EA036958B1 - Composition comprising blautia bacterial strains for reducing the level of pathogenic enterobacteriaceae in the gastrointestinal tract - Google Patents

Abstract

The invention provides the use of a pharmaceutical composition comprising a bacterial strain of the genus Blautia having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 5 for reducing the level of pathogenic Enterobacteriaceae in the gastrointestinal tract of a patient.

Description

Technology area

This invention relates to the field of compositions containing bacterial strains isolated from the digestive tract of mammals, and the use of such compositions for the treatment of disease.

Prior art

The human intestine is believed to be sterile in utero, but immediately after birth it is exposed to a large number of microorganisms from the mother and from the environment. This is followed by a dynamic period of microbial colonization and succession, depending on factors such as mode of birth, environment, nutrition, and host genotype, which collectively affect the composition of the gut microbiota, especially during early life. Subsequently, the microbiota is stabilized and becomes similar to the microbiota of adults [1]. The human intestinal microbiota contains more than 1500 different phylotypes, among which two main bacterial types (phyla), Bacteroidetes and Firmicutes, are quantitatively dominant [2-3]. Successful symbiotic relationships resulting from bacterial colonization of the human intestine have led to the development of a wide range of metabolic, structural, protective and other beneficial functions. The increased metabolic activity of the colonized intestine allows for the degradation of otherwise indigestible food components to form by-products that are an important source of nutrients for the host and provide additional health benefits. Similarly, the immunological importance of the gut microbiota is generally recognized and has been confirmed in microbial-free animals with disorders of the immune system, which is functionally restored after the introduction of commensal bacteria [4-6].

Significant changes in microbiota composition have been documented in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, the levels of Clostridium cluster XIVa and Clostridium cluster XI (F. prausnitzii) in IBD patients are decreased, while the number of E. coli increases, suggesting a shift in the balance between symbionts and pathobionts in the intestine [7-11].

In recognition of the potential beneficial effect that certain bacterial strains can have on the intestines of an animal, different strains have been proposed for use in the treatment of various diseases (see, for example, [12-15]). A number of strains, including mainly the Lactobacillus and Bifidobacterium strains, have been proposed for use in the treatment of various intestinal disorders (see review [16]). Strains of the genus Blautia have also been proposed for use in modulating the bacterial balance of the digestive ecosystem in IBS patients (WO 01/85187). However, the relationships between different bacterial strains and different diseases and the exact effects of specific bacterial strains on the gut and at the systemic level and on any specific types of diseases are poorly understood.

There is a need to study the potential effects of gut bacteria in order to develop new therapies that use gut bacteria.

US 2010/0247489 describes the use of mineral nutrients for the treatment of digestive disorders. US '789 also suggests the possible use of numerous different genera of bacteria, including Enterobacteriaceae, to prevent and reduce the formation of gas in the colon, thus this document suggests that an increase in the number of Enterobacteriaceae spp. in the gastrointestinal tract can be used to treat certain diseases. US '789 does not describe any way to reduce the number of Enterobacteriaceae spp.

WO 2016/086206 suggests that bacteria of the order Clostridiales, including Enterobacteriaceace spp., Can be used to treat or prevent dysbiosis. WO 2016/086206 does not suggest that a decrease in the level of Enterobacteriaceace spp. in the gastrointestinal tract can be used to treat disease, no suggestion is made as to how the level of Enterbacteriaceace spp. can be reduced.

WO 2012/142605 suggests the possibility of treating a number of different diseases with a combination of microorganisms. WO '605 suggests the possible use of a large number of bacterial species, but WO' 605 does not describe how any of the proposed bacterial species can be used to treat any of these diseases.

The essence of the invention

The inventors have developed new therapies for the treatment and prevention of diseases associated with Enterobacteriaceae, in particular E. coli. In particular, the inventors have determined that bacterial strains from the genus Blautia can be effective in reducing the level of Enterobacteriaceae in the gastrointestinal tract. As described in the examples, oral administration of compositions containing Blautia hydrogenotrophica can reduce the level of Enterobacteriaceae in the gastrointestinal tract, including E. coli, and can cure or prevent diseases associated with Enterobacteriaceae or E. coli. Thus, in a first embodiment, the invention provides a composition comprising a bacterial strain of the genus Blautia for use in a method for reducing the level of Enterobacteriaceae in the gastrointestinal tract.

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In preferred embodiments of all aspects of the invention, the Enterobacteriaceae are E. coli. In preferred embodiments of all aspects of the invention, the bacterial strain belongs to Blautia hydrogenotrophica and is preferably a bacterium deposited with DSM accession number 10507/14294.

In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Blautia for use in a method for treating or preventing a disease associated with an Enterobacteriaceae infection, such as E. coli infection. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of diarrhea, gastroenteritis, urinary tract infection, or neonatal meningitis. In certain embodiments, the compositions of the invention are for use in treating or preventing an infection with Enterobacteriaceae, such as E. coli infection.

In further preferred embodiments, the compositions of the invention are intended for use to reduce the level of Enterobacteriaceae in the gastrointestinal tract, preferably E. coli, in the treatment or prevention of IBS, Crohn's disease, ulcerative colitis, functional dyspepsia, diarrhea, gastroenteritis, urinary tract infection or meningitis of the newborn.

The inventors have developed new therapies for the treatment and prevention of diarrhea. In particular, the inventors have determined that bacterial strains from the genus Blautia can effectively reduce diarrhea. As described in the examples, oral administration of compositions containing Blautia hydrogenotrophica can reduce diarrhea in patients with irritable bowel syndrome (IBS).

In preferred embodiments of the invention, the bacterial strain in the composition belongs to Blautia hydrogenotrophica. Closely related strains may also be used, such as bacterial strains having a 16s rRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to the 16s rRNA sequence of the bacterial strain Blautia hydrogenotrophica. Preferably, the bacterial strain has a 16s rRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 5. Most preferably, the bacterial strain in the composition is Blautia hydrogenotrophica strain deposited with DSM access number 10507/14294.

In additional embodiments of the invention, the bacterial strain in the composition belongs to Blautia stercoris. Closely related strains can also be used, such as bacterial strains having a 16s rRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to the 16s pRNA sequence of the bacterial strain Blautia stercoris. Preferably, the bacterial strain has a 16s pRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 1 or 3. Preferably, sequence identity is determined with respect to SEQ ID NO: 3. Preferably, the bacterial strain for use in the invention has the 16s pRNA sequence represented by SEQ ID NO: 3.

In additional embodiments of the invention, the bacterial strain in the composition belongs to Blautia wexlerae. Closely related strains can also be used, such as bacterial strains having a 16s rRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to the 16s rRNA sequence of the Blautia wexlerae bacterial strain. Preferably, the bacterial strain has a 16s pRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 2 or 4. Preferably, sequence identity is determined with respect to SEQ ID NO: 4. Preferably, the bacterial strain for use in the invention has the 16s pRNA sequence represented by SEQ ID NO: 4.

In certain embodiments, a composition of the invention is for oral administration. Oral administration of the strains of the invention can effectively reduce the level of Enterobacteriaceae in the gastrointestinal tract. Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or complete colonization of the intestine.

In certain embodiments, a composition of the invention comprises one or more pharmaceutically acceptable excipients or carriers.

In certain embodiments, a composition of the invention comprises a lyophilized bacterial strain. Lyophilization is an efficient and convenient technique for the preparation of stable compositions that allow delivery of bacteria, and has demonstrated the possibility of obtaining effective compositions in the examples.

In certain embodiments, the invention provides a food product containing the composition described above.

In certain embodiments, the invention provides a vaccine composition comprising a composition as described above.

Additionally, the invention provides a method for reducing the level of Enterobacteriaceae in the gastrointestinal tract and thereby treating or preventing diseases associated with Enterobacteriaceae, comprising administering a composition containing a bacterial strain of the genus Blautia.

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Brief description of graphic materials

FIG. 1. Measurement of the BH population by qPCR (quantitative PCR) showing an increase in BH on days 14 and 28 for HMI-CPK rats (inoculated with human intestinal microbiota with IBS) treated with BH lyophilisate.

FIG. 2. Dosing studies in HIM rats (inoculated with human intestinal microbiota) - quantitative determination of B. hydrogenotrophica by RT-PCR in fecal samples from healthy HIM rats (inoculated with healthy human intestinal microbiota) treated with different concentrations of bacterial species.

FIG. 3. Time of passage of B. hydrogenotrophica after oral administration (10 ⁹ / day) in healthy human HIM rats.

FIG. 4. Comparison of levels of B. hydrogenotrophica present in fecal and cecal samples from HIM-healthy rats (quantified by RT-PCR) after 14 days of administration - B. hydrogenotrophica was administered at 10 ¹⁰ / day / rat.

FIG. 5. Effect of B. hydrogenotrophica administration on microbiota composition in feces of rats associated with the microbiota of a healthy person. Administration of 10 ¹⁰ B. hydrogenotrophica / day for 14 days (results for fecal microbiota from 2 healthy individuals (20 HIM rats) - qPCR analysis).

FIG. 6. Effect of B. hydrogenotrophica administration on microbial populations of HIM-CPK rats.

FIG. 7a. Changes in patient microbiota levels of Blautia hydrogenotrophica during and after treatment with Blautix.

FIG. 7b. Changes in patient microbiota levels of Enterobacteria during and after treatment with Blautix.

