredient.

[0108] More particularly, the composition according to the invention also comprises at least one other dermatological active ingredient acting either on the strengthening of the dermal-epidermal junction, on the cell-matrix adhesion of the skin, and / or on the cell-cell adhesion and / or migration of keratinocytes, or on the skin, depending on the nature of the agent used, e.g. moisturizers, lipid supplements, superfatting agents, exfoliants, keratolytic agents, antioxidants, soothing agents, emollients, soothing agents, cleansing agents, make-up removers, disinfectants, antibacterial agents, antiseptics, antiseborrheic agents, decongestants, reinvigorating agents, cell regeneration activators, or one or more sunscreen agents.

[0109] The compositions of the invention must be in a dermatologically acceptable form, i.e. compatible with the skin, hair and / or hair. In one embodiment, the dermocosmetic composition is administered by topical application.

[0110] Thus, preferably, the dermocosmetic composition according to the invention is formulated in the form of a cream, ointment, salve, mask, serum, milk, lotion, paste, foam, aerosol, stick, powder, solution, suspension, shampoo, conditioner, patch, hydroalcoholic or oily solution, oil-in-water or water-in-oil or multiphase emulsion, aqueous or oily gel, liquid, pasty or solid anhydrous product, and / or a dispersion of oil in an aqueous phase using spheroids, which can be polymeric nanoparticles such as nanospheres and nanocapsules, or lipid vesicles of ionic and / or non-ionic type.

[0111] In one embodiment, the skin cosmetic composition is applied to the skin area in an amount of 0.2-3 mg / cm2.

[0112] In one embodiment, the cosmetic composition according to the present invention is applied once to twice daily for at least 7 days, preferably at least 15 days, even more preferably at least 1 month, and most preferably at least 2 months. Most preferably, the cosmetic composition is applied twice daily for at least 2 months in the method according to the present invention.

[0113] In another aspect, the present invention relates to the use of the Parabacteroides distasonis strain deposited in Agronomy under accession number CNCM I-5828 as a biofertilizer and / or biocontrol agent.

[0114] Biofertilizers are products containing living microorganisms that, when applied to the soil, seeds, or surface of plants, colonize the rhizosphere or internal plant tissues and induce plant growth.

[0115] Biocontrol agents are alternative products to pesticides to manage plant diseases through the use of living entities such as microorganisms.

[0116] In one embodiment, the use as a biofertilizer and / or biocontrol agent is at least 10 9 , preferably at least 10 10This is carried out by a composition comprising a strain of Parabacteroides distasonis deposited under accession number CNCM I-5828, in colony forming units / mL. [Brief description of the drawings]

