Feceobacter przewalskii strain CNCM I-4573 for treatment and prevention of clostridium difficile infection

Abstract

The present invention relates to a bacterial strain of the species Fecalibacterium prausnitzii, which has been preserved in CNCM with a preservation number CNCM I-4573, and / or a culture supernatant thereof, for use in the treatment and / or prevention of a Clostridium difficile infection in an individual. The present invention also relates to a method for producing the bacterial strain of the species Fecalibacterium prausnitzii, and to a method for producing the bacterial strain of the species Fecalibacterium prausnitzii, which has been preserved in CNCM with a preservation number CNCM I-4573.

Description

Technical Field

[0001] This invention relates to a strain of *Faecalibacterium prausnitzii* for the treatment and prevention of *Clostridium difficile* infection in individuals.

[0002] Existing technology

[0003] Clostridium difficile (also known as C. difficile, formerly known as Clostridium difficile) is an anaerobic, spore-forming, toxin-producing Gram-positive bacterium that spreads between humans via the fecal-oral route. Elderly and immunocompromised individuals, whose gut microbiota has been disrupted by antibiotic treatment, are at greatest risk of contracting this highly contagious, life-threatening, and potentially fatal diarrheal disease. With the emergence of highly virulent strains in the early 21st century, Clostridium difficile has become a major, globally distributed intestinal pathogen.

[0004] Even though Clostridium difficile infection can usually occur outside of a hospital, Clostridium difficile is considered a hospital-acquired pathogen.

[0005] However, the disease is now appearing more frequently in individuals previously considered low-risk. These cases tend to occur in younger individuals. Recent antibiotic exposure is a significant risk factor in this population, but the use of acid suppressants and comorbidities such as inflammatory bowel disease, chronic kidney disease, immunodeficiency diseases, malignancies, and solid organ transplantation are also associated with Clostridium difficile (CDI). In some rare cases, infection can even occur spontaneously in otherwise healthy individuals without antibiotic exposure.

[0006] Clostridium difficile accounts for 15-25% of antibiotic-associated bacterial diarrhea cases and is considered a leading cause of healthcare-associated diarrhea in adults. The risk of a first relapse after the initial episode is high and problematic (approximately 20%), and this risk increases further after the first relapse, reaching up to 60%. Symptoms associated with this infection can range from simple diarrhea to pseudomembranous colitis.

[0007] Clostridium difficile has therefore been classified by the European Centre for Disease Prevention and Control (CDC) as one of the top three urgent antibiotic resistance threats, and CDI has become the most common cause of antibiotic-associated diarrhea and gastroenteritis-related deaths in developed countries.

[0008] Currently, the standard treatment for CDI is antibiotics, with vancomycin or fidaxomicin being the most commonly prescribed compounds. Metronidazole is used only when vancomycin or fidaxomicin is unavailable. Fecal microbiota transplantation (FMT) is only used as a last resort after multiple relapses. Probiotics are also used as prophylactic treatment, but their effectiveness is mixed, and vaccines are not yet available. Within healthcare facilities, preventative measures include the cautious use of antibiotics and infection control measures. The use of monoclonal antibodies has also been investigated to enhance host resistance to CDI; however, this approach has yielded mixed results.

[0009] There is a need for novel substances, particularly probiotics, or compositions containing them, for the treatment and / or prevention of Clostridium difficile infection.

[0010] There is a particular need for novel substances, especially probiotics, or compositions containing them, that can reduce the risk of Clostridium difficile infection following disruption of the gut microbiota with antibiotics and / or proton pump inhibitors (PPIs).

[0011] There is a particular need for novel substances, especially probiotics, or compositions containing them, that can reduce the risk of recurrence of Clostridium difficile infection after a given outbreak.

[0012] There is a particular need for novel substances, especially probiotics, or compositions containing them, that can delay the intestinal colonization of Clostridium difficile.

[0013] There is also a need for new substances, particularly probiotics, or compositions containing them, that allow for the reduction of symptoms following Clostridium difficile infection, or even allow for the absence of symptoms following Clostridium difficile infection.