FIG. 8. Results of C _max determination by the hydrogen breath test for day 1, day 2, day 15 and day 16 for IBS patients treated with Blautix (FIG. 8a) and IBS patients treated with placebo (FIG. 8b); fig. 8c is a graph comparing the percentage of Blautix treated patients with a decrease in hydrogen between the mean for days 15 and 16 and the mean for days 1 and 2, with the percentage of patients with a decrease in hydrogen between these time points receiving placebo.

FIG. 9a: Paired data from unadjusted and corrected hydrogen breath tests on Day 1 and Day 15 for IBS patients treated with Blautix; fig. 9b: Graph comparing the mean uncorrected hydrogen breath test results on Day 1 and Day 15 for the Blautix treatment group; fig. 9c: Graph comparing the mean corrected hydrogen breath test results on Day 1 and Day 15 for the Blautix treatment group.

FIG. 10a: Paired data from unadjusted and corrected hydrogen breath tests on Day 1 and Day 15 for IBS patients receiving placebo; fig. 10b: Graph comparing the mean uncorrected hydrogen breath test results on Day 1 and Day 15 for the placebo group; fig. 10c: Graph comparing the adjusted mean adjusted hydrogen breath test results on Day 1 and Day 15 for the placebo group.

FIG. 11. Graphs comparing the average results of the hydrogen breath test on Day 1 and Day 15 for the Bautix treatment group (Verum (drug group with active substances)) and placebo group (Fig. 11a: uncorrected hydrogen level; Fig. 11b: corrected level hydrogen).

FIG. 12. Evaluation of the population of B. hydrogenotrophica by qPCR in fecal samples from HMASPK rats (associated with human microbiota, IBS patient), receiving or not receiving the composition containing B. Hydrogenotrophica (BlautiX) for 28 days.

FIG. 13. Bacterial counts in fecal samples from HMA-CPK rats after administration of B. hydrogenotrophica (BlautiX) versus control solution.

FIG. 14. Changes in patient symptoms during the dosing period (days 1-16) of phase I clinical trials.

FIG. 15. Changes in patients' symptoms during the washout period of phase I clinical trials.

Disclosure of invention

Bacterial strains

The compositions according to the invention contain a bacterial strain of the genus Blautia. The examples demonstrate that bacteria of this genus are useful in reducing the level of Enterobacteriaceae in the gastrointestinal tract, in particular E. coli. Preferred bacterial strains belong to the species Blautia hydrogenotrophica, Blautia stercoris and Blautia wexlerae. Other preferred bacterial strains for use in the invention are Blautia producta, Blautia coccoides and Blautia hansenii. Most preferred are Blautia hydrogenotrophica, especially a bacterium deposited with DSM accession number 10507/14294.

Examples of Blautia strains for use in the invention include Blautia hydrogenotrophica, B. stercoris, B. faecis, B. coccoides, B. glucerasea, B. hansenii, B. luti, B. producta, B. schinkii and B. wexlerae. Blautia spp. Are gram-positive immotile bacteria that can be coccoid or oval, and all are obligate anaerobes that produce acetic acid

- 3 036958 as the main end product of glucose fermentation [17]. Blautia can be isolated from the human intestine, although B. producta was isolated from a septic sample.

Blautia hydrogenotrophica (formerly Ruminococcus hydrogenotrophicus) was isolated from the intestines of mammals, is strictly anaerobic and metabolizes H2 / CO2 to acetate, which may be important for human nutrition and health. A typical Blautia hydrogenotrophica strain is S5a33 = DSM 10507 = JCM 14656. The 16S rRNA gene sequence of Blautia hydrogenotrophica strain S5a36 has GenBank accession number X95624.1 (cited herein as SEQ ID NO: 5). This typical Blautia hydrogenotrophica strain is described in [17] and [18]. Strain S5a33 and strain S5a36 correspond to two subclones of a strain isolated from a fecal sample from a healthy subject. They show the same morphology, physiology and metabolism and have identical 16s rRNA sequences. Thus, in some embodiments, Blautia hydrogenotrophica for use in the invention has a 16s rRNA sequence in accordance with SEQ ID NO: 5.

The bacteria Blautia hydrogenotrophica deposited with accession number DSM 10507 as well as accession number DSM 14294 has been tested in the examples and is also referred to herein as strain BH. The BH strain was deposited in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany) on January 26, 1996 as Ruminococcus hydrogenotrophicus with accession number DSMZ 10507, and 1429 with accession number DSMZ as S5a33 on 14 May 2001. The depositary was INRA Laboratoire de Microbiologie CR de Clermont-Ferrand / Theix 63122 Saint Genes Champanelle, France. Ownership of the deposited materials passed to 4D Pharma Plc by assignment.

The 16S rRNA gene sequence of Blautia stercoris strain GAM6-1T has GenBank accession number HM626177 (referred to herein as SEQ ID NO: 1). A typical example of the Blautia stercoris strain is described in [19]. A typical Blautia wexlerae strain is WAL 14507 = ATCC BAA-1564 = DSM 19850 [17]. The 16S rRNA gene sequence of Blautia wexlerae strain WAL 14507 T has GenBank accession number EF036467 (referred to herein as SEQ ID NO: 2). This typical Blautia wexlerae strain is described in [17].

The preferred Blautia stercoris strain is the strain deposited with accession number NCIMB 42381, which is also referred to herein as strain 830. The 16s rRNA sequence of strain 830 is shown as SEQ ID NO: 3. Strain 830 was deposited with the international depository NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) March 12, 2015 as Blautia stercoris 830, and was assigned accession number NCIMB 42381. GT Biologics Ltd. subsequently changed its name to 4D Pharma Research Limited.

A preferred Blautia wexlerae strain is the strain deposited with accession number NCIMB 42486, which is also referred to herein as strain MRX008. The 16s rRNA sequence of the MRX008 strain is shown as SEQ ID NO: 4. The MRX008 strain was deposited with the international depository NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) November 16, 2015 as Blaqutia / Ruminococcus MRx0008 and assigned accession number NCIMB 42486.

It is expected that bacterial strains closely related to the strain tested in the examples will also effectively reduce the level of Enterobacteriaceae in the gastrointestinal tract, in particular E. coli. In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5, or 99.9% identical to the 16s rRNA sequence of the Blautia hydrogenotrophica bacterial strain.

Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 5.

In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5, or 99.9% identical to the 16s rRNA sequence of the Blautia stercoris bacterial strain. Preferably, the bacterial strain for use in the invention has a 16s pRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 1 or SEQ ID NO: 3. Preferably sequence identity is defined with respect to SEQ ID NO: 3. Preferably, the bacterial strain for use in the invention has a 16s pRNA sequence set forth in SEQ ID NO: 3. In certain embodiments, the bacterial strain for use in the invention has a 16s pRNA sequence of at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to the 16s rRNA sequence of the bacterial strain Blautia wexlerae. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, sequence identity determined with respect to SEQ ID NO: 4. Preferably, the bacterial strain for use in the invention has the 16s pRNA sequence shown by SEQ ID NO: 4.

Bacterial strains that are biotypes of bacteria deposited with accession number

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DSM 10507/14294, or biotypes of bacteria deposited with accession numbers NCIMB 42381 and

NCIMB 42486 is also expected to effectively reduce the level of Enterobacteriaceae in the gastrointestinal tract, in particular E. coli. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.

Strains that are biotypes of the bacterium deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and are suitable for use in the invention can be identified by sequencing other nucleotide sequences of the bacterium deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486. For example, a substantially complete genome can be sequenced and a biotype strain for use in the invention can have at least 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity of at least in 80% of its entire genome (for example, at least 85, 90, 95 or 99% or in its entire genome). For example, in some embodiments, a biotype strain has at least 98% sequence identity in at least 98% of its genome, or at least 99% sequence identity in 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or repeated sequences such as BOX, ERIC, (GTG) ₅ , or REP or [20]. Strains of the biotype may have sequences with at least 95, 96, 97, 98, 99, 99.5 or 99.9% sequence identity with the corresponding sequence of the bacterium deposited with DSM accession number 10507/14294, NCIMB 42381 or NCIMB 42486. B In some embodiments, the biotype strain has a sequence with at least 97, 98, 99, 99.5, or 99.9% sequence identity with the corresponding sequence of the Blautia hydrogenotrophica strain deposited as DSM 10507/14294 and contains a 16s pRNA sequence that is at least at least 99% identical (e.g., at least 99.5%, or at least 99.9% identical) to SEQ ID NO: 5. In some embodiments, the biotype strain has a sequence of at least 97, 98, 99 , 99.5 or 99.9% sequence identity with the corresponding sequence of the Blautia hydrogenotrophica strain deposited as DSM 10507/14294 and has the 16s rRNA sequence according to SEQ ID NO: 5.

Alternatively, strains that are biotypes of bacteria deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and are suitable for use in the invention can be identified using deposit accession number DSM 10507/14294, deposit accession number NCIMB 42381 or deposit with accession number NCIMB 42486 and performing restriction fragment analysis and / or PCR analysis, for example, by methods of fluorescence analysis of amplified fragment length polymorphism (FAFLP) and fingerprinting using PCR amplification of repeated sequences of intergenic palindromes (repetitive DNAelement (repinting) -PCR or protein profiling, or partial 16S or 23s rDNA sequencing. In preferred embodiments, such techniques can be used to identify other strains of Blautia hydrogenotrophica, Blautia stercoris, or Blautia wexlerae.

In certain embodiments, strains that are biotypes of a bacterium deposited with accession number DSM 10507/14294, NCIMB 42381, or NCIMB 42486 and suitable for use in the invention are strains that provide the same pattern as the bacterium deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486, when analyzing by restriction analysis of amplified ribosomal DNA (ARDRA), for example using the restriction enzyme Sau3AI (examples of methods and guidance are given, for example, in [21]). Alternatively, strains of the biotype are identified as having the same carbohydrate fermentation pattern as the bacterium deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486.