[0117] [Figure 1] Parabacteroides distasonis F1-2 reverses colonic hypersensitivity in a dextran sulfate sodium 0.5% colitis-induced mouse model. (a) Visceromotor response to colorectal distension in control (vehicle) mice (n=8), DSS-treated mice (n=10) and Parabacteroides distasonis F1-2-treated mice (n=9). (b) Area under the curve (AUC) was calculated between 60 and 80 mmHg for different groups. Administration of Parabacteroides distasonis F1-2 strain significantly attenuated colonic hypersensitivity induced by DSS treatment in mice. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Dunnett's multiple comparison test. DSS 0.5% + vehicle vs. water + vehicle treated mice: ***p value = 0.0002 (at 80 mmHg); ***p value = 0.0006 (at 60 mmHg); ***p value = 0.0002 (at 40 mmHg). DSS 0.5%+F1-2 vs. water+vehicle treated mice: not significant (40mmHg p-value=0.1945; 60mmHg p-value=0.2234; 80mmHg p-value=0.0589. (b) One-way ANOVA followed by Tukey's multiple comparison test. Water+vehicle vs. DSS 0.5%+vehicle treated mice: ***p-value=0.0002; Water+vehicle vs. DSS 0.5%+F1-2: not significant (p-value=0.2015); DSS 0.5%+vehicle vs. DSS 0.5%+F1-2 treated mice: *p-value=0.0108. [Figure 2a]Administration of Parabacteroides distasonis F1-2, but not Parabacteroides distasonis ATCC 8503, prevents colonic hypersensitivity in DSS-induced colitis. (a) Visceromotor responses to colorectal distension were evaluated in control (water + vehicle) mice (n = 9), DSS 0,5% + vehicle treated mice (n = 11), DSS 0,5% + Parabacteroides distasonis F1-2 (n = 10) or DSS 0,5% + Parabacteroides distasonis ATCC 8503 (n = 10) treated mice at D12. (b) Area under the curve (AUC) was calculated between 60 and 80 mmHg for the different groups. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Tukey's multiple comparison test. Water + vehicle vs. DSS 0.5% + vehicle treated mice: ****p value < 0.0001 (at 80mmHg); ***p value = 0.001 (at 60mmHg); *p value = 0.0243 (at 40mmHg). Water + vehicle vs. DSS 0.5% + ATCC 8503 treated mice: ****p value < 0.0001 (at 80mmHg); **p value = 0.0012 (at 60mmHg); *p value = 0.0343 (at 40mmHg). Water + vehicle vs. DSS 0.5% + F1-2 treated mice: ns (at 80, 60 and 40mmHg). [Figure 2b]Administration of Parabacteroides distasonis F1-2, but not Parabacteroides distasonis ATCC 8503, prevents colonic hypersensitivity in DSS-induced colitis. (a) Visceromotor responses to colorectal distension were evaluated in control (water + vehicle) mice (n = 9), DSS 0,5% + vehicle treated mice (n = 11), DSS 0,5% + Parabacteroides distasonis F1-2 (n = 10) or DSS 0,5% + Parabacteroides distasonis ATCC 8503 (n = 10) treated mice at D12. (b) Area under the curve (AUC) was calculated between 60 and 80 mmHg for the different groups. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Tukey's multiple comparison test. Water + vehicle vs. DSS 0.5% + vehicle treated mice: ****p value < 0.0001 (at 80mmHg); ***p value = 0.001 (at 60mmHg); *p value = 0.0243 (at 40mmHg). Water + vehicle vs. DSS 0.5% + ATCC 8503 treated mice: ****p value < 0.0001 (at 80mmHg); **p value = 0.0012 (at 60mmHg); *p value = 0.0343 (at 40mmHg). Water + vehicle vs. DSS 0.5% + F1-2 treated mice: ns (at 80, 60 and 40mmHg). [Figure 3a]Parabacteroides distasonis F1-2 attenuates colonic hypersensitivity in a post-inflammatory DNBS colitis induced mouse model. (a) Visceromotor response to colorectal distension in EtOH+vehicle mice (n=7), DNBS+vehicle treated mice (n=8) and DNBS+Parabacteroides distasonis F1-2 treated mice (n=10). (b) Area under the curve (AUC) was calculated between 60-80 mmHg for different groups. Parabacteroides distasonis F1-2 administration significantly attenuated colonic hypersensitivity induced by DNBS treatment. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Dunnett's multiple comparison test. EtOH 30%+vehicle vs. DNBS+vehicle treated mice: ***p-value=0.0003 (at 80mmHg); **p-value=0.0038 (at 60mmHg). DNBS+F1-2 vs. EtOH 30%+vehicle treated mice: not significant 80mmHg p-value=0.2504; 60mmHg p-value=0.7811. (b) One-way ANOVA followed by Tukey's multiple comparison test. EtOH 30%+vehicle vs. DNBS+vehicle treated mice: ****p-value<0.0001. EtOH 30%+vehicle vs. DNBS+F1-2 treated mice: ns (p-value=6270). DNBS+vehicle vs. DNBS+F1-2 treated mice: ****p-value<0.0001. [Figure 3b]Parabacteroides distasonis F1-2 attenuates colonic hypersensitivity in a post-inflammatory DNBS colitis induced mouse model. (a) Visceromotor response to colorectal distension in EtOH+vehicle mice (n=7), DNBS+vehicle treated mice (n=8) and DNBS+Parabacteroides distasonis F1-2 treated mice (n=10). (b) Area under the curve (AUC) was calculated between 60-80 mmHg for different groups. Parabacteroides distasonis F1-2 administration significantly attenuated colonic hypersensitivity induced by DNBS treatment. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Dunnett's multiple comparison test. EtOH 30%+vehicle vs. DNBS+vehicle treated mice: ***p-value=0.0003 (at 80mmHg); **p-value=0.0038 (at 60mmHg). DNBS+F1-2 vs. EtOH 30%+vehicle treated mice: not significant 80mmHg p-value=0.2504; 60mmHg p-value=0.7811. (b) One-way ANOVA followed by Tukey's multiple comparison test. EtOH 30%+vehicle vs. DNBS+vehicle treated mice: ****p-value<0.0001. EtOH 30%+vehicle vs. DNBS+F1-2 treated mice: ns (p-value=6270). DNBS+vehicle vs. DNBS+F1-2 treated mice: ****p-value<0.0001. [Figure 4a]Parabacteroides distasonis F1-2 reduces colonic hypersensitivity in a post-infection IBS mouse model. (a) Visceromotor response to colorectal distension in uninfected mice (n=8), infected+vehicle mice (n=9) and Parabacteroides distasonis F1-2 infected treated mice (n=7). (b) Area under the curve between 60 and 80 mmHg for different groups. Colonic hypersensitivity developed by infected mice at the post-infection stage was reversed by Parabacteroides distasonis F1-2 treatment. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Tukey's multiple comparison test. Uninfected + vehicle vs. infected + vehicle treated mice: **p-value = 0.0028 (at 80 mmHg); *p-value = 0.0202 (at 60 mmHg). Infected+vehicle vs. infected+F1-2 treated mice: ##p-value=0.002. (b) Kruskal-Wallis test. Non-infected+vehicle vs. infected+vehicle treated mice: *p-value=0.0267. Infected+vehicle vs. infected+F1-2 treated mice: *p-value=0.0355. Non-infected+vehicle vs. infected+F1-2: not significant (p-value>0.99). [Figure 4b] Parabacteroides distasonis F1-2 reduces colonic hypersensitivity in a post-infection IBS mouse model. (a) Visceromotor response to colorectal distension in uninfected mice (n=8), infected+vehicle mice (n=9) and Parabacteroides distasonis F1-2 infected treated mice (n=7). (b) Area under the curve between 60 and 80 mmHg for different groups. Colonic hypersensitivity developed by infected mice at the post-infection stage was reversed by Parabacteroides distasonis F1-2 treatment. Mean + / - SEM. Statistical analysis: (a) Two-way ANOVA followed by Tukey's multiple comparison test. Uninfected + vehicle vs. infected + vehicle treated mice: **p-value = 0.0028 (at 80 mmHg); *p-value = 0.0202 (at 60 mmHg). Infected+vehicle vs. infected+F1-2 treated mice: ##p-value=0.002. (b) Kruskal-Wallis test. Non-infected+vehicle vs. infected+vehicle treated mice: *p-value=0.0267. Infected+vehicle vs. infected+F1-2 treated mice: *p-value=0.0355. Non-infected+vehicle vs. infected+F1-2: not significant (p-value>0.99). [Figure 5a]Parabacteroides distasonis F1-2 reduces the level of nociceptor activation. (a) Experimental protocol. Schematic of activation signals observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimuli (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Neuronal cells were first stimulated with capsaicin (1 μM) or IS, then incubated with Tyrode or Parabacteroides distasonis F1-2. Neuronal cells were stimulated a second time with capsaicin or IS mixed with Parabacteroides distasonis F1-2, then finally stimulated with KCl (50 mM). IS consisted of bradykinin, serotonin, histamine, PGE2 and KCl. (b) % of neuronal cell subpopulations activated by capsaicin. (c) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for capsaicin positive responding cells in each group. (d) % of neuronal cell subpopulations activated by IS. (e) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for IS positive responding cells in each group. Statistical analysis: Chi-square test ****p-value<0,0001. GPCR: G protein-coupled receptor. IS: inflammatory soap. [Figure 5b]Parabacteroides distasonis F1-2 reduces the level of nociceptor activation. (a) Experimental protocol. Schematic of activation signals observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimuli (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Neuronal cells were first stimulated with capsaicin (1 μM) or IS, then incubated with Tyrode or Parabacteroides distasonis F1-2. Neuronal cells were stimulated a second time with capsaicin or IS mixed with Parabacteroides distasonis F1-2, then finally stimulated with KCl (50 mM). IS consisted of bradykinin, serotonin, histamine, PGE2 