[0014] There is also a need for novel substances, particularly probiotics, or compositions containing them, that allow for limiting the intestinal colonization of said bacteria, more quickly eliminating Clostridium difficile from the gut, and advantageously reducing or even eliminating post-infection symptoms. Summary of the Invention

[0015] The object of this invention is to provide novel substances, particularly probiotics and compositions, for the treatment and / or prevention of Clostridium difficile infection in an individual.

[0016] In the context of this invention, the terms "prevent," "prevention," and "preventing" mean reducing the risk or likelihood of a given phenomenon occurring (i.e., in this invention, the development of Clostridium difficile infection, particularly post-C. difficile infection symptoms) to a lower level. Specifically, the terms "prevent," "prevention," and "preventing" mean reducing the risk or likelihood of the first episode of Clostridium difficile infection (CDI) in an individual to a lower level, and / or reducing the risk or likelihood of recurrence of CDI to a lower level. For the prevention of the first episode, the target population may specifically correspond to any patient for whom antibiotic treatment and / or treatment including the administration of a proton pump inhibitor (PPI) is prescribed, particularly any patient for whom antibiotic treatment is prescribed.

[0017] CDI relapse is defined as diarrhea recurrence within 8 weeks after completion of effective anti-C. difficile treatment and a positive Clostridium difficile toxin test.

[0018] As used herein, the terms “treating,” “treat,” or “treatment” include relieving and / or eliminating symptoms associated with a particular condition or symptom, as well as the complete disappearance of the condition or symptom in question.

[0019] This invention is based on the inventors' discovery of the ability of a strain of *Bacillus prednisei* deposited in CNCM with accession number CNCM I-4573 to prevent and / or treat *Clostridium difficile* infection in individuals.

[0020] According to the inventors' experimental results, a specific strain of *Clostridium difficile* (deposited in the CNCM on January 21, 2012 by the French National Institute for Agricultural Research (Institut National de la Recherche Agronomique, INRA) with accession number CNCM I-4573) has the unexpected ability to treat and prevent *Clostridium difficile* infection in individuals in vivo, particularly the unexpected ability to delay *Clostridium difficile* intestinal colonization, rapidly clear *Clostridium difficile* from the intestine, and reduce or even prevent the onset of any symptoms following *Clostridium difficile* infection.

[0021] According to the first subject matter, the present invention relates to bacterial strains of the species *Bacillus prednisei* deposited in CNCM with accession number CNCM I-4573 and / or its culture supernatant, for the treatment and / or prevention of *Clostridium difficile* infection in individuals.

[0022] Therefore, the strain identified by the inventors is a probiotic strain that can be used in the applications shown herein.

[0023] Individuals according to the invention may be selected from the group consisting of: humans; Suidae, especially pigs, and more especially piglets; Bovinae, especially cattle, and more especially bulls, and more especially steers; Canidae, especially dogs; Equidae, especially horses; and Phasianidae, especially poultry, and more especially chickens; preferably mammals, and more especially humans (J. Scott Weese; Journal of Veterinary Diagnostic Investigation 2020, Vol. 32(2) 213-221).

[0024] Bacterial strains of *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573 can be applied to the individuals in a live, inactive, and / or dead form, preferably in a live or inactive form, and more particularly in a live form.

[0025] Live bacterial forms refer to culturable and / or metabolically active bacterial strains, especially culturable and metabolically active bacterial strains.

[0026] The inactive form of the bacterial strain according to the invention can be, for example, a lyophilized product prepared using methods well known to those skilled in the art.

[0027] According to the implementation plan, the individual may develop Clostridium difficile infection after using at least one antibiotic and / or at least one proton pump inhibitor (PPI). The bacterial strain of *Propionibacterium frenulum* deposited at CNCM with accession number CNCM I-4573 and / or its culture supernatant can be administered simultaneously and / or separately (particularly simultaneously) with the at least one antibiotic and / or the at least one PPI. The bacterial strain of *Propionibacterium frenulum* deposited at CNCM with accession number CNCM I-4573 and / or its culture supernatant can be administered simultaneously with the at least one antibiotic and / or the at least one PPI, and / or after the at least one antibiotic and / or the at least one PPI.

