OA19329A

OA19329A - Compositions comprising bacterial strains

Info

Publication number: OA19329A
Application number: OA1201900026
Authority: OA
Inventors: Imke MULDER; Alex STEVENSON; Fergus Shanahan; Paul O'toole; Helene SAVIGNAC; Ian JEFFERY
Original assignee: 4D Pharma Plc
Priority date: 2016-12-12
Filing date: 2017-12-12
Publication date: 2020-06-29

Abstract

Provided are compositions comprising a bacterial strain of the genus Blautia, for use in method of increasing the microbiota diversity and/or inducing stability of the microbiota of subject.

Description

The compositions of the invention comprise a bacterial strain of the genus Blautia. The examples demonstrate that bacteria of this genus are useful for increasing the microbiota diversity and/or inducing the stability of the microbiota. The preferred bacterial strains are of the species Blautia hydrogenotrophica, Blautia stercoris and Blautia wexlerae. Most preferred is Blautia hydrogenotrophica, particularly the bacterium deposited under accession number DSM 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. The Blautia species are Gram-reaction-positive, non-motile bacteria that may be either coccoid or oval and ail are obligate anaerobes that produce acetic acid as the major end product of glucose fermentation [25]. Blautia may be isolated from the human gut, although B. producta was isolated from a septicaemia sample.

Blautia hydrogenotrophica (previously known as Ruminococcus hydrogenotrophicus) has been isolated from the guts of mammals, is strictly anaérobie, and métabolisés H₂/CO₂ to acetate, which may be important for human nutrition and health. The type strain of Blautia hydrogenotrophica is S5a33 = DSM 10507 = JCM 14656. The GenBank accession number for the 16S rRNA gene sequence of Blautia hydrogenotrophica strain S5a36 is X95624.1 (disclosed herein as SEQ ID NO:5). This exemplary Blautia hydrogenotrophica strain is described in [25] and [26], The S5a33 strain and the S5a36 strain correspond to two subclones of an acetogenic strain isolated from a faecal sample of a healthy subject. They show identical morphology, physiology and metabolism and hâve identical 16S rRNA sequences. Thus, in some embodiments, the Blautia hydrogenotrophica for use in the invention has the 16S rRNA sequence of SEQ ID NO:5.

The Blautia hydrogenotrophica bacterium deposited under accession number DSM 10507 and also under accession number DSM 14294 was tested in the examples and is also referred to herein as strain BH. Strain BH was deposited with the Deutsche Sammlung von Mikroorganismen [German Microorganism Collection] (Mascheroder Weg 1b, 38124 Braunschweig, Germany) on 10th May 2001 as “Ruminococcus hydrogenotrophicus” under accession number DSM 10507 and also under accession number DSM 14294. The depositor was INRA Laboratoire de Microbiologie CR de Clermont-Ferrand/Theix 63122 Saint Genès Champanelle, France. Ownership ofthe bacterium deposited as DSM 10507 and DSM 14294 has passed via assignment to 4D Pharma Pic.

The GenBank accession number for the 16S rRNA gene sequence of Blautia stercoris strain GAM6-1^T is HM626177 (disclosed herein as SEQ ID NO:1). An exemplary Blautia stercoris strain is described in [27], The type strain of Blautia wexlerae is WAL 14507 = ATCC BAA-1564 = DSM 19850 [25], The GenBank accession number for the 16S rRNA gene sequence of Blautia wexlerae strain WAL 14507 T is EF036467 (disclosed herein as SEQ ID NO:2). This exemplary Blautia wexlerae strain is described in [25].

A preferred Blautia stercoris strain is the strain deposited under accession number NCI MB 42381, which is also referred to herein as strain 830. A 16S rRNA sequence for the 830 strain is provided in SEQ ID NO:3. Strain 830 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 12th March 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 under accession number NCIMB 42486, which is also referred to herein as strain MRX008. A 16S rRNA sequence for the MRX008 strain is provided in SEQ ID NO:4. Strain MRX008 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA,

Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 16th November 2015 as “Blautia/Ruminococcus and was assigned accession number NCIMB 42486.

Bacterial strains closely related to the strain tested in the examples are also expected to be effective for increasing the microbiota diversity and/or inducing the stability of the microbiota. In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia hydrogenotrophica. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence that has the sequence of SEQ ID NO:5.