Other Blautia strains suitable for use in the compositions and methods of the invention, such as biotypes of bacteria deposited with accession number DSM 10507/14294, NCIMB 42381, or NCIMB 42486, can be identified using any suitable method or strategy, including the assays described in examples. For example, strains for use in the invention can be identified by culturing bacteria and administering to rats for testing by a distension assay. In particular, bacterial strains having similar growth characteristics, metabolic patterns and / or surface antigens to bacteria deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 may be suitable for use in the invention. A useful strain will have comparable microbiota modulating activity with DSM 10507/14294, NCIMB 42381, or NCIMB 42486. In particular, a biotype strain will produce effects on Enterobacteriaceae comparable to those shown in the examples, which can be identified using culture and administration protocols described in the examples.

A particularly preferred strain according to the invention is the Blautia hydrogenotrophica strain deposited with accession number DSM 10507/14294. It is a typical BH strain that

- 5,036958 was used for testing in the examples and demonstrated an effective reduction in the level of Enterobacteriaceae in the gastrointestinal tract, in particular E. coli. Thus, the invention provides a cell, such as an isolated cell, of the Blautia hydrogenotrophica strain deposited with accession number DSM 10507/14294, or a derivative thereof, for use in therapy, in particular in the diseases described herein.

Derivatives of the strain deposited with accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 may be a daughter strain (progeny) or a strain grown (subcloned) from the parent strain. The derived strain according to the invention can be modified, for example, at the genetic level, without loss of biological activity. In particular, the derived strain according to the invention is therapeutically active. The derived strain will have a microbiota modulating activity comparable to the original strain DSM 10507/14294, NCIMB 42381 or NCIMB 42486. In particular, the derived strain will produce effects on Enterobacteriaceae comparable to those shown in the examples, which can be identified using the protocols cultivation and introduction described in the examples. The derivative DSM 10507/14294 will usually be the biotype of strain DSM 10507/14294. Derivative NCIMB 42381 will usually be the biotype of strain NCIMB 42381. Derivative NCIMB 42486 will usually be the biotype of strain NCIMB 42486.

References to cells of the Blautia hydrogenotrophica strain deposited with accession number DSM 10507/14294 encompass any cells having the same safety and therapeutic efficacy characteristics as strains deposited with accession number DSM 10507/14294, and such cells are within the scope of the invention. References to cells of the Blautia stercoris strain deposited with accession number NCIMB 42381 encompass any cells having the same safety and therapeutic efficacy characteristics as strains deposited with accession number NCIMB 42381, and such cells are within the scope of the invention. References to cells of the Blautia wexlerae strain deposited with accession number NCIMB 42486 encompass any cells having the same safety and therapeutic efficacy characteristics as strains deposited with accession number NCIMB 42486, and such cells are within the scope of the invention.

In preferred embodiments, the bacterial strains in the compositions of the invention are viable and capable of partially or completely colonizing the intestine.

Therapeutic Uses

In certain embodiments, the compositions of the invention are intended for use to reduce the level of Enterobacteriaceae in the gastrointestinal tract. Increased levels of Enterobacteriaceae in the gastrointestinal tract are associated with numerous pathological conditions and diseases, and the examples demonstrate that the compositions of the invention can effectively reduce the level of Enterobacteriaceae in the gastrointestinal tract.

In preferred embodiments of all aspects of the invention, the Enterobacteriaceae is E. coli.

Thus, preferably, the compositions of the invention are intended for use to reduce the level of E. coli in the gastrointestinal tract.

In certain embodiments, Enterobacteriaceae is a pathogenic strain such as E. coli O157: H7, O104: H4, O121, O26, O103, O111, O145, O104: H21, or O104: H4.

In alternative embodiments, Enterobacteriaceae is a commensal or non-pathogenic strain. Elevated levels of such Enterobacteriaceae can contribute to conditions such as IBS and Crohn's disease, and the examples demonstrate that compositions of the invention can have the effect of reducing the number of Enterobacteriaceae in the context of IBS.

In certain embodiments, the compositions of the invention are for use in the treatment or prevention of a disease associated with an Enterobacteriaceae infection, such as E. coli infection. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of diarrhea, gastroenteritis, urinary tract infection, or neonatal meningitis. In such embodiments, the Enterobacteriaceae may be a pathogenic strain.

In certain embodiments, compositions of the invention are for use in the treatment or prevention of a disease associated with elevated levels of Enterobacteriaceae, such as E. coli. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of diarrhea, gastroenteritis, urinary tract infection, or neonatal meningitis. In such embodiments, the Enterobacteriaceae may be a commensal or non-pathogenic strain.

In some embodiments, the pathogenesis of the disease or condition involves the intestine. In some embodiments, the pathogenesis of the disease or condition does not involve the intestine. In some embodiments, the pathogenesis of the disease or condition is not localized to the intestine. In some embodiments, treatment or prevention is performed at a site other than the intestine. In some embodiments, the treatment or prevention is performed in the intestine as well as at a site away from the intestine. In certain embodiments, the disease or condition is systemic.

In certain embodiments, the compositions of the invention are for use in treating or preventing an infection with Enterobacteriaceae, such as E. coli infection. In preferred embodiments, the compositions of the invention are for use in the treatment or prevention of an infection of the gastrointestinal tract, and in particular the cecum. In such embodiments, the Enterobacteriaceae may be a pathogenic strain.

In some embodiments, the level of Enterobacteriaceae is decreased in the subject's stool. In some embodiments, the level of Enterobacteriaceae is reduced in a stool sample from a subject. In some embodiments, the level of Enterobacteriaceae is decreased in the distal intestine of the subject. In some embodiments, the level of Enterobacteriaceae is reduced in the cecum. In some embodiments, the level of Enterobacteriaceae is decreased in the colon. In some embodiments, the level of Enterobacteriaceae is decreased in the rectum. In some embodiments, the level of Enterobacteriaceae is decreased in the small intestine.

In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Blautia for use in the treatment of infections caused by pathogenic Enterobacteriaceae.

In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Blautia for use in the treatment of E. coli infection.

In preferred embodiments, the compositions of the invention are for use to reduce the level of Enterobacteriaceae in the gastrointestinal tract, preferably E. coli, in the treatment or prevention of a disease associated with elevated levels of Enterobacteriaceae, such as IBS, Crohn's disease, ulcerative colitis. functional dyspepsia, diarrhea, gastroenteritis, urinary tract infection, or neonatal meningitis. It is known that Enterobacteriaceae, and in particular E. coli, are potential initiators or are known to aggravate Crohn's disease and ulcerative colitis [22-24], therefore the effect demonstrated in the examples for the compositions of the invention may be useful in the treatment of these states.

In certain embodiments, the compositions of the invention are for use in treating or preventing IBS, Crohn's disease, ulcerative colitis, functional dyspepsia, diarrhea, gastroenteritis, urinary tract infection, or neonatal meningitis by reducing the level of Enterobacteriaceae in the gastrointestinal tract.

In preferred embodiments, the compositions of the invention comprise a bacterium deposited with accession number DSM 10507/14294 and are intended for use to reduce the level of Enterobacteriaceae in the gastrointestinal tract in the treatment of Crohn's disease, ulcerative colitis, functional dyspepsia, or most preferably IBS. In further preferred embodiments, the compositions of the invention comprise a bacterium deposited with accession number DSM 10507/14294 and are intended for use in the treatment or prevention of Crohn's disease, ulcerative colitis, functional dyspepsia, or most preferably IBS, by reducing the level of Enterobacteriaceae in the gastrointestinal tract. ...

In certain embodiments, the composition is administered in combination with an antibiotic such as fluoroquinolone, azithromycin, ciprofloxacin, or rifaximin. In certain embodiments, the composition is administered in combination with a dehydrating agent, such as an oral rehydration solution. The composition of the invention can be administered concurrently with an antibiotic or dehydrating agent, or sequentially with an antibiotic or dehydrating agent.

In certain embodiments, the compositions are for use in patients who exhibit, or are expected to exhibit, increased levels of Enterobacteriaceae in the gastrointestinal tract, for example, as compared to a healthy subject or a population of healthy subjects.

In certain embodiments, the compositions of the invention are for use in a method for treating, preventing, or reducing colonization of the gastrointestinal tract by Enterobacteriaceae.

In some embodiments, a composition of the invention is for use in a method for reducing the level of Enterobacteriaceae in the gastrointestinal tract in a subject having an increased level of hydrogen in their respiration compared to a healthy subject. In some embodiments, the composition of the invention is for use in reducing the respiratory hydrogen level of a subject that exhibits, or is expected to exhibit, an increased level of Enterobacteriaceae in the gastrointestinal tract. The subject is preferably a subject diagnosed with IBS, Crohn's disease, ulcerative colitis, functional dyspepsia, diarrhea, gastroenteritis, urinary tract infection or neonatal meningitis and / or an infection with pathogenic Enterobacteriaceae, eg E. coli. The examples show that treatment with a composition according to the invention reduces the level of hydrogen detected in breath hydrogen tests. Accordingly, hydrogen levels are preferably assessed using a hydrogen breath test. The hydrogen breath test is well known in the art and thus the skilled person will know how to perform such a test. In some embodiments, lactulose is administered to the patient as a test substrate.