and KCl. (b) % of neuronal cell subpopulations activated by capsaicin. (c) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for capsaicin positive responding cells in each group. (d) % of neuronal cell subpopulations activated by IS. (e) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for IS positive responding cells in each group. Statistical analysis: Chi-square test ****p-value<0,0001. GPCR: G protein-coupled receptor. IS: inflammatory soap. [Figure 5c]Parabacteroides distasonis F1-2 reduces the level of nociceptor activation. (a) Experimental protocol. Schematic of activation signals observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimuli (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Neuronal cells were first stimulated with capsaicin (1 μM) or IS, then incubated with Tyrode or Parabacteroides distasonis F1-2. Neuronal cells were stimulated a second time with capsaicin or IS mixed with Parabacteroides distasonis F1-2, then finally stimulated with KCl (50 mM). IS consisted of bradykinin, serotonin, histamine, PGE2 and KCl. (b) % of neuronal cell subpopulations activated by capsaicin. (c) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for capsaicin positive responding cells in each group. (d) % of neuronal cell subpopulations activated by IS. (e) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for IS positive responding cells in each group. Statistical analysis: Chi-square test ****p-value<0,0001. GPCR: G protein-coupled receptor. IS: inflammatory soap. [Figure 5d]Parabacteroides distasonis F1-2 reduces the level of nociceptor activation. (a) Experimental protocol. Schematic of activation signals observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimuli (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Neuronal cells were first stimulated with capsaicin (1 μM) or IS, then incubated with Tyrode or Parabacteroides distasonis F1-2. Neuronal cells were stimulated a second time with capsaicin or IS mixed with Parabacteroides distasonis F1-2, then finally stimulated with KCl (50 mM). IS consisted of bradykinin, serotonin, histamine, PGE2 and KCl. (b) % of neuronal cell subpopulations activated by capsaicin. (c) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for capsaicin positive responding cells in each group. (d) % of neuronal cell subpopulations activated by IS. (e) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for IS positive responding cells in each group. Statistical analysis: Chi-square test ****p-value<0,0001. GPCR: G protein-coupled receptor. IS: inflammatory soap. [Figure 5e]Parabacteroides distasonis F1-2 reduces the level of nociceptor activation. (a) Experimental protocol. Schematic of activation signals observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimuli (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Neuronal cells were first stimulated with capsaicin (1 μM) or IS, then incubated with Tyrode or Parabacteroides distasonis F1-2. Neuronal cells were stimulated a second time with capsaicin or IS mixed with Parabacteroides distasonis F1-2, then finally stimulated with KCl (50 mM). IS consisted of bradykinin, serotonin, histamine, PGE2 and KCl. (b) % of neuronal cell subpopulations activated by capsaicin. (c) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for capsaicin positive responding cells in each group. (d) % of neuronal cell subpopulations activated by IS. (e) Overall kinetic signal activation measured as normalized fluorescence 340nm / 380nm for IS positive responding cells in each group. Statistical analysis: Chi-square test ****p-value<0,0001. GPCR: G protein-coupled receptor. IS: inflammatory soap. [Figure 6a]The neuroinhibitory potential of Parabacteroides distasonis F1-2 may involve G protein-coupled receptors. (a) Bradykinin dose-response curves for ND7 / 23 expressing bradykinin receptors incubated with or without Parabacteroides distasonis F1-2 or Parabacteroides distasonis ATCC 8503 strain. (b) Experimental protocol. Schematic of Ca2+ activation signals of ND7 / 23 neuronal cells loaded with calcium probe (FLIPR Calcium 6 QF) stimulated with bradykinin (40 nM) after 45 min incubation with Parabacteroides distasonis F1-2. (c) Activation signals of ND7 / 23 neuronal cells stimulated with bradykinin after 45 min incubation with Parabacteroides distasonis F1-2 with extracellular Ca2+ or (d) in the absence of extracellular Ca2+. (e) Relative response to control. Bar graphs represent maximum peak values ​​at normalized F-F0 compared to control. (f) Relative response to bradykinin stimulation of ND7 / 23 with or without overnight pretreatment with pertussis toxin. Statistical analysis: (a) Mean + / - SEM. Two-way ANOVA followed by Dunnett's multiple comparison test. Parabacteroides distasonis F1-2 vs. vehicle: *p-value = 0,0191 (at 10-11 M); **p-value = 0,0016 (at 10-10 M); ***p-value = 0,0002 (at 10-9 M); *p-value = 0,0205 (at 10-8 M); *p-value = 0,0222 (at 10-7 M); **p-value = 0,0091 (at 10-5 M); *p-value = 0,0165 (at 10-4 M). Parabacteroides distasonis ATCC 8503 vs. vehicle: *p-value=0,0157 (at 10-11 M); *p-value=0,0266 (at 10-10 M); **p-value=0,0042 (at 10-9 M). Each point represents the mean of four independent readings. (e) One-way ANOVA followed by Tukey's multiple comparison test. Vehicle (+) Ca2+ vs. F1-2 (+) Ca2+: ***p-value=0,0003. Vehicle (-) Ca2+ vs. F1-2 (-) Ca2+: ****p-value<0,0001. Each bar represents the mean of three independent readings. (f) Mean + / - SEM. Two-way ANOVA followed by Sidak's multiple comparison test. ****p-value<0,0001.Each bar represents the average of six independent readings. [Figure 6b]The neuroinhibitory potential of Parabacteroides distasonis F1-2 may involve G protein-coupled receptors. (a) Bradykinin dose-response curves for ND7 / 23 expressing bradykinin receptors incubated with or without Parabacteroides distasonis F1-2 or Parabacteroides distasonis ATCC 8503 strain. (b) Experimental protocol. Schematic of Ca2+ activation signals of ND7 / 23 neuronal cells loaded with calcium probe (FLIPR Calcium 6 QF) stimulated with bradykinin (40 nM) after 45 min incubation with Parabacteroides distasonis F1-2. (c) Activation signals of ND7 / 23 neuronal cells stimulated with bradykinin after 45 min incubation with Parabacteroides distasonis F1-2 with extracellular Ca2+ or (d) in the absence of extracellular Ca2+. (e) Relative response to control. Bar graphs represent maximum peak values ​​at normalized F-F0 compared to control. (f) Relative response to bradykinin stimulation of ND7 / 23 with or without overnight pretreatment with pertussis toxin. Statistical analysis: (a) Mean + / - SEM. Two-way ANOVA followed by Dunnett's multiple comparison test. Parabacteroides distasonis F1-2 vs. vehicle: *p-value = 0,0191 (at 10-11 M); **p-value = 0,0016 (at 10-10 M); ***p-value = 0,0002 (at 10-9 M); *p-value = 0,0205 (at 10-8 M); *p-value = 0,0222 (at 10-7 M); **p-value = 0,0091 (at 10-5 M); *p-value = 0,0165 (at 10-4 M). Parabacteroides distasonis ATCC 8503 vs. vehicle: *p-value=0,0157 (at 10-11 M); *p-value=0,0266 (at 10-10 M); **p-value=0,0042 (at 10-9 M). Each point represents the mean of four independent readings. (e) One-way ANOVA followed by Tukey's multiple comparison test. Vehicle (+) Ca2+ vs. F1-2 (+) Ca2+: ***p-value=0,0003. Vehicle (-) Ca2+ vs. F1-2 (-) Ca2+: ****p-value<0,0001. Each bar represents the mean of three independent readings. (f) Mean + / - SEM. Two-way ANOVA followed by Sidak's multiple comparison test. ****p-value<0,0001.Each bar represents the average of six independent readings. [Figure 6c]The neuroinhibitory potential of Parabacteroides distasonis F1-2 may involve G protein-coupled receptors. (a) Bradykinin dose-response curves for ND7 / 23 expressing bradykinin receptors incubated with or without Parabacteroides distasonis F1-2 or Parabacteroides distasonis ATCC 8503 strain. (b) Experimental protocol. Schematic of Ca2+ activation signals of ND7 / 23 neuronal cells loaded with calcium probe (FLIPR Calcium 6 QF) stimulated with bradykinin (40 nM) after 45 min incubation with Parabacteroides distasonis F1-2. (c) Activation signals of ND7 / 23 neuronal cells stimulated with bradykinin after 45 min incubation with Parabacteroides distasonis F1-2 with extracellular Ca2+ or (d) in the absence of extracellular Ca2+. (e) Relative response to control. Bar graphs represent maximum peak values ​​at normalized F-F0 compared to control. (f) Relative response to bradykinin stimulation of ND7 / 23 with or without overnight pretreatment with pertussis toxin. Statistical analysis: (a) Mean + / - SEM. Two-way ANOVA followed by Dunnett's multiple comparison test. Parabacteroides distasonis F1-2 vs. vehicle: *p-value = 0,0191 (at 10-11 M); **p-value = 0,0016 (at 10-10 M); ***p-value = 0,0002 (at 10-9 M); *p-value = 0,0205 (at 10-8 M); *p-value = 0,0222 (at 10-7 M); **p-value = 0,0091 (at 10-5 M); *p-value = 0,0165 (at 10-4 M). Parabacteroides distasonis ATCC 8503 vs. vehicle: *p-value=0,0157 (at 10-11 M); *p-value=0,0266 (at 10-10 M); **p-value=0,0042 (at 10-9 M). Each point represents the mean of four independent readings. (e) One-way ANOVA followed by Tukey's multiple comparison test. Vehicle (+) Ca2+ vs. F1-2 (+) Ca2+: ***p-value=0,0003. Vehicle (-) Ca2+ vs. F1-2 (-) Ca2+: ****p-value<0,0001. Each bar represents the mean of three independent readings. (f) Mean + / - SEM. Two-way ANOVA followed by Sidak's multiple comparison test. ****p-value<0,0001.Each bar represents the average of six independent readings. [Figure 6d]The neuroinhibitory potential of Parabacteroides distasonis F1-2 may involve G protein-coupled receptors. (a) Bradykinin dose-response curves for ND7 / 23 expressing bradykinin receptors incubated with or without Parabacteroides distasonis F1-2 or Parabacteroides distasonis ATCC 8503 strain. (b) Experimental protocol. Schematic of Ca2+ activation signals of ND7 / 23 neuronal cells loaded with calcium probe (FLIPR Calcium 6 QF) stimulated with bradykinin (40 nM) after 45 min incubation with Parabacteroides distasonis F1-2. (c) Activation signals of ND7 / 23 neuronal cells stimulated with bradykinin after 45 min incubation with Parabacteroides distasonis F1-2 with extracellular Ca2+ or (d) in the absence of extracellular Ca2+. (e) Relative response to control. Bar graphs represent maximum peak values ​​at normalized F-F0 compared to control. (f) Relative response to bradykinin stimulation of ND7 / 23 with or without overnight pretreatment with pertussis toxin. Statistical analysis: (a) Mean + / - SEM. Two-way ANOVA followed by Dunnett's multiple comparison test. Parabacteroides distasonis F1-2 vs. vehicle: *p-value = 0,0191 (at 10-11 M); **p-value = 0,0016 (at 10-10 M); ***p-value = 0,0002 (at 10-9 M); *p-value = 0,0205 (at 10-8 M); *p-value = 0,0222 (at 10-7 M); **p-value = 0,0091 (at 10-5 M); *p-value = 0,0165 (at 10-4 M). Parabacteroides distasonis ATCC 8503 vs. vehicle: *p-value=0,0157 (at 10-11 M); *p-value=0,0266 (at 10-10 M); **p-value=0,0042 (at 10-9 M). Each point represents the mean of four independent readings. (e) One-way ANOVA followed by Tukey's multiple comparison test. Vehicle (+) Ca2+ vs. F1-2 (+) Ca2+: ***p-value=0,0003. Vehicle (-) Ca2+ vs. F1-2 (-) Ca2+: ****p-value<0,0001. Each bar represents the mean of three independent readings. (f) Mean + / - SEM. Two-way ANOVA followed by Sidak's multiple comparison test. ****p-value<0,0001.Each bar represents the average of six independent readings. [Figure 6e]The neuroinhibitory potential of Parabacteroides distasonis F1-2 may involve G protein-coupled receptors. (a) Bradykinin dose-response curves for ND7 / 23 expressing bradykinin receptors incubated with or without Parabacteroides distasonis F1-2 or Parabacteroides distasonis ATCC 8503 strain. (b) Experimental protocol. Schematic of Ca2+ activation signals of ND7 / 23 neuronal cells loaded with calcium probe (FLIPR Calcium 6 QF) stimulated with bradykinin (40 nM) after 45 min incubation with Parabacteroides distasonis F1-2. (c) Activation signals of ND7 / 23 neuronal cells stimulated with bradykinin after 45 min incubation with Parabacteroides distasonis F1-2 with extracellular Ca2+ or (d) in the absence of extracellular Ca2+. (e) Relative response to control. Bar graphs represent maximum peak values ​​at normalized F-F0 compared to control. (f) Relative response to bradykinin stimulation of ND7 / 23 with or without overnight pretreatment with pertussis toxin. Statistical analysis: (a) Mean + / - SEM. Two-way ANOVA followed by Dunnett's multiple comparison test. Parabacteroides distasonis F1-2 vs. vehicle: *p-value = 0,0191 (at 10-11 M); **p-value = 0,0016 (at 10-10 M); ***p-value = 0,0002 (at 10-9 M); *p-value = 0,0205 (at 10-8 M); *p-value = 0,0222 (at 10-7 M); **p-value = 0,0091 (at 10-5 M); *p-value = 0,0165 (at 10-4 M). Parabacteroides distasonis ATCC 8503 vs. vehicle: *p-value=0,0157 (at 10-11 M); *p-value=0,0266 (at 10-10 M); **p-value=0,0042 (at 10-9 M). Each point represents the mean of four independent readings. (e) One-way ANOVA followed by Tukey's multiple comparison test. Vehicle (+) Ca2+ vs. F1-2 (+) Ca2+: ***p-value=0,0003. Vehicle (-) Ca2+ vs. F1-2 (-) Ca2+: ****p-value<0,0001. Each bar represents the mean of three independent readings. (f) Mean + / - SEM. Two-way ANOVA followed by Sidak's multiple comparison test. ****p-value<0,0001.Each bar represents the average of six independent readings. [Figure 6f]The neuroinhibitory potential of Parabacteroides distasonis F1-2 may involve G protein-coupled receptors. (a) Bradykinin dose-response curves for ND7 / 23 expressing bradykinin receptors incubated with or without Parabacteroides distasonis F1-2 or Parabacteroides distasonis ATCC 8503 strain. (b) Experimental protocol. Schematic of Ca2+ activation signals of ND7 / 23 neuronal cells loaded with calcium probe (FLIPR Calcium 6 QF) stimulated with bradykinin (40 nM) after 45 min incubation with Parabacteroides distasonis F1-2. (c) Activation signals of ND7 / 23 neuronal cells stimulated with bradykinin after 45 min incubation with Parabacteroides distasonis F1-2 with extracellular Ca2+ or (d) in the absence of extracellular Ca2+. (e) Relative response to control. Bar graphs represent maximum peak values ​​at normalized F-F0 compared to control. (f) Relative response to bradykinin stimulation of ND7 / 23 with or without overnight pretreatment with pertussis toxin. Statistical analysis: (a) Mean + / - SEM. Two-way ANOVA followed by Dunnett's multiple comparison test. Parabacteroides distasonis F1-2 vs. vehicle: *p-value = 0,0191 (at 10-11 M); **p-value = 0,0016 (at 10-10 M); ***p-value = 0,0002 (at 10-9 M); *p-value = 0,0205 (at 10-8 M); *p-value = 0,0222 (at 10-7 M); **p-value = 0,0091 (at 10-5 M); *p-value = 0,0165 (at 10-4 M). Parabacteroides distasonis ATCC 8503 vs. vehicle: *p-value=0,0157 (at 10-11 M); *p-value=0,0266 (at 10-10 M); **p-value=0,0042 (at 10-9 M). Each point represents the mean of four independent readings. (e) One-way ANOVA followed by Tukey's multiple comparison test. Vehicle (+) Ca2+ vs. F1-2 (+) Ca2+: ***p-value=0,0003. Vehicle (-) Ca2+ vs. F1-2 (-) Ca2+: ****p-value<0,0001. Each bar represents the mean of three independent readings. (f) Mean + / - SEM. Two-way ANOVA followed by Sidak's multiple comparison test. ****p-value<0,0001.Each bar represents the average of six independent readings. [Figure 7a] The neuronal inhibitory capacity of Parabacteroides distasonis F1-2 may involve the bradykinin receptor. (a) Experimental protocol. Schematic of the activation signal observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimulus (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Ca2+ responses were measured in neuronal cells from DRG mice as normalized fluorescence 340 nm / 380 nm ratio. (b) % of the subpopulations of neuronal cells activated by IS(+), IS(-) or bradykinin. (c) % of the subpopulations of neuronal cells activated by bradykinin or histamine receptor 1 specific agonist (H1R) (2-pyridylethylamine). F1-2: Parabacteroides distasonis F1-2; Stim: Stimulation; IS: Inflammatory Soap; Brady: Bradykinin; H1R: Histamine Receptor 1 specific agonist. Statistical analysis: Chi-square test. ****p-value<0,0001. [Figure 7b]The neuronal inhibitory capacity of Parabacteroides distasonis F1-2 may involve the bradykinin receptor. (a) Experimental protocol. Schematic of the activation signal observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimulus (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Ca2+ responses were measured in neuronal cells from DRG mice as normalized fluorescence 340 nm / 380 nm ratio. (b) % of the subpopulations of neuronal cells activated by IS(+), IS(-) or bradykinin. (c) % of the subpopulations of neuronal cells activated by bradykinin or histamine receptor 1 specific agonist (H1R) (2-pyridylethylamine). F1-2: Parabacteroides distasonis F1-2; Stim: Stimulation; IS: Inflammatory Soap; Brady: Bradykinin; H1R: Histamine Receptor 1 specific agonist. Statistical analysis: Chi-square test. ****p-value<0,0001. [Figure 7c] The neuronal inhibitory capacity of Parabacteroides distasonis F1-2 may involve the bradykinin receptor. (a) Experimental protocol. Schematic of the activation signal observed by ratiometric calcium imaging with two subpopulations. The first one corresponds to neuronal cells responding to both the first and second stimulus (black). The second one corresponds to neuronal cells responding only to the first stimulus (gray). Ca2+ responses were measured in neuronal cells from DRG mice as normalized fluorescence 340 nm / 380 nm ratio. (b) % of the subpopulations of neuronal cells activated by IS(+), IS(-) or bradykinin. (c) % of the subpopulations of neuronal cells activated by bradykinin or histamine receptor 1 specific agonist (H1R) (2-pyridylethylamine). F1-2: Parabacteroides distasonis F1-2; Stim: Stimulation; IS: Inflammatory Soap; Brady: Bradykinin; H1R: Histamine Receptor 1 specific agonist. Statistical analysis: Chi-square test. ****p-value<0,0001. EXAMPLES