[0028] The bacterial strains and / or their culture supernatants used according to the present invention may be contained in a composition containing a physiologically acceptable medium, preferably in an oral or rectal composition, and more particularly preferably in a pharmaceutical product.

[0029] The term "physiologically acceptable medium" is intended to refer to a medium that is compatible with the body of the individual to whom the composition must be administered. For example, it is a non-toxic solvent, such as water. In particular, the medium is compatible with oral administration.

[0030] The compositions of the present invention are preferably for oral or rectal administration, particularly for oral administration.

[0031] The oral composition of the present invention may be selected from the group consisting of: food, beverage, pharmaceutical, nutritional product, food additive, food supplement and dairy product, and particularly in the form of a pharmaceutical.

[0032] The composition according to the invention for the stated purpose may be suitable as a medicine equivalent to 10 4 Up to 10 15 Total Cell Count (TCC), more preferably 10 8 Up to 10 13 The daily dose of the *Proteus vulgaris* strain of the present invention administered by TCC, for example at a dose equivalent to 10... 13 TCC daily dose administration of the bacterial strain according to the present invention. Attached Figure Description

[0033] Figure 1 The assessment of weight loss in mice treated with or without *Clostridium difficile* I-4573 according to the invention is shown. Mice were weighed to monitor the development of clinical signs following *Clostridium difficile* infection. Two groups of mice were tested: mice treated with PBS-glycerol 16% (the medium); and mice treated with *Clostridium difficile* strain I-4573.

[0034] Horizontal axis: Days (D0 to D20, where D0 is the day the previously antibiotic-treated mice were fed Clostridium difficile R20291). Vertical axis: Mouse body weight (as a percentage of their body weight at D0).

[0035] Figure 2 The 7-day survival rate of mice treated with PBS-glycerol 16% (the medium) or with Bacillus prednisii strain I-4573 is shown.

[0036] Horizontal axis: Days (D0 to D7). Vertical axis: Probability of survival (0 to 1).

[0037] Figure 3 The colonization of Clostridium difficile R20291 in mice (10 mice per group) over time (D0 to D20—D0 being the day the mice previously treated with antibiotics were fed Clostridium difficile R20291).

[0038] The horizontal axis (arrows), from left to right: D0, D2, D3, D4, D5, D6, D8, D10, D14, and D20. The vertical axis: percentage of mice with detectable Clostridium difficile in their feces. Invention Details

[0040] The inventors have conducted in-depth research to identify the ability of specific strains of *Pseudomonas prednisone* to treat and / or prevent *Clostridium difficile* infection in individuals.

[0041] In fact, the inventors have unexpectedly determined that the strain of *Clostridium difficile* I-4573 has the ability to not only delay the intestinal colonization of *Clostridium difficile* in individuals, but also to rapidly clear *Clostridium difficile* from the intestines, and to significantly reduce symptoms following *Clostridium difficile* infection, particularly to prevent the occurrence of any symptoms following *Clostridium difficile* infection.

[0042] The strain of *Proteus vulgaris* of the present invention

[0043] Prevotella is a major member of the Firmicutes phylum and is one of the most abundant symbiotic bacteria in the large intestine microbiota of healthy individuals.

[0044] *Prevotella foetida* is an oxygen-sensitive (EOS) bacterium, making it difficult to culture, especially under anaerobic conditions (Duncan et al., 2002, Int. J. Syst. Evol. Microbiol. 52(Pt 6): 2141-6 and Lopez-Siles et al., Appl. Environ Microbiol. January 2012; 78 (2):420-8). *Prevotella foetida* is one of the most abundant butyrate-producing bacteria known in the human digestive tract, and short-chain butyrate fatty acids are crucial for intestinal physiology, systemic function, and beneficial effects on human health (Macfarlane and Macfarlane (2011), J. Clin. Gastroenterol. 45 Suppl: S120-7).

[0045] As a medicine, a suitable daily dose of the bacterial strain according to the present invention may be 10. 7 Up to 10 14 The present invention comprises a colony-forming unit (CFU) of the *Pleurotus erythrorhizon* strain.