The breath hydrogen test is also a useful tool for monitoring the efficacy or likely efficacy of reducing Enterobacteriaceae and treating or preventing a composition of the invention. For example, a decrease in the level of hydrogen detectable in the breath of a subject after treatment with a composition of the invention may indicate that the treatment has a level-lowering, therapeutic or prophylactic effect. Accordingly, in some embodiments, the methods and use of the invention further comprise monitoring the breathing hydrogen level of the subject during and / or after treatment with a composition of the invention and thereby assessing the effectiveness or likely effectiveness of level reduction, treatment, or prevention. For example, hydrogen levels can be monitored at one or more (eg, 1, 2, 3, 4, or more than 4) points in time, for example, including time points before treatment, at the beginning of treatment, during treatment, at the end of treatment and / or after treatment, if desired. In some embodiments, the level of hydrogen in the subject's breath at the end and / or after the dosing period (during which the subject is administered the composition) is compared to the level at the beginning and / or prior to the dosing period, and a decrease in the level indicates the effectiveness or likely effectiveness of reducing the level, treatment or prevention. For example, in embodiments in which the dosing period is 16 days, it may be desirable to take measurements on day 1 and day 16, or, for example, on day 1, day 2, day 15 and day 16. In some embodiments, multiple measurements are taken. and determining the average of the measurements thus obtained (eg, average for day 1 and day 2 and average for day 15 and day 16). In some embodiments, the hydrogen reduction of C _max levels for at least 40 ppm (mn ^-1) indicates that the decline in the treatment or prevention are effective or probably effective. In some embodiments, the respiration hydrogen level of the subject is measured only once, for example, at the end of or after treatment, and the determination that the level has a value equal to or close to a predetermined level indicates that a decrease in the level, treatment or prevention, were probably effective. The breath hydrogen test is a standard assay and thus predetermined levels are known in the art.

Treatment or prevention can refer, for example, to decreasing the severity of symptoms or reducing the frequency of exacerbations, or reducing the set of factors (triggers) that are problematic for the patient.

Enterobacteriaceae levels can be detected in a patient's feces using standard techniques such as the qPCR techniques used in the examples.

Administration methods

Preferably, the compositions of the invention are intended for administration to the gastrointestinal tract to provide delivery and / or partial or complete colonization of the intestine with the bacterial strain of the invention. Typically, the compositions of the invention are administered orally, but they can be administered rectally, intranasally, or by buccal or sublingual routes.

In certain embodiments, compositions of the invention may be administered as a foam, spray, or gel.

In certain embodiments, the compositions of the invention may be administered as a suppository, such as a rectal suppository, for example, in the form of cocoa butter, synthetic tallow (eg, suppocire, witepsol), glycerol gelatin, polyethylene glycol, or glycerin soap composition (soap glycerin).

In certain embodiments, a composition of the invention is administered into the gastrointestinal tract through a tube such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or an opening such as an opening in the chest wall cells that provide access to the stomach, jejunum, and other suitable access openings.

The compositions of the invention can be administered in a single dose, or they can be administered sequentially in a treatment regimen. In certain embodiments, the compositions of the invention are for daily administration. The examples demonstrate that daily administration provides successful colonization and clinical benefit.

In certain embodiments, the compositions of the invention are administered regularly, eg, daily, every other day, or weekly, over an extended period of time, such as for at least one week, two weeks, one month, two months, six months, or one year. The examples demonstrate that administration of BH may not lead to permanent colonization of the intestine, therefore, regular administration over long periods of time can provide great therapeutic benefits.

- 8 036958

In some embodiments, compositions of the invention are administered for 7, 14, 16, 21, or days, or no more than 7, 14, 16, 21, or 28 days. For example, in some embodiments, compositions of the invention are administered over a period of 16 days.

In certain embodiments, treatment in accordance with the invention is accompanied by an assessment of the patient's gut microbiota. Treatment can be repeated if delivery and / or partial or complete colonization with the strain of the invention is not achieved and thus no effect is observed, or treatment can be discontinued if delivery and / or partial or complete colonization is successful and the effect is observed ...

In certain embodiments, a composition of the invention can be administered to a pregnant animal, eg, a mammal, such as a human, to prevent the development of elevated levels of Enterobacteriaceae in the infant / young in utero and / or after birth.

The compositions of the invention can be administered to a patient diagnosed with elevated levels of Enterobacteriaceae or a disease or condition associated with elevated levels of Enterobacteriaceae, or who has been identified as at risk of elevated levels of Enterobacteriaceae. The compositions can also be administered as a prophylactic measure to prevent the development of elevated levels of Enterobacteriaceae in a healthy patient.

The compositions of the invention can be administered to a patient who has been identified as having abnormal intestinal microbiota. For example, the patient may have reduced or no colonization of Blautia, and in particular Blautia hydrogenotrophica, Blautia stercoris or Blautia wexlerae.

The compositions of the invention can be administered in the form of a food product such as a nutritional supplement.

In general, the compositions of the invention are intended to treat humans, although they can be used to treat animals, including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for improving the growth and performance of animals. When administered to animals, gavage may be employed.

In some embodiments, the subject to which the composition is to be administered is an adult. In some embodiments, the subject to which the composition is to be administered is a human infant.

Compositions

In general, the composition of the invention contains bacteria. In preferred embodiments of the invention, the composition is prepared in lyophilized form. For example, a composition according to the invention may contain granules or gelatin capsules, for example hard gelatin capsules, containing the bacterial strain of the invention.

Preferably, the composition according to the invention contains lyophilized bacteria. Lyophilization of bacteria is a well-established procedure and recommendations are given, for example, in [25-27]. The examples demonstrate that lyophilisate compositions are particularly effective.

Alternatively, the composition of the invention may comprise a live, active bacterial culture. The examples demonstrate that the bacterial cultures of the invention are therapeutically effective.

In some embodiments, the bacterial strain in the composition of the invention has not been inactivated, eg, has not been inactivated by heat. In some embodiments, the bacterial strain in the composition of the invention has not been killed, for example, has not been killed by heat. In some embodiments, the bacterial strain in the composition of the invention has not been attenuated, such as not attenuated by heat. For example, in some embodiments, the bacterial strain in the composition of the invention has not been killed, inactivated, and / or attenuated. For example, in some embodiments, the bacterial strain in the composition of the invention is live. For example, in some embodiments, the bacterial strain in the composition of the invention is viable. For example, in some embodiments, a bacterial strain in a composition of the invention is capable of partially or completely colonizing the intestine. For example, in some embodiments, the bacterial strain in the composition of the invention is viable and capable of partially or completely colonizing the intestine.

In some embodiments, the composition comprises a mixture of live bacterial strains and killed bacterial strains.

In preferred embodiments, the composition of the invention is encapsulated to allow delivery of the bacterial strain to the intestine. Encapsulation protects the composition from degradation prior to delivery to the target area by, for example, rupture due to chemical or physical influences such as pressure, enzymatic activity, or physical destruction, which can be initiated by changes in pH. Any suitable encapsulation method can be used. Typical examples of encapsulation techniques include entrapment in a porous matrix, attachment or adsorption to surfaces of a solid support, self-aggregation by flocculation or with crosslinking agents, and mechanical retention behind a microporous membrane or microcapsule. Encapsulation guidelines that may be useful for preparing compositions of the invention are given, for example, in references [28-29].

The composition can be administered orally and can be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Blautia are anaerobic. Other ingredients (such as, for example, vitamin C) can be included as oxygen scavengers and prebiotic substrates to improve delivery and / or partial or complete colonization and in vivo survival. Alternatively, the probiotic composition of the invention can be administered orally as a food or nutritional product such as a fermented milk product based on milk or whey, or as a pharmaceutical product.

The composition can be formulated as a probiotic.

The composition of the invention comprises a therapeutically effective amount of a bacterial strain of the invention. A therapeutically effective amount of the bacterial strain is sufficient to have a beneficial effect on the patient. A therapeutically effective amount of the bacterial strain may be sufficient to provide delivery and / or partial or complete colonization of the patient's intestine.

A suitable daily dose of bacteria, for example for an adult, may be from about 1 x 10 ³ to about 1 x 10 ¹¹ colony forming units (CFU); for example, from about 1 x 10 ⁷ to about 1 x 10 ¹⁰ CFU; in another example, from about 1 x 10 ⁶ to about 1 x 10 ¹⁰ CFU; in another example, from about 1 x 10 ⁷ to about 1 x 10 ¹¹ CFU; in another example, from about 1 x 108 to about 1 x 10 ¹⁰ CFU; in another example, from about 1 x 108 to about 1 x 10 ¹¹ CFU.

In certain embodiments, the dose of bacteria is at least 10 ⁹ cells per day, such as at least 10 ¹⁰ , at least 10 ^11, or at least 10 ¹² cells per day.

In certain embodiments, the composition comprises a bacterial strain in an amount of from about 1x10 ⁶ to about 1x10 ¹¹ CFU / g, based on the weight of the composition; for example, from about 1 x 108 to about 1 x 10 ¹⁰ CFU / g. The dose can be, for example, 1, 3, 5 and 10 g.

Typically, a probiotic, such as a composition of the invention, is optionally combined with at least one suitable prebiotic compound. The prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide or sugar alcohol that is not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of this invention comprises a prebiotic compound in an amount of from about 1 to about 30 wt% based on the total weight of the composition (eg, 5 to 20 wt%). Carbohydrates can be selected from the group consisting of fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomaltoligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosanoligosaccharides (or COS), aravidiachiumsaccharides, modified araviducans, modified , D-tagatose, acacia fibers, carob, oats and citrus fibers. In one aspect, the prebiotics are short-chain fructo-oligosaccharides (hereinafter referred to as cc-FOS (FOSs-c.c.)); these cc-FOS are non-digestible carbohydrates, usually obtained by conversion of beet sugar, and contain a sucrose molecule to which three glucose molecules are linked.