[0118] In the following examples, colonic hypersensitivity in an animal model is measured to assess the efficacy of strains according to the invention against chronic visceral pain in humans.

[0119] Example 1: Parabacteroides distasonis F1-2 strain has analgesic effects in a dextran sulfate sodium acute colitis mouse model (inflammatory visceral pain mouse model).

[0120] Parabacteroides distasonis culture method

[0121] The strain Parabacteroides distasonis (isolate F1-2) was cultivated in a complex anaerobic medium containing neutralized soy peptone (10 g / L, OXOID), yeast extract (15 g / L, OXOID), KH2PO4 (0.9 g / L), K2HPO4 (0.9 g / L), NaCl (0.9 g / L), sodium acetate (2.7 g / L), MgSO4, 7H2O (0.09 g / L), L-cysteine ​​hydrochloride (1 g / L), D-glucose (20 g / L), NaHCO3 (4 g / L), maltose (0.5 g / L), cellobiose (0.5 g / L) and adjusted to pH 6.9. For inoculum preparation, 1 mL of Parabacteroides distasonis F1-2 frozen stock culture was subcultured in a 50 mL flask containing 25 mL of anaerobic medium (described above) and incubated for 24 h at 37 °C under strict anaerobic conditions. A second subculture was required, which consisted of transferring the 24-h first subculture (OD600nm of 3-4) in 225 mL of anaerobic medium and incubating for 20 h at 37 °C under strict anaerobic conditions. Cultivation with growing cells was performed in a 5 L bioreactor by inoculating 5% (v / v) of the second subculture (OD600nm = 3) in fresh anaerobic medium. Anaerobic conditions were maintained by continuous injection of a gas mixture (N2 (85%), H2 (5%) and CO2 (10%)) that allowed to maintain a low redox potential (-400 mV) and homogenization was ensured by two Rushton blades at a stirring speed of 150 rpm. Bioreactor cultivation was carried out for 24 h at a controlled temperature of 37 °C, reaching an OD600nm of 6. Biomass of Parabacteroides distasonis F1-2 was harvested by centrifugation of the whole culture at 5000 g for 30 min at 4 °C under anaerobic conditions to maintain cell viability and stored at -80 °C in a PBS + glycerol (20% (v / v)) solution. The dry matter of the biomass was adjusted to 19% with PBS + glycerol (20%) to provide a standardized bacterial preparation. Bacterial titers were determined according to batch production by counting on Brucella blood agar (from anaerobic biosystems) at 1,2 × 10 10 CFU / mL ~ 1,5 × 10 10 was estimated to be between CFU / mL.

[0122] animal

[0123] Fifty-seven male C57 / Blk6 mice (6–9 weeks old) were obtained from Janvier Labs (53 Le Genest-Saint-Isle, France).

[0124] Animal treatment

[0125] Acute colitis was induced by 0.5% (w / v) dextran sodium sulfate (DSS) (mol wt = 36,000-50,000 Da) dissolved in drinking water ad libitum for 12 days. 10 CFU / mL of Parabacteroides distasonis F1-2 strain was administered by force every day. The weight loss of the animals was controlled during DSS administration, and feces were collected every day of the experiment and frozen at -80°C for further analysis. The test groups were as follows: control (water + vehicle group (PBS + glycerol (20%)), DSS + vehicle (PBS + glycerol (20%)) and DSS + F1-2 group. Colonic hypersensitivity was measured at the end of the experiment. In a second experiment, the analgesic effect of Parabacteroides distasonis F1-2 strain was compared with that of Parabacteroides distasonis ATCC 8503 reference strain according to the same procedure.

[0126] Measurement of colonic hyperresponsiveness by colorectal distension

[0127] Mouse colonic sensitivity induced by DSS treatment was evaluated by quantifying the visceromotor response in response to colorectal distension (CRD). Mice were acclimated 30 min before the experiment to reduce movement artifacts due to restraint stress. A polyethylene balloon with a connected catheter was inserted into the distal colon 1 cm from the base of the balloon to the mouse anus during light isoflurane anesthesia. The catheter was fixed to the tail with tape. The balloon was connected to a pressure transducer to control the intraballoon pressure during the CRD procedure. A barostat was used to manage air infiltration and balloon pressure control. The CRD procedure allowed the assessment of visceral pain-related responses and consisted of repeated stepwise distensions of 20, 40, 60 and 80 mmHg with a pulse duration of 20 s and 4 min intervals between each other.

[0128] Results - Conclusion

[0129] To evaluate the antinociceptive effect of strain Parabacteroides distasonis F1-2 on colonic hypersensitivity, a model of colitis induced by 0.5% dextran sodium sulfate was used. Parabacteroides distasonis F1-2 was administered by force every day throughout the period of DSS treatment, thus for 12 consecutive days. DSS 0.5% treatment significantly increased colonic hypersensitivity (***p value = 0.0002 at 80 mmHg) (Figure 1a) (***p value = 0.0002) (Figure 1b), whereas Parabacteroides distasonis F1-2 oral administration significantly reduced colonic hypersensitivity compared to the control. In fact, there was no significant difference between the water + vehicle and DSS 0.5% + F1-2 mouse groups (Figures 1a and 1b), causing a partial reversal of the treatment-induced pain phenotype.

[0130] In a second experiment, following the same procedure, the antinociceptive effect of Parabacteroides distasonis F1-2 strain was compared with Parabacteroides distasonis ATCC 8503 reference strain in the same visceral pain mouse model. Parabacteroides distasonis F1-2 strain exerted a significant antinociceptive effect in mice treated with dextran sulfate sodium (water + vehicle vs. DSS 0.5% + F1-2: not significant) (Figure 2a), whereas Parabacteroides distasonis ATCC 8503 reference strain had no beneficial effect on colonic hypersensitivity (water + vehicle vs. DSS 0.5% + ATCC 8503: ****p value < 0.0001 at 80 mmHg) (Figures 2a and 2b).

[0131] Example 2: Parabacteroides distasonis strain F1-2 has analgesic effects in a dinitrobenzenesulfonic acid colitis mouse model.

[0132] Parabacteroides distasonis culture method

[0133] See Example 1: Parabacteroides distasonis culture methods.

[0134] animal

[0135] Twenty-five male C57 / Blk6 mice (6–9 weeks old) were obtained from Janvier Labs (53 Le Genest-Saint-Isle, France).

[0136] Animal treatment

[0137] Colitis was induced by intrarectal instillation of dinitrobenzenesulfonic acid (DNBS) (100 mg / Kg) dissolved in 30% (v / v) ethanol solution on days 1 and 23. For 10 days (days 17–26), 0.2 mL of 1.52 × 10 10CFU / mL of Parabacteroides distasonis F1-2 strain was administered by force every day. Four days after each rectal instillation of DNBS, the animals were tracked for weight loss to control the efficacy of treatment. The test groups were as follows: control (EtOH 30%+vehicle (PBS+glycerol)), DNBS+vehicle (PBS+glycerol) and DNBS+F1-2. Colonic hypersensitivity was measured at the end of the experiment.

[0138] Measurement of colonic hyperresponsiveness by colorectal distension

[0139] See Example 1: Measurement of colonic hyperresponsiveness by colorectal distension

[0140] Results - Conclusion

[0141] To evaluate the antinociceptive effects of Parabacteroides distasonis, the F1-2 strain was tested in a murine model of postinflammatory visceral pain induced by rectal instillation of DNBS. DNBS induced a transient increase in intestinal permeability and inflammatory response between 1 and 3 days after instillation, whereas colonic hypersensitivity was affected over a longer period of time.

[0142] The beneficial effect of Parabacteroides distasonis F1-2 was observed in a post-inflammatory DNBS colitis-induced mouse model (Figure 3). Indeed, DNBS rectal instillation caused colonic hypersensitivity (Figure 3a) (***p-value=0.0003 at 80mmHg and **p-value=0.0038 at 60mmHg), whereas Parabacteroides distasonis F1-2 administered for 10 days reversed the pain phenotype (Figure 3a) (EtOH 30%+vehicle vs. DNBS+F1-2; ns). This was also the case when we calculated the AUC (Figure 3b).