[0046] As a medicine, the daily dosage can also range from 10. 4 Up to 10 15 Total Cell Count (TCC), more preferably 10 8 Up to 10 13The *Proteus vulgaris* strain of the present invention described in TCC, for example, at an amount equivalent to 10... 13 Daily dose of TCC.

[0047] The *Prevotella foetida* strain specifically implemented in this invention is a bacterial strain of the *Prevotella foetida* species deposited in CNCM with accession number CNCM I-4573.

[0048] As described above, the *Pleurotus ostreatus* strain and / or its culture supernatant of the present invention are used for the treatment and / or prevention of *Clostridium difficile* infection in individuals.

[0049] The *F. preibryophyte* bacterial strain of the present invention, deposited at CNCM with accession number I-4573, can exist in an isolated form. According to the present invention, "isolated form" means isolated from any fecal material or intestinal sample. Therefore, the bacterial strain of the present invention is preferably not administered to patients in the form of fecal microbiota transplantation (FMT). Methods for isolating bacteria from the gut microbiota are well known to those skilled in the art, for example, as described by Foditsch et al. (PLoS One. 2014; 9(12): e116465).

[0050] Individuals according to the invention can be selected from the group consisting of: humans; pigs, especially piglets; bovines, especially cattle, especially bulls, especially beef cattle; canines, especially dogs; equines, especially horses; and pheasants, especially poultry, especially chickens. In particular, individuals according to the invention are mammals, especially humans.

[0051] The strains described in this invention are probiotics whose activity is located in the gut. The probiotics according to this invention indicate that when a sufficient amount of the bacteria is ingested in live form, it can have a beneficial effect on an individual's health.

[0052] Therefore, bacterial strains of the *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573 can be applied to individuals in need in live, inactive, and / or dead forms, and preferably in live form to the intestine.

[0053] The bacterial strains described in this invention can be administered to the digestive tract of an individual to be treated in various ways (i.e., orally or rectally). The bacteria according to the invention are preferably administered orally.

[0054] According to a preferred embodiment, the bacterial strain of the present invention is contained in a composition containing a physiologically acceptable medium. Such a composition is preferably for oral or rectal administration, particularly oral administration, and especially for administration in the form of a pharmaceutical product.

[0055] Composition

[0056] The present invention also relates to compositions according to the invention for the stated purpose, namely compositions comprising at least the *Prevotella foetida* strain I-4573 described herein in a physiologically acceptable medium.

[0057] The compositions according to the invention for the stated purpose are intended for use in the digestive tract, particularly the intestines.

[0058] Therefore, the composition according to the present invention for the stated purpose can be selected from oral or rectal compositions. The compositions of the present invention are preferably oral or rectal compositions, more preferably oral compositions.

[0059] The composition used for the purposes described in this invention is an oral composition intended for oral administration to an individual.

[0060] The composition used for the purposes described in this invention is a rectal composition intended for rectal administration to an individual.

[0061] The oral composition may be in the form of suspension, tablet, pill, capsule, granule or powder.

[0062] The oral composition according to the invention for the stated purpose may be selected from the group consisting of: food, beverage, pharmaceutical, nutritional product, food additive, food supplement or dairy product, and particularly pharmaceutical.

[0063] According to a preferred embodiment, the composition according to the invention for the stated purpose is a pharmaceutical product.

[0064] Medications intended for oral administration may be in the form of capsules, gel capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes, lozenges, chewing gum, oral solutions or emulsions, syrups or gels.

[0065] For example, the medicine according to the invention for oral administration can be in capsules, particularly in the form of a lyophilized product, preferably together with a pharmaceutically acceptable excipient.

[0066] Advantageously, the compositions according to the invention for the stated purpose are intended for oral administration and may be provided with an anti-gastric acid coating to ensure that the bacterial strains of the invention contained in the composition can penetrate the damaged stomach. Therefore, the release of the bacterial strains can occur first in the upper intestine.

[0067] The pharmaceutical products according to the present invention for oral use may further comprise sweeteners, stabilizers, antioxidants, additives, flavoring agents and / or dyes.

[0068] Its formulation is carried out by means of commonly used methods for producing sugar-coated tablets, gel capsules, gels, controlled-release hydrogels, emulsions, tablets or capsules.