Compositions of the invention may contain pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients are given in reference [30]. Suitable carriers or diluents for therapeutic use are well known in the pharmaceutical industry and are described, for example, in the source [31]. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerin, and water. The choice of pharmaceutical carrier, excipient, or diluent can be made in accordance with the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may contain, as or in addition to the carrier, excipient or diluent, any suitable binder (s), lubricant (s), suspending agent (s), coating agent (s), solubilizer (s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, bulk lactose, betalactose, corn sugars, natural and synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, colorants and even flavoring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, cysteine, and p-hydroxybenzoic acid esters, for example, in some embodiments, the preservative is selected from sodium benzoate, sorbic acid, and p-10 036958 hydroxybenzoic acid esters. Antioxidants and suspending agents can also be used. An additional example of a suitable carrier is sucrose. An additional example of a preservative is cysteine.

The compositions of the invention can be formulated as a food product. For example, a food product can provide a nutritional benefit in addition to a therapeutic effect of the invention, such as a nutritional supplement. Likewise, the food composition can be formulated to enhance the taste of the composition of the invention or to make the composition more palatable by making it more similar to a conventional food ingredient rather than a pharmaceutical composition. In certain embodiments, the composition of the invention is formulated as a milk-based product. The term milk-based product means any liquid or semi-solid milk or whey product having different fat content. A milk-based product can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, milk reconstituted from milk powder and whey without any processing, or a processed product such as yogurt, sour milk, curdled milk , sour milk, sour whole milk, buttermilk, and other sour dairy products. Another important group includes dairy drinks such as whey drinks, fermented milk products, condensed milk, breast milk substitutes (infant), or baby milks; flavored milks, ice cream; milk-containing foods such as sweets.

In some embodiments, the compositions of the invention comprise one or more bacterial strains of the genus Blautia and do not contain bacteria of any other genus, or contain only a minimal (de minimis) or biologically insignificant amount of bacteria of another genus.

In certain embodiments, the compositions of the invention comprise a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may contain only minimal or biologically insignificant amounts of other bacterial strains or species. Such compositions can be a culture that is substantially free of other species of organism. In some embodiments, such compositions may be a lyophilisate substantially free of other organisms.

In certain embodiments, the compositions of the invention comprise one or more bacterial strains of the genus Blautia, for example Blautia hydrogenotrophica, and do not contain any other bacterial genus, or contain only minimal or biologically insignificant amounts of bacteria of another genus. In certain embodiments, the compositions of the invention comprise one Blautia species, eg, Blautia hydrogenotrophica, and do not contain any other bacterial species, or contain only minimal or biologically insignificant amounts of another species of bacteria. In certain embodiments, the compositions of the invention comprise a single Blautia strain, such as Blautia hydrogenotrophica, and do not contain any other bacterial strains or species, or contain only minimal or biologically insignificant amounts of bacteria of a different strain or species.

In some embodiments, the compositions of the invention comprise more than one bacterial strain or species. For example, in some embodiments, compositions of the invention comprise more than one strain of the same species (e.g., more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 , 35, 40 or 45 strains) and optionally does not contain any other species of bacteria. In some embodiments, the compositions of the invention comprise less than 50 strains of the same species (e.g., less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 strains) and optionally does not contain any other species of bacteria. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1 -6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30 , 6-15, 16-25 or 31-50 strains of the same species and optionally does not contain any other species of bacteria. In some embodiments, the compositions of the invention comprise more than one species from the same genus (e.g., more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23 , 25, 30, 35 or 40 species) and optionally does not contain bacteria of any other kind. In some embodiments, the compositions of the invention comprise less than 50 species from the same genus (e.g., less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3 and optionally does not contain bacteria of any other kind In some embodiments, the compositions of the invention comprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8 , 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 210, 2-5 , 6-30, 6-15, 16-25, or 31-50 species are of the same genus and optionally does not contain bacteria of any other genus The invention includes any combination of the above.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises a Blautia bacterial strain in a microbial consortium. For example, in some embodiments, the Blautia bacterial strain is present in combination with one or more (e.g., at least 2, 3, 4, 5, 10, 15, or 20) other bacterial strains from other genera with which it can exist symbiotically in vivo in the intestine. For example, in some embodiments, the composition comprises a bacterial

- 11 036958 strain Blautia hydrogenotrophica in combination with a bacterial strain from another genus. In some embodiments, the microbial consortium comprises two or more bacterial strains obtained from a faecal sample from one organism, such as a human. In some embodiments, the microbial consortium does not occur together in nature. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from fecal samples from at least two different organisms. In some embodiments, the two different organisms are of the same species, such as two different humans. In some embodiments, the two different organisms are a human infant and an adult. In some embodiments, the two different organisms are a human and a non-human mammal.

In some embodiments, the composition of the invention further comprises a bacterial strain having the same safety and therapeutic efficacy characteristics as the Blautia hydrogenotrophica strain deposited with accession number DSM 10507/14294, but is not the Blautia hydrogenotrophica strain deposited with accession number DSM 10507 / 14294, or non-Blautia hydrogenotrophica, or non-Blautia.

In some embodiments, when the composition of the invention contains more than one bacterial strain, species, or genus, the individual bacterial strains, species, or genera can be designed for separate, simultaneous, or sequential administration. For example, the composition can contain all of more than one bacterial strain, species or genus, or bacterial strains, species or genera can be stored separately and administered separately, simultaneously or sequentially. In some embodiments, more than one bacterial strains, species, or genera are kept separate but mixed prior to use.

In some embodiments, the bacterial strain for use in the invention is obtained from the faeces of an adult. In some embodiments, when the composition of the invention contains more than one bacterial strain, all bacterial strains are obtained from adult feces or, if other bacterial strains are present, they are present only in minimal amounts. The bacteria can be grown in culture after being obtained from adult feces and used in the composition of the invention.

In some embodiments, one or more Blautia bacterial strains is / are the only therapeutically active agent (s) in the composition of the invention. In some embodiments, the bacterial strain (s) in the composition is / are the only therapeutically active agent (s) in the composition of the invention.

Compositions for use in accordance with the invention may or may not require marketing authorization.

In certain embodiments, the invention provides a pharmaceutical composition as defined above, wherein said bacterial strain is lyophilized. In certain embodiments, the invention provides the aforementioned pharmaceutical composition wherein said bacterial strain has been spray dried. In certain embodiments, the invention provides a pharmaceutical composition as defined above in which the bacterial strain has been lyophilized or spray dried and in which it is alive. In certain embodiments, the invention provides the aforementioned pharmaceutical composition in which the bacterial strain has been lyophilized or spray dried and in which it is viable. In certain embodiments, the invention provides a pharmaceutical composition as defined above in which the bacterial strain has been lyophilized or spray dried and in which it is capable of partially or completely colonizing the intestine. In certain embodiments, the invention provides a pharmaceutical composition as defined above in which the bacterial strain is lyophilized or spray dried and in which it is viable and capable of partially or completely colonizing the intestine.

In some cases, the lyophilized or spray dried bacterial strain is reconstituted prior to administration. In some cases, the reduction is carried out using the diluent described in this document.

Compositions of the invention may contain pharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceutical composition comprising a bacterial strain of the invention; a pharmaceutically acceptable excipient, carrier or diluent; moreover, the amount of bacterial strain is sufficient to reduce levels of Enterobacteriaceae and / or treat a disorder associated with increased levels of Enterobacteriaceae, when administered to a subject in need thereof; the disorder being associated with increased levels of Enterobacteriaceae, such as Crohn's disease, ulcerative colitis, functional dyspepsia, or more preferably IBS, or diarrhea, gastroenteritis, urinary tract infection, or neonatal meningitis.

In certain embodiments, the invention provides a pharmaceutical composition as defined above, wherein the amount of the bacterial strain is from about 1 x 10 ³ to about 1 x 10 ¹¹ colony forming units per gram of composition weight.

In certain embodiments, the invention provides a pharmaceutical composition as defined above wherein the composition is administered at a dose of 1, 3, 5, or 10 g.

In certain embodiments, the invention provides a pharmaceutical composition as defined above, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.

In certain embodiments, the invention provides a pharmaceutical composition as defined above comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol.

In certain embodiments, the invention provides the above pharmaceutical composition comprising a diluent selected from the group consisting of ethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceutical composition comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, bulk lactose, beta-lactose, corn sugar, gum arabic, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.

In certain embodiments, the invention provides the above pharmaceutical composition further comprising at least one of a preservative, an antioxidant, and a stabilizer.

In certain embodiments, the invention provides the above pharmaceutical composition comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters.

In certain embodiments, a pharmaceutical composition of the invention is provided, wherein the composition does not contain any minerals, or more specifically does not contain any metals with an atomic number greater than 33, for example, the composition does not contain any minerals from the group consisting of selenium, molybdenum, tungsten, selenium compounds, molybdenum compounds and tungsten compounds.

In certain embodiments, the invention provides a pharmaceutical composition as defined above, wherein said bacterial strain is lyophilized.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is stored in a sealed container at a temperature of about 4 or about 25 ° C, and the container is placed in an atmosphere with 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units persist after a period of time of at least about 1, 3, 6 months; 1, 1.5, 2, 2.5 or 3 years.