[0143] Example 3: Parabacteroides distasonis strain F1-2 has analgesic effects in a mouse model of post-IBS visceral pain.

[0144] Parabacteroides distasonis culture method

[0145] See Example 1: Parabacteroides distasonis culture methods.

[0146] animal

[0147] Twenty-four male C57 / Blk6 mice (6–9 weeks old) were obtained from Janvier Labs (53 Le Genest-Saint-Isle, France).

[0148] Animal treatment

[0149] After overnight static growth in flasks containing Luria broth, Citrobacter rodentium (ATCC 51459 TM DBS100) cells were collected by centrifugation and suspended in PBS buffer at 5 × 10 9 On day 0, each mouse was administered 1 × 10 9 CFU of Citrobacter rodentium were administered to the mice. The infectious phase occurred before disappearing completely on day 16. Clearance of the pathogen was confirmed by counting fecal Citrobacter rodentium on MacConkey agar. Parabacteroides distasonis F1-2 strain was administered daily by force from day 16 to day 23 (3.03 × 10 9 CFU / mouse). The test groups were as follows: control (uninfected + vehicle (PBS + glycerol)) group (n=8), infected mice + vehicle (PBS + glycerol) (n=9) and infected mice + Parabacteroides distasonis F1-2 strain (n=7). Colonic hypersensitivity was measured at the end of the experiment.

[0150] Measurement of colonic hyperresponsiveness by colorectal distension

[0151] See Example 1: Measurement of colonic hyperresponsiveness by colorectal distension

[0152] Results - Conclusion Since colonic hypersensitivity can occur after a gastrointestinal infection episode, the analgesic potential of Parabacteroides distasonis F1-2 strain was tested in a post-infectious IBS mouse model induced by infection with the murine pathogen Citrobacter rodentium. After 16 days of infection, the pathogen was completely cleared (data not shown) and mice were force-fed with Parabacteroides distasonis F1-2 bacterial strain daily for 8 days. In this model, despite the complete clearance of the pathogen, mice developed a pain phenotype at the post-infection stage. Indeed, significant colonic hypersensitivity was found in the infected group (Figure 4a) (**p-value = 0.0028 at 80 mmHg and *p-value = 0.0202 at 60 mmHg) (Figure 4b) (*p-value = 0.0267). A significant analgesic effect exerted by Parabacteroides distasonis F1-2 was observed. Indeed, the colonic hypersensitivity developed by infected mice was completely reversed by Parabacteroides distasonis F1-2 treatment (Figure 4a) (##p-value=0.002 at 80 mmHg) (Figure 4b) (uninfected+vehicle vs. infected+F1-2: ns).

[0153] Example 4: Parabacteroides distasonis F1-2 reduces the level of nociceptor activation.

[0154] Parabacteroides distasonis culture method

[0155] See Example 1: Parabacteroides distasonis culture methods.

[0156] Culturing primary neuronal DRG cells

[0157] Dorsal root ganglia from C57 / Blk6 mice (male) were explanted, ventral and dorsal roots and connective tissue were removed, and digested with an enzyme mix containing collagenase type III (5 mg / mL, Worthington) and dispase (10 mg / mL; Gibco) for 45 min at 37°C. The DRG suspension was centrifuged at 175g for 15 s, after which the supernatant was removed and replaced by 1 mL of Dulbecco's modified Eagle's medium (DMEM; SIGMA). The DRG suspension was triturated with a fire-polished Pasteur pipette and spun at 175g for 5 s. The supernatant containing the dissociated cells was saved at each trituration step. Trituration was repeated six times using three fire-polished Pasteur pipettes of decreasing diameter. The cell solution was spun at 175g for 5 min. The supernatant was removed and replaced by 600 μL of DMEM supplemented with 10% (v / v) fetal bovine serum, sodium pyruvate (1 mM; Gibco), L-glutamine (2 mM), and penicillin (100 μg / mL), streptomycin (100 μg / mL), vitamins, amino acids (MEM NEAA, Gibco), and NGF (6.25 pg / mL). The cell suspension was seeded onto 12 poly-L-lysine (100 μg / mL) and laminin (200 μg / mL) coated glass sheets in a culture dish. The cells were incubated at 37 °C for 30 min, after which 1 mL of DMEM supplemented medium was added to each well. The cells were incubated at 37 °C overnight.

[0158] Assessing neuronal cell activation by ratiometric calcium imaging

[0159] Intracellular free Ca 2+ followed by Ca 2+Ratiometric calcium imaging was performed using the ratiometric dye Fura-2 acetoxymethyl ester (Fura 2-AM, Invitrogen). DRG neurons were loaded with Fura-2 solution (4 μM) supplemented with pluronic acid (1 μg / mL) for 45 min at 37 °C with slight agitation (40 rpm). All imaging experiments were performed at room temperature in the dark. After loading, glass coverslips were mounted in an imaging / perfusion chamber equipped with a perfusion valve system mounted and observed by an inverted microscope. Neuronal cells were alternately illuminated with wavelengths of 340 nm and 380 nm. Exposure time to excitation was 400 ms for each wavelength. Image pairs were acquired every 2 s. Calcium imaging experiments were performed after rinsing with Tyrode's solution, with 1.99–2.4 × 10 8 The experiment consisted of two stimulations spaced by a 5 min incubation of Parabacteroides distasonis F1-2 viable cells / mL of Tyrode's solution. The Tyrode's solution consisted of: NaCl (140 mM), KCl (3 mM), MgCl2 (1 mM), CaCl2 (2 mM), D-glucose (10 mM), HEPES (10 mM) adjusted to pH 7.4. Osmolarity was also adjusted to 300 mOsm. Finally, a KCl (50 mM) stimulation was evaluated at the end of the experiment to assess cell viability. The KCl solution consisted of: NaCl (93 mM), KCl (50 mM), MgCl2 (1 mM), CaCl2 (2 mM), D-glucose (10 mM), HEPES (10 mM) adjusted to pH 7.4. Osmolarity was also adjusted to 300 mOsm. In this experiment, capsaicin and Parabacteroides distasonis F1-2 strain were diluted in Tyrode's physiological solution before use.

[0160] Results - Conclusion To understand the molecular mechanisms occurring between Parabacteroides distasonis F1-2 and nociceptors, neurons from DRG mice were cultured and loaded with Fura-2 to track neuronal activation under nociceptive stimulation. In these experiments, primary neuronal cells from DRG mice were first stimulated with stimulating solution and then incubated with vehicle or Parabacteroides distasonis F1-2 bacterial strain for 5 min. Then, neuronal cells were stimulated a second time with stimulating solution mixed with vehicle or Parabacteroides distasonis F1-2 bacterial strain. A final KCl (50 mM) stimulation was performed to control cell viability at the end of the experiment (Figure 5a).

[0161] In the first experiment, the stimulant consisted of a capsaicin solution that specifically targets neuronal cells expressing the TRPV1 channel, which are involved in visceral pain. 2+ The responses (measured as normalized fluorescence 340 nm / 380 nm ratio) were followed by ratiometric calcium imaging. Quantification of the mean maximum ΔF / F0 of capsaicin-positive nociceptive neurons revealed two respective groups in each condition. Indeed, in each condition tested, two subpopulations within TRPV1-positive neurons were identified. The first one corresponds to TRPV1-positive neurons that responded to both the first and second stimuli, represented in black (Figure 5b), while the second subpopulation corresponds to TRPV1 neurons that responded only to the first stimulus, represented in grey (Figure 5b). Incubation of neuronal cells with Parabacteroides distasonis F1-2 significantly increased the percentage of the second subpopulation by turning off 26.34% of TRPV1 cells against 4.5% of the control (Figure 5b) (****p-value < 0,0001). This result was also reflected by a slight decrease in the calcium signal in the overall kinetics (Figure 5c).