[0069] The composition for the stated purpose containing the bacterial strain described in this invention can be administered rectally.

[0070] Rectal administration can be in particular in the form of suppositories, enemas, or foams (especially enemas).

[0071] The compositions according to the present invention for the stated purpose may also be in the form of nutritional compositions.

[0072] The nutritional composition according to the invention for the stated purpose may be in the form of yogurt, cereal bars, breakfast cereals, desserts, frozen foods, soups, pet foods, liquid suspensions, powders, tablets, chewing gum, or candies.

[0073] In particular, the compositions of the present invention for the said uses can be adapted as pharmaceuticals at an amount equivalent to 10 7 Up to 10 14 The daily dose of colony-forming units (CFU) of the strain according to the invention is preferably equivalent to 10 11 Daily dose of CFU.

[0074] For example, the composition according to the invention for the stated purpose can be administered to an individual in need of it in a single daily dose of 1 g, containing the I-4573 bacterial strain described in this invention, the amount of which is equivalent to 10 7 Up to 10 14 CFU (preferably 10) 11 The dosage of CFU.

[0075] In another example, the composition according to the invention for the stated purpose can be administered to an individual in need of it at a single daily dose of 0.2 g, containing the I-4573 bacterial strain described in this invention, the amount of said bacterial strain equivalent to 10 7 Up to 10 14 CFU (preferably 10) 11 The amount of CFU.

[0076] As mentioned earlier, the daily dose of a drug can also range from 10. 4 Up to 10 15 Total Cell Count (TCC) (more preferably from 10) 8 Up to 10 13 The *Porphyromonas foetida* strain of the present invention (TCC), for example, at an amount equivalent to 10... 13 Daily dose of TCC.

[0077] The composition according to the invention for the stated purpose can be administered to the individual in need of it 1 to 10 times daily, wherein each dose contains the I-4573 bacterial strain of the invention, in an amount, for example, 5.10 g / dL. 8 Up to 5.10 11 TCC, which allows the total daily dose of the I-4573 bacterial strain described in this invention to be administered to the individual as described above.

[0078] Specifically, the composition according to the invention for the stated purpose can be administered to an individual for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 14 days, 15 days, 3 weeks, 1 month, or more than 1 month. When the composition according to the invention for the stated purpose is administered for more than 1 day, the same daily dose of the *Prevotella foetida* strain described in the invention can be administered daily, or the daily dose can vary with the duration of treatment. In particular, the daily dose of the *Prevotella foetida* strain described in the invention can be higher in the first few days of treatment and then gradually reduced.

[0079] The compositions according to the invention may further comprise at least one of the following: antioxidants, fish oil, DHA, EPA, vitamins, minerals, phytonutrients, proteins, lipids, probiotics, and combinations thereof.

[0080] The individual according to the invention can be accordingly:

[0081] - Individuals who will begin or have already begun antibiotic treatment; and / or

[0082] - Individuals who are at the end of antibiotic treatment or have just finished antibiotic treatment; and / or

[0083] - Individuals who will begin or have already begun proton pump inhibitor (PPI) therapy; and / or

[0084] - Individuals who are at the end of proton pump inhibitor (PPI) therapy or have just finished PPI therapy; and / or

[0085] - Individuals who have received treatment for a previous Clostridium difficile infection episode (e.g., the first episode of Clostridium difficile infection), such as with vancomycin and / or fendamycin; and / or

[0086] - Individuals experiencing their first episode or recurrent Clostridium difficile infection.

[0087] According to the invention, Clostridium difficile infection to be treated and / or prevented is particularly likely to occur after the individual in question has used at least one antibiotic and / or PPI. Therefore, the individual according to the invention is particularly likely to be an individual who is about to begin or has already begun antibiotic and / or PPI treatment or who suffers from dysbiosis.

[0088] Therefore, the bacterial strains, their culture supernatants, or compositions comprising them according to the present invention can be administered simultaneously and / or separately (particularly simultaneously) with at least one antibiotic (antibiotic) and / or at least one and / or PPI (probiotic) for antibiotic treatment, for example, separately on the same day, or even separately with at least one day or more between the administration of the bacterial strain and the administration of the antibiotic / PPI.