In some embodiments, a composition of the invention is provided in a sealed container containing the composition described herein. In some embodiments, the sealed container is a sachet or vial. In some embodiments, a composition of the invention is provided in a syringe containing the composition described herein.

The composition of this invention may, in some embodiments, be provided as a pharmaceutical composition. For example, the composition can be in the form of a tablet or capsule. In some embodiments, the capsule is a gel-cap.

In some embodiments, the compositions of the invention are administered orally. Oral administration can include swallowing, whereby the formulation enters the gastrointestinal tract, and / or buccal, lingual, or sublingual administration, whereby the formulation enters the bloodstream directly from the mouth.

Pharmaceutical compositions suitable for oral administration include solid plugs, solid microdispersed materials, semi-solid and liquid materials (including multiphase or dispersed systems) such as tablets; soft or hard capsules containing multiparticulate materials or nanoparticulate materials, liquids (eg, aqueous solutions), emulsions or powders; sucking tablets (including those filled with liquid); gum; gels; fast-dispersing dosage forms; films; caplets (ovules); sprays; and buccal / mucoadhesive patches.

In some embodiments, the pharmaceutical composition is an enteric formulation, i.e. a gastro-resistant composition (eg, resistant to gastric pH), suitable for delivering a composition of the invention to the intestine by oral administration. Enteric compositions can be especially useful where the bacteria or other component of the composition is acid sensitive, for example prone to degradation under gastric conditions.

In some embodiments, the enteric composition comprises an enteric coating. In some embodiments, the composition is an enteric coated dosage form. For example, the composition can be an enteric coated tablet or an enteric coated capsule and the like. The enteric coating may be a conventional enteric coating, for example a conventional tablet, capsule and the like coating. for oral delivery. The composition may include a film coating, such as a thin film layer of an enteric polymer, such as an acid insoluble polymer.

In some embodiments, the enteric composition is enteric in nature, eg, gastro-resistant, without the need for an enteric coating. Thus, in some embodiments, the composition is an enteric composition that does not contain an enteric coating. In some embodiments, the composition is a capsule made from a thermogelling material. In some embodiments, the thermosetting material is a cellulosic material such as methylcellulose, hydroxymethylcellulose, or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule includes a shell that does not contain any film-forming polymer. In some embodiments, the capsule includes a shell and the shell contains hydroxypropyl methylcellulose and does not contain any film-forming polymer (see, for example, [32]). In some embodiments, the composition is inherently an enteric capsule (eg, Vcaps® from Capsugel).

In some embodiments, the composition is a soft capsule. Soft capsules are capsules that can, due to the addition of softeners such as, for example, glycerin, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, have a certain elasticity and deformability. Soft capsules can be obtained, for example, based on gelatin or starch. Gelatin soft capsules are commercially available from a variety of suppliers. Depending on the route of administration, such as, for example, oral or rectal, soft capsules can have various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be prepared by conventional methods such as, for example, using the Scherer process (pressing method), the Accogel process (pressing method), or a drip or blowing process.

Cultivation methods

Bacterial strains for use in the present invention can be cultured using standard microbiological techniques, as described, for example, in references [33-35].

The solid or liquid medium used for the culture can be, for example, YCFA (yeast extract-casein fatty acid hydrolyzate) agar or YCFA medium. YCFA medium may contain (per 100 ml, approximate values): casiton (1.0 g), yeast extract (0.25 g), NaHCO3 (0.4 g), cysteine (0.1 g), K ₂ HPO ₄ (0.045 g), KH2PO4 (0.045 g), NaCl (0.09 g), (NH ₄ ) ₂ SO ₄ (0.09 g), MgSO ₄ -7H ₂ O (0.009 g), CaCl ₂ (0.009 g) , resazurin (0.1 mg), hemin (1 mg), biotin (1 mcg), cobalamin (1 mcg), paminobenzoic acid (3 mcg), folic acid (5 mcg) and pyridoxamine (15 mcg).

General Provisions

In the practice of this invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology available to those skilled in the art will be used. Such techniques are described in detail in the literature. See, for example, sources [36-43], etc.

The term comprising encompasses an inclusive as well as consisting of, for example, a composition containing X, may consist exclusively of X, or may further include something, such as X + Y.

The term about in relation to the numerical value x is optional and means, for example, x ± 10%.

An expression as such does not completely exclude, for example, a composition that is essentially free of Y may be completely free of Y. If necessary, an expression as such may be omitted from the definition of the invention.

References to percent sequence identity between two nucleotide sequences means that when aligned, the percentage of nucleotides when comparing the two sequences will be the same. Such alignment and percent homology or sequence identity can be determined using computer programs known to those skilled in the art, for example, described in section 7.7.18 of the source [44]. The preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12 and a gap extension penalty of 2, the BLOSUM 62 matrix. The Smith-Waterman homology search algorithm is disclosed in [ 45].

Unless specifically indicated, a multi-step process or method may include

- 14 036958 additional stages at the beginning or at the end of the process, or may include additional intermediate stages. Also the steps can be combined, skipped or performed in an alternative order, if necessary.

Various embodiments of the invention are described herein. It should be understood that the features specified in each embodiment may be combined with other specified features to provide additional options for implementation. In particular, embodiments identified herein as suitable, typical, or preferred may be combined with each other (unless they are mutually exclusive).

Modes for carrying out the invention

Example 1. Administration of bacterial inoculums to rats carrying the intestinal microbiota of a human patient with IBS

Summary.

Rats are inoculated with the faecal microbiota of a human IBS patient. The rats are then injected with compositions containing the bacterial strains of the invention and assayed for colonization by Blautia hydrogenotrophica.

Strain.

Blautia hydrogenotrophica (BH) strain DSM 10507/14294.

Compositions and introduction.

Lyophilisate BH - administered by gastric tube.

The control solution is administered by gavage.

Rats.

Inoculated with intestinal microbiota of a person with IBS.

Research scheme.

Day 14 - Rats are inoculated with intestinal microbiota of a human with IBS.

Days 0-28 - Daily dose of BH lyophilisate or control solution.

Days 0, 14 and 28 - qPCR of the BH population in fecal samples.

Results.

FIG. 1 depicts the results of qPCR analysis of the BH population in fecal samples from rats treated with control solution (CPK) or BH lyophilisate (CPK + BH). An increase in the BH population was observed on days 14 and 28 in rats receiving BH lyophilisate, confirming successful colonization.

Findings.

The introduction of compositions containing Blautia hydrogenotrophica resulted in successful colonization.

Example 2. Administration of bacterial lyophilisate to healthy rats and effect on Enterobacteriaceae.

The effects of administration of a lyophilisate of strain DSM 10507/14294 Blautia hydrogenotrophica (BH) on HIM-healthy rats (inoculated with intestinal microbiota of a healthy person) were investigated and the results are shown in FIG. 2-5. Additional experimental details are provided above in the description of the figures. FIG. 2 depicts that a suitable dose of BH in rats is 10 ⁹ cells per day or more. FIG. 3 depicts that in these experiments, BH did not permanently colonize the digestive tract of rats. FIG. 4 depicts that BH is present predominantly in the cecum. Surprisingly, FIG. 5 depicts that BH administration induces a statistically significant decrease in Enterobacteriaceae in rat feces, as detected by qPCR.

Example 3. Administration of a bacterial lyophilisate in a rat model of IBS.

Were further investigated the effects of the introduction of lyophilisate strain DSM 10507/14294 Blautia hydrogenotrophica (BH) in a rat model of IBS. Microbial-free rats were inoculated with fecal samples from C-IBS (constipated) or U-CPK (unsubtyped) patients. The results are shown in FIG. 6, and additional experimental details are set forth above in the description of the figures.

FIG. 6 confirms that administration of BH lyophilisate causes a statistically significant reduction in Enterobacteriaceae, which are predominantly represented by E. coli. As expected, an increase in BH is also observed.

Example 4. Comparison of Blautia hydrogenotrophica in the microbiota of patients.

Phase I clinical trials have been conducted in which Blautia hydrogenotrophica (Blautix, strain deposited with accession number DSM 10507/14294) was administered to people with irritable bowel syndrome (IBS). Patients received Blautix over the dosing period (days 1-16), with a washout period on days 19-23. Blautix has been found to be safe and well tolerated. Blautia hydrogenotrophica (Blautix) in patient microbiota was measured on day 1, day 16 and at the end of the study (EOS), with the following results.

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Table 1

A patient Prela rath Blautix - Day 1 Blautix - Day 16 Blautix - EOS Average St. Dev. Average Art. off Average Art. off Placebo Good wow 4.30E + 07 8.4 0E + 07 4.79Е + 07 7. 63E + 07 3.59E + 07 6.23E + 07 Blautix Healthy 2.92E + 07 2.25E + 07 6.35E + 07 1.02E + 08 2.58E + 07 3.34E + 07 Placebo IBS 3.5 8E + 07 3.45E + 07 2.44E + 07 3.13E + 07 2.09E + 07 2.47E + 07 Blautix IBS 3.17E + 07 6.28E + 07 8.53E + 07 1.57E + 08 3.71E + 07 5.56E + 07

Analysis revealed trends in Blautia hydrogenotrophica levels in patient stool samples (see FIG. 7a). Participants receiving Blautix had increased levels of bacteria in their stool samples after the dosing period (day 16). This demonstrates the successful delivery of Blautix to the intestines. Levels returned to normal after a washout period (end of studies; EOS). Thus, Blautix does not permanently colonize the intestines after dosing is stopped. Both observations are consistent with the preclinical model.

Example 5. Comparison of Enterobacteria in the microbiota of patients.