[0162] In the second experiment, the stimulant consisted of an inflammatory mixture solution containing inflammatory mediators involved in visceral pain such as bradykinin, histamine, serotonin and prostaglandin 2. The results showed that incubation of neuronal cells with Parabacteroides distasonis F1-2 significantly increased the percentage of neuronal cells inactivated at the second stimulus applied by 29,77% off versus 17,39% in the control (Figure 5d) (****p-value < 0,0001) and led to a calcium signal that was reduced in overall kinetics (Figure 5e).

[0163] Example 5: Neuroinhibitory activity of Parabacteroides distasonis F1-2 involves G protein-coupled receptors.

[0164] Parabacteroides distasonis culture method

[0165] See Example 1: Parabacteroides distasonis culture methods.

[0166] Culture and treatment of ND7 / 23 immortalized neuronal cells

[0167] ND7 / 23 cells were generated by fusion of cultured neonatal rat DRG neurons with N18TG2 mouse neuroblastoma cells

[19] . This immortalized cell line has been characterized as a sensory neuron model [20, 21]. ND7 / 23 cells were cultured in 15 mL of Dulbecco's modified Eagle's medium (DMEM; SIGMA) supplemented with 10% (v / v) fetal bovine serum, sodium pyruvate (110 mg / mL), L-glutamine (2 mM), and penicillin (100 μg / mL), streptomycin (100 μg / mL) in 75 cm2 culture flasks. At 80% confluence, cells were detached from the substrate, centrifuged at 175 g for 5 min, and suspended in fresh ND7 / 23 culture medium at 800,000 cells / mL. ND7 / 23 suspensions were seeded onto 96 microplate wells (Greiner bio-one 655096) at 80,000 cells / well (100 μL / well) and incubated overnight at 37 °C. Experiments with the ND7 / 23 cell line were performed over a period of several months, spanning on average a minimum of 30-40 passages. The number of cell passages did not significantly affect the response to bradykinin stimulation. To test the involvement of Gαi in the direct interaction between Parabacteroides distasonis strain F1-2 and neuronal cells, ND7 / 23 cells were cultured in the presence or absence of pertussis toxin (PTX, 250 ng / mL, SIGMA) and incubated overnight at 37°C before use.

[0168] Neuromodulatory properties of F1-2 on ND7 / 23 immortalized neuronal cells After 24 hours of incubation, the ND7 / 23 cells were confluent. The culture medium was removed and replaced with 81 μL of calcium probe (Kit FLIPR Calcium 6-QF, Molecular Devices, λ485 nm (excitation) and λ525 nm (emission)) and 81 μL of HBSS (Hank's Balanced Salt Solution)-HEPES buffer 20 mM (adjusted to pH 7.4, 300 mOsm). The cells were incubated with the calcium probe for 2 hours at 37 °C, according to the supplier's recommendations. After incubation, the calcium probe was removed and Ca 2+The HBSS-HEPES buffer was replaced with HBSS-HEPES buffer containing or not.

[0169] To evaluate the effect of F1-2 on bradykinin EC50, ND7 / 23 cells were cultured at 1 × 10 8 Parabacteroides distasonis F1-2 viable cells / mL or vehicle (PBS+glycerol) were incubated for 45 min before stimulation with bradykinin (at various final concentrations). Data analysis: The maximum signal to stimulation in the second part of the experiment was determined relative to the F-F0 normalized kinetic signal. The maximum activation signal to bradykinin compared to the control was expressed for each bradykinin concentration tested.

[0170] Results - Conclusion

[0171] In the first experiment, immortalized ND7 / 23 sensory neuron cells were stimulated with various bradykinin concentrations (10 pM-100 μM) after incubation with Parabacteroides distasonis F1-2 or vehicle for 45 min. The maximum peak of calcium signal represented on the dose-response curve showed that Parabacteroides distasonis F1-2 reduced the activation level of ND7 / 23 neuron cells for all bradykinin concentrations tested (Figure 6a). These results indicated that Parabacteroides distasonis F1-2 can interact with bradykinin receptors by reducing the activation signal in response to stimulation (Figure 6a). The neuroinhibitory effect of Parabacteroides distasonis F1-2 appears to be higher than that of the Parabacteroides distasonis ATCC 8503 reference strain (Figure 6a).

[0172] Neuronal activation is the result of neuronal depolarization induced in part by intracellular release of calcium provided by the endoplasmic reticulum (RE) but also by calcium influx from the extracellular compartment. Indeed, in our experiments, bradykinin stimulation leads to activation of cellular pathways involving GPCRs, resulting in an increase in intracellular calcium concentration from the RE. Immortalized ND7 / 23 sensory neuronal cells were incubated with Parabacteroides distasonis F1-2 bacterial strain for 45 min before stimulation with bradykinin (40 nM), with or without extracellular calcium (Figure 6b). The deprivation of extracellular calcium allowed us to measure neuronal activation specifically related to intracellular calcium flux. The results showed that Parabacteroides distasonis F1-2 strain reduced calcium signals on ND7 / 23 neuronal cells (Figures 6c and 6d). The strongest inhibitory effect was observed when neuronal cells were deprived of extracellular calcium (vehicle (+)Ca 2+ Against F1-2(+)Ca 2+ ***p value = 0.0003). Vehicle (-) Ca 2+ Against F1-2(-)Ca 2+ :****p<0.0001) (Figure 6e), suggesting that G-coupled protein receptors may be involved in bacteria-neuron interactions.

[0173] The involvement of Gαi-coupled protein receptor activation in the neuronal inhibitory effect of Parabacteroides distasonis F1-2 was evaluated by treating ND7 / 23 sensory neuronal cells with pertussis toxin (PTX). After incubation with Parabacteroides distasonis F1-2 strain for 45 min, neuronal cells were stimulated with bradykinin (40 nM). Even when Gαi was blocked by PTX, the inhibitory effect of Parabacteroides distasonis F1-2 was observed in the presence or absence of extracellular calcium (Figure 6f), indicating that the neuronal inhibitory effect of this strain is not the result of Gαi activation.

[0174] Example 6: The neuroinhibitory activity of Parabacteroides distasonis F1-2 may involve the bradykinin receptor.

[0175] Parabacteroides distasonis culture method

[0176] See Example 1: Parabacteroides distasonis culture methods.

[0177] Culturing primary neuronal DRG cells

[0178] See Example 4: Cultivation of primary neuronal DRG cells

[0179] Assessing neuronal cell activation by ratiometric calcium imaging

[0180] See Example 4: Assessment of neuronal cell activation by ratiometric calcium imaging

[0181] Results - Conclusion

[0182] The contribution of bradykinin receptors in the neuroinhibitory ability of Parabacteroides distasonis F1-2 was evaluated in primary neuronal cells from DRG mice. Primary neuronal cells were first stimulated with a stimulating solution and then incubated with vehicle or Parabacteroides distasonis F1-2 for 5 min. Neuronal cells were then stimulated a second time with stimulating agents mixed with vehicle or Parabacteroides distasonis F1-2 strain. A final KCl (50 mM) stimulation was performed to control cell viability at the end of the experiment (Figure 7a). In the first experiment, the stimulating agents consisted of an inflammatory soap (IS) solution targeting G-coupled protein receptors with (IS(+)Brady) or without (IS(-)Brady) bradykinin, or bradykinin alone. Ca in each neuronal cell was measured using a 10-μL stimulating agent. 2+The responses (measured as normalized fluorescence 340 nm / 380 nm ratio) were followed by ratiometric calcium imaging. Quantification of the mean maximum ΔF / F0 of IS-positive nociceptive neurons revealed two respective groups in each condition. Indeed, in each condition tested, two subpopulations within the responsive neurons to the applied stimuli were identified. The first one corresponds to the responsive neurons that responded to both the first and second stimuli, represented in black (Figure 7b), whereas the second subpopulation corresponds to the neurons that responded only to the first stimulus, represented in grey (Figure 7b). The neuroinhibitory potential of Parabacteroides distasonis F1-2, reflected by a significant increase in neuronal cells inactivated with the second stimulus, was only observed when the stimulus contained bradykinin. Indeed, when neuronal cells were stimulated with IS containing bradykinin, incubation of Parabacteroides distasonis F1-2 cells led to an increase in inactivated neuronal cells, turning off 29,96% of responsive cells against 13,62% in the control (Figure 7b) (****p-value<0,0001). Surprisingly, the inhibitory effect was lost when IS depleted of bradykinin under bradykinin stimulation (Figure 7b), and a neuroinhibitory effect was observed, leading to an increase in inactivated neuronal cells, turning off 82,93% against 39,45% in the control (Figure 7b) (****p-value<0,0001).