[0089] In a specific embodiment, the bacterial strain or its culture supernatant according to the present invention for the intended use is administered to the individual in the same composition as the at least one antibiotic and / or PPI.

[0090] In a specific embodiment, the bacterial strain or its culture supernatant according to the present invention for the intended use is administered to the individual in a separate composition with the at least one antibiotic and / or PPI.

[0091] When the bacterial strain or its culture supernatant used according to the present invention is administered in separate compositions with the at least one antibiotic and / or PPI, the compositions may be administered to the individual simultaneously or separately via the same route or via different routes.

[0092] The term "simultaneously" can be understood to mean that the bacterial strain, its culture supernatant, or composition containing it according to the present invention can be administered at the same time or on the same day or several days as the antibiotic and / or PPI.

[0093] The term "separate" can be understood as meaning that the bacterial strains, their culture supernatants, or compositions containing them according to the present invention can be applied at least a few days apart (e.g., at least three days apart).

[0094] The bacterial strains, their culture supernatants, or compositions comprising them according to the present invention for the purposes described herein can be administered simultaneously and / or separately with at least one antibiotic (antibiotic) and / or at least one PPI drug for antibiotic treatment. For example, the bacterial strains, their culture supernatants, or compositions comprising them according to the present invention for the purposes described herein can be administered simultaneously with the administration of the antibiotic / PPI drug, or can be administered after the antibiotic and / or PPI treatment has ended.

[0095] The dosage and frequency of administration of the bacterial strain or its culture supernatant used for the purposes according to the present invention can be adjusted based on the host response.

[0096] For illustrative purposes only, the frequency of application of the bacterial strains, their culture supernatants, or compositions comprising them according to the present invention for the stated purpose may be daily for 5 to 15 consecutive days. Alternatively, the application may be once every 2, 3, 4, 5, 6, or 7 days for a duration of several months.

[0097] In cases where an individual is about to begin or has already begun treatment as described above (particularly antibiotic and / or PPI treatment), the frequency of administration of the bacterial strain, its culture supernatant, or composition comprising it according to the invention for the purpose may be daily administration throughout the individual's treatment (particularly throughout the entire antibiotic and / or PPI treatment), and the administration of the bacterial strain, its culture supernatant, or composition comprising it according to the invention for the purpose:

[0098] - It can be started one day or a few days before the above treatments (especially antibiotics and / or PPI treatment); and / or

[0099] - It can be ended one or a few days after the above treatments (especially antibiotics and / or PPI treatment) are completed.

[0100] As is known in the art, adjustments can be made as needed for protein degradation, systemic delivery versus local delivery, as well as for age, weight, general health condition, sex, diet, timing of administration, drug interactions, and severity of illness, and these can be readily determined by those skilled in the art through routine experiments.

[0101] The antibiotic drug, or antibiotic and / or related PPI drug according to the present invention, can be any drug that alters, inhibits, or disrupts the growth of the gut microbiota.

[0102] In a non-restrictive manner, such antibiotics may be selected from, for example, the group consisting of: penicillins, tetracyclines, cephalosporins, quinolones, lincomycin, macrolides, sulfonamides, glycopeptides, aminoglycosides, carbapenems, and mixtures thereof, and particularly from the group consisting of: amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole and trimethoprim, clavulanic acid, ofloxacin, levofloxacin, and mixtures thereof.

[0103] For example, antibiotics can be selected from the group consisting of: amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole and trimethoprim, clavulanic acid, levofloxacin, vancomycin, fendamycin, metronidazole, kanamycin, gentamicin, colistin, clindamycin and mixtures thereof.

[0104] In a non-restrictive manner, such a PPI may be selected, for example, from the group consisting of: omeprazole, lansoprazole, pantoprazole, rabeprazole, esomeprazole, and dexlansoprazole.

[0105] This invention also relates to the following:

[0106] - Bacterial strains of *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573; and / or

[0107] - its culture supernatant; and / or

[0108] - A composition containing bacterial strains or their culture supernatant in a physiologically acceptable medium.

[0109] Use for the treatment and / or prevention of Clostridium difficile infection in an individual.