Enterobacteria were also measured in the microbiota of patients during phase I clinical trials on day 1, day 16, and at the end of the studies, with the following results.

A patient A drug Enterobacteria - Day 1 Enterobacteria - Day 16 Enterobacteria - EOS Average Art. off Average Art. off Average Art. off Place bo Healthy 5.89E + 08 1.00E + 09 3.3 2E + 0 8 6.2 6E + 08 2.8 5E + 08 4.28E + 08 Blaut Healthy 9.96E + 08 1.06E + 09 4.87E + 08 6.64E + 08 2.70E + 08 3.76E + 08 Place bo IBS 7.85E + 08 1.28E + 09 3.01E + 08 4.92E + 08 2.45E + 09 2.45E + 09 Blaut ix IBS 8.3 8 E + 09 1.9 6E + ten 1.12E + 09 1.45E + 09 6.17E + 08 9.91E + 08

The analysis revealed trends in Enterobacteria levels in patient stool samples (see FIG. 7b). IBS patients treated with Blautix showed a decrease in Enterobacteria. This is consistent with the observations of the preclinical model.

Example 6. Results of a breath hydrogen test.

Respiratory hydrogen levels are a biomarker of Blautix activity - its mechanism of action (MoA) is linked to the metabolism of endogenous H2 to produce acetate. People were injected with lactulose and hydrogen (H ₂ ) (C _max ) levels were determined in samples taken at four time points: day 1, day 2, day 15, and day 16. Uncorrected hydrogen results were converted to corrected hydrogen results.

Some patients were excluded from the analysis. There were three reasons why subjects were not included in the hydrogen breath test: 1) they had a CMAX hydrogen breath test <20 on one of the four sampling days and were therefore considered not responding; 2) they were methane producers (defined as producing more methane than hydrogen in the breath test), this affects the hydrogen response; and / or 3) a highly outliers in the hydrogen breath test (232 ppm) were obtained. Subjects 3.12 (Blautix), 3.24 (Blautix),

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4.07 (Blautix) were excluded as not responding. Subjects 3.03 (Blautix) and 3.08 (placebo) were excluded as methane producers (4.07, indicated as excluded above, was also a methane producer).

Subject 4.09 (placebo) was excluded due to highly outliers.

The corrected hydrogen analysis results for the end of the dosing period (days 15/16) were compared to the baseline (days 1/2). 10 of 12 patients (83%) treated with Blautix experienced a decrease in hydrogen levels during this period (Figs. 8a and 8c). In contrast, 3 out of 6 (50%) placebo patients had reduced hydrogen levels (Figs. 8b and 8c).

FIG. 9 depicts uncorrected and corrected hydrogen results for the Blautix treatment group (Verum (active ingredient group)) together with a statistical analysis of the results. The mean values for uncorrected and corrected H _{2 were found to} differ between day 1 and day 15. After 13.5 days of the experiment, a statistically significant (p <0.05) decrease in H _{2 was} detected in the breath test with C _max determination after stimulation with lactulose ... In contrast, in the placebo group, the mean values for day 1 and day 15 were equivalent (p> 0.05) (Fig. 10). Thus, the mean in the experimental group (also called the VERUM group) declines between day 1 and day 15, while the mean for the placebo group is equivalent on day 1 and day 15 for both uncorrected hydrogen measurements and the results of the determination of hydrogen (Fig. 11).

Example 7. Tests for stability.

The composition described herein, containing at least one bacterial strain described herein, is stored in a sealed container at a temperature of 25 or 4 ° C, and the container is placed in an atmosphere with 30, 40, 50, 60, 70, 75, 80, 90 or 95% RH. After 1, 2, 3, 6 months; For 1, 1.5, 2, 2.5 or 3 years, at least 50, 60, 70, 80 or 90% of the bacterial strain should be retained, as measured by colony forming units according to standard protocols.

Example 8. Ability of B. hydrogenotrophica to alter the microbiota in a rat model associated with human microbiota (HMA rats).

Summary.

Groups of 16 microbial-free rats (including 8 rats in the control group and 8 rats in the experimental group) were inoculated with the fecal microbiota of a human IBS patient (HMASPK rats). Three consecutive experiments were conducted using fecal samples from 3 different IBS patients. Two other groups of rats (n = 10) were inoculated with fecal samples from a healthy subject (n = 2 subjects; 2 groups of HMA-healthy rats) as controls. Thus, 24 rats associated with the IBS microbiota (control), 24 rats associated with the IBS microbiota treated with Blautix, and 20 rats associated with the healthy human microbiota were used. Half of the HMA-CPK rats were then injected for 28 days with the composition containing the bacterial B. hydrogenotrophica strain of the invention, while the other half received the control solution. On day 28 after administration, the levels of those microorganisms from the fecal microbiota for which changes were previously observed in patients with IBS were analyzed.

Strain.

Blautia hydrogenotrophica (BH), strain DSM 10507V14294.

Composition and introduction.

The BH lyophilisate was suspended in a sterile mineral salt solution to a concentration of 10 ¹⁰ bacteria / ml. 2 ml of this suspension was injected daily into an HMA-CPK rat using a gastric tube for 28 days.

The control solution was a sterile mineral salt solution that was administered daily (2 ml per rat) by gavage to the control group of HMA-CPK rats.

Rats.

Male non-microbial rats (10 weeks old) were inoculated with the fecal microbiota of a human IBS patient (HMA-IBS rats). Sixteen rats were inoculated with the same human feces inoculum. Three consecutive experiments were performed with fecal samples from three different subjects with IBS. Two other groups of ten rats were inoculated with fecal samples from 2 healthy subjects (norm-sensitivity control groups).

Research scheme.

Day 14 - Inoculation of germ-free rats with human fecal microbiota.

Days 0-28 - daily dose of BH lyophilisate (experimental group) or control solution (control group) by gavage

Days 0, 14 and 28 - collection of faecal samples for microbial analysis: qPCR to assess the population of BH and other commensal groups of microorganisms and the calculation of functional groups of microorganisms using selective media and strictly anaerobic method.

Results.

FIG. 12 shows the results of qPCR analysis of a population of B. hydrogenotrophica in 17 036958 samples of feces from HMA-CPK rats treated with a control solution or BH lyophilisate. A significant increase in the BH population was observed at the end of the dosing period (D28) in rats receiving the lyophilisate

BH, which confirms the successful delivery of BH to the colon.

FIG. 13 depicts the effect of B. hydrogenotrophica administration on certain microorganisms in the faecal microbiota, for which changes were previously observed in patients with IBS. The level of Enterobacteriaceae species decreased after the introduction of B. hydrogenotrophica.

findings

The introduction of Blautia hydrogenotrophica resulted in a decrease in Enterobacteriaceae.

Example 9 Symptom Changes in Patients During Phase I Clinical Trials

Phase I clinical trials have been conducted in which Blautia hydrogenotrophica (Blautix, strain deposited with accession number DSM 10507 as well as accession number DSM 14294) was administered to people with irritable bowel syndrome (IBS). Patients received Blautix over the dosing period (days 1-16), with a washout period on days 19-23. Blautix has been found to be safe and well tolerated.

Four symptoms were monitored, one of which was diarrhea. The studies recorded whether patients experienced an improvement, no change, or worsening of each of these symptoms. Results for patients receiving Blautix were compared with those obtained for patients receiving placebo. Symptom control was performed at three time points: day 1, day 15/16 and at the end of the trial. The results are shown in FIG. 14 and 15.

When comparing patient reported symptoms on Day 16 to a baseline of Day 1, 82% of 17 IBS patients treated with Blautix reported an improvement in symptoms (FIG. 14). Improvement of symptoms, one of which was diarrhea, supports the use of Blautix to treat or prevent diarrhea.

50% of patients receiving placebo reported improvement in symptoms (Fig. 14). High response rates to placebo are well established in clinical trials of IBS. Xifaxan was recently approved for the treatment of IBS on the basis of significantly smaller improvements over placebo (see: http://www.accessdata.fda.gov/spl/data/5ab6fceb4d22-4480-81fc-8bc28c16770d/5ab6fceb -4d22-4480-81fc-8bc28c 16770d.xml).

Based on the description provided herein, a worsening of symptoms was expected at study completion (days 19-23) compared to symptoms at completion of dosing (day 16). This worsening of symptoms was observed in phase I clinical trials: 41% of IBS patients reported worsening of symptoms after discontinuation of Blautix (Fig. 15). Worsening of symptoms, one of which was diarrhea, after discontinuation of Blautix thus also supports the use of Blautix in the treatment or prevention of diarrhea.

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Sequences.