[0183] Bradykinin receptors are GPCRs that recruit Gαi or Gαq proteins. Neuronal inhibition requires α activation in the case of Gαi protein-coupled receptors, whereas inhibition of the α subunit is required in the case of Gαq protein-coupled receptors. To evaluate the interaction specificity between Parabacteroides distasonis F1-2 strain and bradykinin receptors, similar experiments were performed by replacing the bradykinin stimulus with a specific agonist (2-pyridylethylamine) of histamine receptor 1 or H1R, which is a Gαq protein-coupled receptor as well as the bradykinin receptor. Although Parabacteroides distasonis F1-2 significantly increased the percentage of non-responsive cells to the second bradykinin stimulation (Fig. 7c) (****p-value < 0,0001), no neuroinhibitory effect was observed with 2-pyridylethylamine stimulation (Fig. 6c), suggesting that H1 receptors are not involved in the direct interaction of Parabacteroides distasonis F1-2 with neuronal cells. Furthermore, the results suggest that Parabacteroides distasonis F1-2 does not specifically interact with Gαq protein, since no inhibitory effect was observed with H1R stimulation.

[0184] Name of depository institution:Collection nationale de cultures de micro-organismes (CNCM)

[0185] Depository institution: Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15

[0186] Accession number: CNCM I-5828

[0187] Deposit date: February 16, 2022 Description of sequence listing SEQ ID NO: 1: Parabacteroides distasonis strain F1-2 according to the invention, gene for 16S ribosomal RNA. References 1. Marchesi,J.R.& Ravel,J.The vocabulary of microbiome research:a proposal.Microbiome 3,31 (2015). 2. Eckburg,P.B.et al.Diversity of the human intestinal microbial flora.Science 308,1635-1638 (2005). 3. Sender,R.,Fuchs,S.& Milo,R.Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans.Cell 164,337-340 (2016). 4. Sender,R.,Fuchs,S.& Milo,R.Revised Estimates for the Number of Human and Bacteria Cells in the Body.PLOS Biology 14,e1002533 (2016). 5. Rinninella,E.et al.What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age,Environment,Diet,and Diseases.Microorganisms 7,E14 (2019). 6. Casen,C.et al.Deviations in human gut microbiota:a novel diagnostic test for determining dysbiosis in patients with IBS or IBD.Aliment Pharmacol Ther 42,71-83 (2015). 7. Li,J.,Butcher,J.,Mack,D.& Stintzi,A.Functional impacts of the intestinal microbiome in the pathogenesis of inflammatory bowel disease.Inflamm Bowel Dis 21,139-153 (2015). 8. Lloyd-Price,J.et al.Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases.Nature 569,655-662 (2019). 9. Caenepeel,C.,Sadat Seyed Tabib,N.,Vieira-Silva,S.& Vermeire,S.Review article:how the intestinal microbiota may reflect disease activity and influence therapeutic outcome in inflammatory bowel disease.Aliment Pharmacol Ther 52,1453-1468 (2020). 10. Aghazadeh,R.et al.Inflammatory bowel disease in Iran:a review of 457 cases.J Gastroenterol Hepatol 20,1691-1695 (2005). 11. Ceuleers,H.et al.Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome:The role of proteases.World J Gastroenterol 22,10275-10286 (2016). 12. Jones,R.C.W.,Xu,L.& Gebhart,G.F.The mechanosensitivity of mouse colon afferent fibers and their sensitization by inflammatory mediators require transient receptor potential vanilloid 1 and acid-sensing ion channel 3.J Neurosci 25,10981-10989 (2005). 13. Akbar,A.et al.Expression of the TRPV1 receptor differs in quiescent inflammatory bowel disease with or without abdominal pain.Gut 59,767-774 (2010). 14. Akbar,A.et al.Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain.Gut 57,923-929 (2008). 15. Miranda,A.et al.The role of transient receptor potential vanilloid 1 in mechanical and chemical visceral hyperalgesia following experimental colitis.Neuroscience 148,1021-1032 (2007). 16. Gottesman-Katz,L.,Latorre,R.,Vanner,S.,Schmidt,B.L.& Bunnett,N.W.Targeting G protein-coupled receptors for the treatment of chronic pain in the digestive system.Gut 70,970-981 (2021). 17. Sakamoto,M.& Benno,Y.Reclassification of Bacteroides distasonis,Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen.nov.,comb.nov.,Parabacteroides goldsteinii comb.nov.and Parabacteroides merdae comb.nov.International Journal of Systematic and Evolutionary Microbiology 56,1599-1605 (2006). 18. Hiippala,K.et al.Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp.from A Healthy Fecal Donor.Nutrients 12,935 (2020). 19. Wood,J.N.et al.Novel cell lines display properties of nociceptive sensory neurons.Proceedings of the Royal Society of London.Series B:Biological Sciences 241,187-194 (1990). 20. Haberberger,R.V.,Barry,C.& Matusica,D.Immortalized Dorsal Root Ganglion Neuron Cell Lines.Front.Cell.Neurosci.14,(2020). 21. Kobrinsky,E.M.,Pearson,H.A.& Dolphin,A.C.Low- and high-voltage-activated calcium channel currents and their modulation in the dorsal root ganglion cell line ND7-23.Neuroscience 58,539-552 (1994).

Claims

1. Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

2. The Parabacteroides distasonis strain according to claim 1, wherein the 16s rRNA gene sequence of the Parabacteroides distasonis strain is as set forth in SEQ ID NO:

1.

3. 3. The Parabacteroides distasonis strain according to claim 1, wherein the strain is isolated from the faeces of a healthy human.

4. 10. A dietary supplement composition comprising the bacterial strain of claim 1.

5. 5. The dietary supplement composition of claim 4, further comprising at least one acceptable nutritional ingredient, preferably selected from probiotics, prebiotics, synbiotics, parabiotics, metabiotics, vitamins, minerals, herbs, amino acids and enzymes.

6. A composition comprising the bacterial strain of claim 1 for use as a medicine.

7. 7. A composition for use according to claim 6 for use in the treatment and / or prevention of visceral pain.

8. 8. The composition for use according to claim 7, wherein the visceral pain is induced by inflammatory bowel disease, including Crohn's disease and ulcerative colitis, or by irritable bowel syndrome.

9. The composition comprises at least 10 ９ , preferably at least 10 １０ A dietary supplement composition according to any one of claims 4 to 5, or a composition for use according to any one of claims 6 to 8, comprising colony forming units / mL of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

10. A dietary supplement composition according to any one of claims 4 to 5 or a composition for use according to any one of claims 6 to 8, wherein the composition comprises only live bacteria.

11. A dietary supplement composition according to any one of claims 4 to 5, or a composition for use according to any one of claims 6 to 8, wherein the composition comprises only killed bacteria and / or fractions of bacteria.

12. 9. The composition for use according to any one of claims 6 to 8, further comprising at least one other acceptable active pharmaceutical ingredient, and / or at least one acceptable pharmaceutical excipient, and / or at least one acceptable pharmaceutical carrier.

13. The composition for use according to any one of claims 6 to 8, wherein the composition is for oral, intravenous, enteral or subcutaneous administration, preferably oral administration.

14. 14. The composition for use according to claim 13, wherein said oral administration is by gelatin capsule, capsule, tablet, powder, granule, oral solution or suspension.

15. Cells of the Parabacteroides distasonis strain deposited under accession number CNCM I-5828.

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