[0110] The present invention also relates to a method for treating and / or preventing Clostridium difficile infection in an individual, comprising administering to said individual:

[0111] - Bacterial strains of *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573; and / or

[0112] - its culture supernatant; and / or

[0113] - A composition containing bacterial strains or their culture supernatant in a physiologically acceptable medium.

[0114] This invention also relates to the following:

[0115] - Bacterial strains of *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573; and / or

[0116] - its culture supernatant; and / or

[0117] - A composition containing bacterial strains or their culture supernatant in a physiologically acceptable medium.

[0118] Used for the manufacture of medicines for the treatment and / or prevention of Clostridium difficile infection in an individual.

[0119] The present invention will now be described in more detail through the following embodiments, which are given by way of illustration only.

[0120] Unless otherwise stated, all percentages refer to weight percentages. Example

[0121] The ability of the *Pleurotus erythrophagus* strain according to the present invention, deposited in CNCM with accession number CNCM I-4573, to regulate the intestinal colonization of *Clostridium difficile* in vitro and in vivo, particularly in mice exhibiting induced dysbiosis.

[0122] Twenty 6-week-old female conventional C57 Black 6 (C57BL / 6) mice were identified and housed in isolators with sterile food and water to protect them from external environmental influences. The mice were divided into two groups:

[0123] -10 mice were treated with PBS-glycerol 16% (the medium); and

[0124] -10 mice treated with Bacillus prenanti strain CNCM I-4573.

[0125] To induce gut microbiota dysbiosis, all mice were given an antibiotic mixture in their drinking water for 6 days prior to infection (D-6) and 3 days prior to infection (D-6 to D-3). This antibiotic mixture consisted of 0.4 mg / mL kanamycin, 0.035 mg / mL gentamicin, 0.0567 mg / mL colistin, 0.215 mg / mL metronidazole, and 0.045 mg / mL vancomycin. On D-1, all mice were given a single 200 μL intraperitoneal (IP) dose of clindamycin at a dose of 50 mg / kg.

[0126] From D-6 to D+14, for each group, mice were fed 200 μL of the bacterial strain corresponding to their group once daily. At D0, all mice were given 500 μL of a 2×10⁻⁶ strain. 5 Clostridium difficile strain R20291 (DSM 27147) was administered via gavage for infection. For this purpose, Clostridium difficile strain R20291 (DSM 27147) was pre-cultured the day before, and Gram staining was performed the following morning to verify the purity of the suspension. The culture was then diluted with 27 mL of BHI and 3 mL of the pre-culture. After approximately 7 hours of anaerobic culture, the suspension was counted and adjusted to 2 × 10⁻⁶ CFU / mL in 50 mL of regenerated 1X PBS. 5 bacteria / mL. Mice were weighed daily for 8 days after infection with Clostridium difficile, and then weighed on days 10, 14, and 20.

[0127] To monitor intestinal colonization by Clostridium difficile, feces from mice were collected on days 1, 2, 3, 5, 8, 12, 14, and 20 and resuspended in PBS to obtain a final concentration of 10 mg / mL. The suspension was then diluted to 10 μL. -1 10 -2 and 10-3 The solution was spread onto BHI agar + 3% blood + Clostridium difficile selective supplement (Biomérieux®) and incubated at 37°C under anaerobic conditions for 48 hours. The undiluted suspension was also spread under the same conditions.

[0128] result

[0129] Mice were given a mixture of antibiotics to induce dysbiosis, allowing Clostridium difficile R20291 (DSM 27147) to better colonize the gut. Mice were fed either Bacillus prednisone propionibacterium CNCM I-4573 or PBS-glycerol 16% (the medium) each day of the experiment (D-6 to D-14). Clinical scores (weight loss and decreased activity, diarrhea, mortality) were assessed after infection with the toxin-producing Clostridium difficile strain R20291 (DSM 27147), and colonization was monitored by counting bacteria in feces.

[0130] 1) Clinical assessment

[0131] Assessment of clinical scores ( Figure 1 No weight loss, reduced activity, or diarrhea was observed in mice treated with the *Proteus vulgaris* strain described in this invention.