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

Bacterial strains that are biotypes of the bacterium deposited under accession number DSM 10507/14294 or biotypes of the bacteria deposited under accession numbers NCIMB 42381 and NCIMB 42486 are also expected to be effective for increasing the microbiota diversity and/or inducing the stability of the microbiota. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.

Strains that are biotypes of a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and that are suitable for use in the invention may be identified by sequencing other nucléotide sequences for a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may hâve at least 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g.

across at least 85%, 90%, 95% or 99%, or across its whole genome). For example, in some embodiments, a biotype strain has at least 98% sequence identity across at least 98% of its genome or at least 99% sequence identity across 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or répétitive sequences such as BOX, ERIC, (GTG)₅, or REP or [28], Biotype strains may hâve sequences with at least 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486. In some embodiments, a biotype strain has a sequence with at least 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294 and comprises a 16S rRNA sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ ID NO:5. In some embodiments, a biotype strain has a sequence with at least 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294 and has the 16S rRNA sequence of SEQ ID NO:5.

Alternatively, strains that are biotypes of a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and that are suitable for use in the invention may be identified by using the accession number DSM 10507/14294 deposit, the accession number NCIMB 42381 deposit, or the accession number NCIMB 42486 deposit, and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and répétitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Blautia hydrogenotrophica, Blautia stercoris or Blautia wexlerae strains.

In certain embodiments, strains that are biotypes of a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and that are suitable for use in the invention are strains that provide the same pattern as a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example,[29]). Alternatively, biotype strains are identified as strains that hâve the same carbohydrate fermentation patterns as a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486.

Other Blautia strains that are useful in the compositions and methods of the invention, such as biotypes of a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486, may be identified using any appropriate method or strategy, including the assays described in the examples. For instance, strains for use in the invention may be identified by culturing bacteria and administering to rats to test in the distension assay. In particular, bacterial strains that hâve similar growth patterns, metabolic type and/or surface antigens to a bacterium deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 may be useful in the invention. A useful strain will hâve comparable microbiota modulatory activity to the DSM 10507/14294, NCIMB 42381 or NCIMB 42486 strain. In particular, a biotype strain will elicit comparable effects on the microbiota to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.

A particularly preferred strain of the invention is the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294. This is the exemplary BH strain tested in the examples and shown to be effective for increasing the microbiota diversity and/or inducing the stability of the microbiota. Therefore, the invention provides a cell, such as an isolated cell, of the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294, or a dérivative thereof, for use in therapy, in particular for the diseases and disorders described herein.

A dérivative ofthe strain deposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A dérivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a dérivative strain of the invention is therapeutically active. A dérivative strain will hâve comparable microbiota modulatory activity to the original DSM 10507/14294, NCIMB 42381 or NCIMB 42486 strain. In particular, a dérivative strain will elicit comparable effects on the microbiota to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples. A dérivative of the DSM 10507/14294 strain will generally be a biotype of the DSM 10507/14294 strain. A dérivative of the NCIMB 42381 strain will generally be a biotype of the NCIMB 42381 strain. A dérivative of the NCIMB 42486 strain will generally be a biotype of the NCIMB 42486 strain.

Référencés to cells of the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294 encompass any cells that hâve the same safety and therapeutic efficacy characteristics as the strains deposited under accession number DSM 10507/14294, and such cells are encompassed by the invention. Référencés to cells of the Blautia stercoris strain deposited under accession number NCIMB 42381 encompass any cells that hâve the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42381, and such cells are encompassed by the invention. Référencés to cells of the Blautia wexlerae strain deposited under accession number NCIMB 42486 encompass any cells that hâve the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42486, and such cells are encompassed by the invention.

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

Therapeutic uses

In certain embodiments, the compositions of the invention are for use in increasing the microbiota diversity and/or inducing the stability of the microbiota. Reduced diversity of the microbiota and/or reduced stability of the microbiota are associated with numerous pathological diseases and disorders, and the examples demonstrate that the compositions of the invention may be effective for increasing the microbiota diversity and/or inducing the stability of the microbiota. Accordingly, the disease or disorder to be treated or prevented using a composition of the invention is preferably a disease or disorder associated with a level of microbiota diversity that is reduced relative to the microbiota diversity of a healthy subject and/or a disease or disorder that is associated with reduced stability of the microbiota. Thus, in some embodiments, the disease or disorder may be associated with a level of microbiota diversity that is reduced relative to the microbiota diversity of a healthy subject and also be associated with reduced stability of the microbiota.