SEQ ID NO : 1 (Gen 16S ribosomal RNA strain GAM6-1 Blautia stercoris, partial sequence - HM626177) 1 tgcaagtcga ggactgagcg gcgaagcgct tacgacagaa ccttcggggg aagatgtaag 61 gcggacgggt agttggaaac gagtaacgcg tgggtaacct gcctcataca gggggataac 121 ggctgctaat actccggtgg accgcataag cgcacggtat cgcatgatac agtgtgaaaa 181 tatgagatgg caaggcgacg acccgcgtct gattagctag ttggaggggt aacggcccac 241 atcagtagcc gcccagactc ggcctgagag ggtgaacggc cacattggga ctgagacacg 301 ctacgggagg cagcgacgcc cagcagtggg gaatattgca caatggggga aaccctgatg 361 gcgtgaagga aaatgacggt agaagtatct cggtatgtaa acttctatca gcagggaaga 421 acctgactaa gtagggggca gaagccccgg ctaactacgt gccagcagcc gcggtaatac 481 agcgttatcc gtctgatgtg ggatttactg ggtgtaaagg gagcgtagac ggaagagcaa 541 aaaggctggg tgccggagag gcttaacccc aggactgcat tggaaactgt ttttcttgag 601 gtaagcggaa caccagtggc ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa 661 gaaggcggct agcaaacagg tactggacgg taactgacgt tgaggctcga aagcgtgggg 721 attagatacc ggagcaaagc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg

- 19 036958

781 tcttcggtgc cgcagcaaac gcaataagta ttccacctgg ggagtacgtt cgcaagaatg 841 aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt taattcgaag 901 caacgcgaag aaccttacca agtcttgaca tcgatctgac cggttcgtaa tggaaccttt 961 ccttcgggac agagaagaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1021 gggttaagtc ccgcaacgag cgcaacccct atcctcagta gccagcaggt gaagctgggc 1081 actctgtgga gactgccagg gataacctgg aggaaggcgg ggacgacgtc aaatcatcat 1141 gccccttatg atttgggcta cacacgtgct acaatggcgt aaacaaaggg aagcgagccc 1201 gcgaggggga gcaaatccca aaaataacgt cccagttcgg actgcagtct gcaactcgac 1261 tgcacgaagc tggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa

tacgttcccg

1321 ggtcttgtac acaccgcccg tcacaccatg ggagtcagta acgcccgaag tc

SEQ ID NO: 2 (16S ribosomal RNA gene of WAL 14507 Blautia wexlerae, partial sequence - EF036467)

1 caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg 61 gacgggtgag taacgcgtgg gtaacctgcc ttatacaggg ggataacagt cagaaatggc 121 tgctaatacc gcataagcgc acagagctgc atggctcagt gtgaaaaact ccggtggtat 181 aagatggacc cgcgttggat tagcttgttg gtggggtaac ggcccaccaa ggcgacgatc 241 catagccggc ctgagagggt gaacggccac attgggactg agacacggcc cagactccta 301 cgggaggcag cagtggggaa tattgcacaa tgggggaaac cctgatgcag cgacgccgcg 361 tgaaggaaga agtatctcgg tatgtaaact tctatcagca gggaagatag tgacggtacc 421 tgactaagaa gccccggcta actacgtgcc agcagccgcg gtaatacgta

- 20 036958

gggggcaagc 481 gttatccgga tgatgtgaaa tttactgggt gtaaagggag cgtagacggt gtggcaagtc 541 ggcatgggct cggaggggta caacctgtgg actgcattgg aaactgtcat acttgagtgc 601 agcggaattc cagtggcgaa ctagtgtagc ggtgaaatgc gtagatatta ggaggaacac 661 ggcggcttac aaacaggatt tggacggtaa ctgacgttga ggctcgaaag cgtggggagc 721 agataccctg ggcaaagcca gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt 781 ttcggtgccg caagaatgaa tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg 841 actcaaagga attcgaagca attgacgggg acccgcacaa gcggtggagc atgtggttta 901 acgcgaagaa ggatctttcc ccttaccaag tcttgacatc cgcctgaccg atccttaacc 961 ttcgggacag gagatgttgg gcgagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1021 gttaagtccc ggtgggcact gcaacgagcg caacccctat cctcagtagc cagcatttaa 1081 ctggggagac tcatcatgcc tgccagggat aacctggagg aaggcgggga tgacgtcaaa 1141 ccttatgatt cgagattgtg tgggctacac acgtgctaca atggcgtaaa caaagggaag 1201 agatggagca acccgactac aatcccaaaa ataacgtccc agttcggact gtagtctgca 1261 acgaagctgg gttcccgggt aatcgctagt aatcgcggat cagaatgccg cggtgaatac 1321 cttgtacaca gtgacctaac ccgcccgtca caccatggga gtcagtaacg cccgaagtca 1381 tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt

aacaaggt

SEQ ID NO: 3 (Consensus sequence of 16s pRNA of Blautia stercoris strain 830)

TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTT

- 21 036958

ACGACAGAACCTTCGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGT AACCTGCCTCATACAGGGGGATAACAGTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTA TCGCATGATACAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTG GAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCACATTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAA ACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGG GAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATA CGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTC TGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTTGAGTGCCGGA GAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCG AAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTG CCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAA TTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTATTCGAAGCAACGCGAAGAACCTTAC CAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAACCTTTCCTTCGGGACAGAGAAGACAGG TGGTGCATGGTTGTCGTCAGCTCGT GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGG AGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAA TGGCGTAAACAAAGGGAAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTC GGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTC GCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCC GAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAACTGGGGTGAAGT CTAGGGGGT

SEQ ID NO: 4 (Consensus sequence of 16s pRNA of Blautia wexlerae strain MRX008)

TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGG TAACCTGCCTTATACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGA GCTGCATGGCTCAGTGTGAAAAACTCCGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTT GGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCGGCCTGAGAGGGTGAACGGCCACATT GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGA AACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAG GGAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGT CTGATGTGAAAGGCATGGGCTCAACCTGTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGG AGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGC GAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTA

- 22 036958

GATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGG TGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGG AATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT TACCAAGTCTTGACATCCGCCTGACCGATCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGAC AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC AACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCAGGGATAACCT GGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTAC AATGGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAAAATAACGTCCCAGT TCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATG CCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGC

C C GAAG T CAG T GAC C TAAC T G CAAAGAAG GAG C T G C C GAA

SEQ ID NO: 5 (Gene 16S ribosomal RNA strain S5a36 Blautia hydrogenotrophica, partial sequence - X95624.1)

1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagcgatag agaacggaga 61 tttcggttga agttttctat tgactgagtg gcggacgggt gagtaacgcg tgggtaacct 121 gccctataca gggggataac agttagaaat gactgctaat accgcataag cgcacagctt 181 cgcatgaagc ggtgtgaaaa actgaggtgg tataggatgg acccgcgttg gattagctag 241 ttggtgaggt aacggcccac caaggcgacg atccatagcc ggcctgagag ggtgaacggc 301 cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg gaatattgca 361 caatggggga aaccctgatg cagcgacgcc gcgtgaagga agaagtatct cggtatgtaa 421 acttctatca gcagggaaga aagtgacggt acctgactaa gaagccccgg ctaattacgt 481 gccagcagcc gcggtaatac gtaaggggca agcgttatcc ggatttactg ggtgtaaagg 541 gagcgtagac ggtttggcaa gtctgatgtg aaaggcatgg gctcaacctg tggactgcat 601 tggaaactgt cagacttgag tgccggagag gcaagcggaa ttcctagtgt agcggtgaaa 661 tgcgtagata ttaggaggaa caccagtggc gaaggcggcc tgctggacgg

- 23 036958 taactgacgt

721 tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa 781 cgatgaatac taggtgtcgg gtggcaaagc cattcggtgc cgcagcaaac gcaataagta 841 ttcccacctg gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg gggacccgca 901 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aaatcttgac 961 atccctctga ccgggaagta atgttccctt ttcttcggaa cagaggagac aggtggtgca 1021 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1081 tattcttagt agccagcagg tagagctggg cactctaggg agactgccag ggataacctg 1141 gaggaaggtg gggatgacgt caaatcatca tgccccttat gatttgggct acacacgtgc 1201 tacaatggcg taaacaaagg gaagcgaagg ggtgacctgg agcaaatctc aaaaataacg 1261 tctcagttcg gattgtagtc tgcaactcga ctacatgaag ctggaatcgc tagtaatcgc 1321 gaatcagaat gtcgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccat 1381 gggagtcagt aacgcccgaa gtcagtgacc caaccnaaag gagggagctg

ccgaaggtgg

1441 gactgataac tggggtga

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Claims (12)

1. The use of a pharmaceutical composition containing a bacterial strain of the genus Blautia having a 16s rRNA gene sequence at least 95% identical to SEQ ID NO: 5 to reduce the level of pathogenic Enterobacteriaceae in the gastrointestinal tract of a patient. 2. Use according to claim 1, wherein the Enterobacteriaceae is E. coli. 3. Use according to claim 1 or 2, characterized in that the patient has irritable bowel syndrome, Crohn's disease, ulcerative colitis, functional dyspepsia, diarrhea, gastroenteritis, urinary tract infection or neonatal meningitis. 4. Use according to claim 3, characterized in that the patient has diarrhea, gastroenteritis, urinary tract infection or neonatal meningitis. 5. Use according to claim 3, characterized in that the patient has irritable bowel syndrome. 6. The use of a pharmaceutical composition containing a bacterial strain of the genus Blautia having a 16s rRNA gene sequence at least 95% identical to SEQ ID NO: 5 for the treatment of infections caused by pathogenic Enterobacteriaceae in the gastrointestinal tract of a patient. 7. Use according to claims 1-6, characterized in that said bacterial strain is a Blautia hydrogenotrophica strain having a 16s rRNA gene sequence at least 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 5. 8. Use according to claims 1 to 7, characterized in that said bacterial strain is a Blautia hydrogenotrophica strain DSMZ 14294 having a 16s rRNA gene sequence of SEQ ID NO: 5. 9. Use according to claims 1-8, characterized in that the composition is intended for oral administration. - 26 036958 10. Use according to claims 1-9, characterized in that the composition contains one or more pharmaceutically acceptable excipients, in particular carriers. 11. Use according to claims 1-10, characterized in that the bacterial strain is lyophilized. 12. Use according to claims 1-10, characterized in that the bacterial strain is native.