[0132] On the other hand, in the corresponding control group (PBS-mediated mice), weight loss was observed starting from day 2, followed by recovery to normal weight, reduced activity, and diarrhea on day 6 post-infection.

[0133] Monitor the survival rate of animals after being challenged. For example... Figure 2 As shown, after infection with Clostridium difficile, two mice in the PBS-glycerol 16% group (control-media) died, and one mouse treated with Bacillus prednisone CNCM I-4573 died 5 days after infection.

[0134] 2) Monitoring planting

[0135] For monitoring the colonization of Clostridium difficile in mice ( Figure 3 All mice were colonized by Clostridium difficile on day 4.

[0136] Mice in the *Proteus vulgaris* strain group described in this invention colonized well, but they did not appear to show symptoms. Colonization was delayed compared to the control group (vector group), and *Clostridium difficile* was eliminated more quickly (starting from D6, compared to D14 in the control group). At D10, mice were still colonized by *Clostridium difficile*. Four out of four mice treated with PBS-glycerol 16% (vector control group) were colonized by *Clostridium difficile*, while only one out of six mice in the *Proteus vulgaris* strain treatment group described in this invention was colonized by *Clostridium difficile*.

[0137] Treatment based on the application of the *Pleurotus erythrorhizium* strain described in this invention unexpectedly demonstrated the ability to provide protection against *Clostridium difficile* infection. Although all mice were colonized, they did not exhibit clinical symptoms (stable weight, normal feces, and normal activity). Colonization was delayed, and surprisingly, faster elimination of *Clostridium difficile* was observed.

Claims

1. Faecalibacterium prausnitidum, deposited in CNCM with accession number CNCM I-4573. z ii) Bacterial strains of the species and / or their culture supernatants, used for the treatment and / or prevention of Clostridium difficile infection in an individual.

2. The bacterial strain and / or its culture supernatant for use according to claim 1, characterized in that, The individuals are selected from the group consisting of: humans; Suidae, especially pigs, and even more so piglets; Bovinae, especially cattle, and even more so bulls, and even more so beef cattle; Canidae, especially dogs; Equidae, especially horses; and Phasianidae, especially poultry, and even more so chickens.

3. The bacterial strain and / or its culture supernatant for use according to claim 1 or 2, characterized in that, The individuals in question are mammals, and more specifically, humans.

4. The bacterial strain and / or its culture supernatant according to any one of claims 1 to 3, characterized in that, The bacterial strain of *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573 is applied to the individual in a live, inactive, and / or dead form, preferably in a live or inactive form, and more particularly in a live form.

5. The bacterial strain and / or its culture supernatant according to any one of claims 1 to 4, characterized in that, The Clostridium difficile infection occurred after the individual used at least one antibiotic and / or at least one proton pump inhibitor (PPI).

6. The bacterial strain and / or its culture supernatant according to any one of claims 1 to 5, characterized in that, The bacterial strain of *Proteus vulgaris* species deposited in CNCM with accession number CNCM I-4573 and / or its culture supernatant, shall be administered simultaneously with the at least one antibiotic and / or the at least one PPI, and / or after the at least one antibiotic and / or the at least one PPI.

7. The bacterial strain and / or its culture supernatant for use according to any one of claims 1 to 6, wherein the bacterial strain of *Proteus vulgaris* species deposited at CNCM with accession number CNCMI-4573 is contained in a composition containing a physiologically acceptable medium.

8. The bacterial strain and / or its culture supernatant for use according to claim 7, characterized in that, The bacterial strain of *Proteus vulgaris* species deposited at CNCM with accession number CNCMI-4573 is included in the oral composition or rectal composition, particularly in the oral composition.

9. The bacterial strain and / or its culture supernatant for use according to claim 7 or 8, characterized in that, The bacterial strain is contained in the drug.

10. The bacterial strain and / or its culture supernatant according to any one of claims 7 to 9, characterized in that, The composition is suitable as a medicine in an amount equivalent to 10 4 Up to 10 15 The daily dose of bacterial strains with a total cell count (TCC) is preferably administered at an amount equivalent to 10. 8 Up to 10 13 TCC, and more specifically equivalent to 10 13 The form of daily doses of TCC.

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