In certain embodiments, the compositions of the invention are for use in treating or preventing a disease or disorder selected from IBS, IBD, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases and one or more metabolic diseases/disorders. Treatment or prévention of other diseases and disorders is also envisaged. In certain embodiments, the compositions of the invention are for use in treating or preventing IBS or IBD. In certain embodiments, the compositions of the invention are for use in treating or preventing IBS. In certain embodiments, the compositions of the invention are for use in treating or preventing IBD. In certain embodiments, the compositions of the invention are for use in treating or preventing one or more allergie diseases. In certain embodiments, the compositions of the invention are for use in treating or preventing obesity. In certain embodiments, the compositions of the invention are for use in treating or preventing one or more infectious diseases. In certain embodiments, the compositions of the invention are for use in treating or preventing one or more autoimmune diseases. In certain embodiments, the compositions of the invention are for use in treating or preventing one or more metabolic diseases/disorders. Preferably, the treatment or prévention comprises increasing the microbiota diversity and/or inducing the stability ofthe microbiota in the subject.

In certain embodiments, the one or more infectious diseases is selected from a viral, bacterial or fungal disease. In certain embodiments, the one or more allergie diseases is asthma. In certain embodiments, the one or more metabolic diseases/disorders is selected from diabètes, e.g. type 2 diabètes, and obesity. In certain embodiments, the one or more autoimmune diseases is selected from multiple sclerosis and rheumatoid arthritis.

In certain embodiments, the compositions of the invention are for use in treating or preventing IBS, IBD, obesity, type 2 diabètes, one of more infectious diseases, one or more allergie diseases, one or more autoimmune diseases or one or more metabolic diseases/disorders by increasing the microbiota diversity in the microbiota. In certain embodiments, the compositions of the invention are for use in treating or preventing IBS or IBD by inducing the stability of the microbiota. In certain embodiments, the compositions of the invention are for use in treating or preventing IBS by inducing the stability ofthe microbiota

In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Blautia, for use in the treatment or prévention of IBD, IBS, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases or one or more metabolic diseases/disorders, wherein the treatment or prévention comprises increasing the microbiota diversity and/or inducing the stability of the microbiota in the subject.

In some embodiments, the invention provides a composition comprising a bacterial strain of the genus Blautia for use in treating or preventing a disease or disorder selected from IBS, IBD, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases and one or more metabolic diseases/disorders. In some embodiments, the invention provides a method of treating or preventing a disease or disorder selected from IBS, IBD, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases and one or more metabolic diseases/disorders, comprising administering a composition comprising a bacterial strain of the genus Blautia.

In preferred embodiments, the compositions of the invention comprise the bacterium deposited under accession number DSM 10507/14294 and are for use in increasing the microbiota diversity and/or inducing the stability of the microbiota in the subject in the treatment of IBD, IBS, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases or one or more metabolic diseases/disorders. In further preferred embodiments, the compositions of the invention comprise the bacterium deposited under accession number DSM 10507/14294 and are for use in treating or preventing IBD, IBS, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases or one or more metabolic diseases/disorders by increasing the microbiota diversity and/or inducing the stability ofthe microbiota.

In some embodiments, the pathogenesis of the disease or disorder affects the intestine. In some embodiments, the pathogenesis of the disease or disorder does not affect the intestine. In some embodiments, the pathogenesis of the disease or disorder is not localised at the intestine. In some embodiments, the treating or preventing occurs at a site other than at the intestine. In some embodiments, the treating or preventing occurs at the intestine and also at a site other than at the intestine. In certain embodiments, the disease or disorder is systemic.

In certain embodiments, the compositions are for use in subjects that exhibit, or are expected to exhibit, reduced levels of microbiota diversity, for example, when compared to a healthy subject, or a population of healthy subjects. For example, in some embodiments, the composition is for use in treating a subject having less than 101 different bacterial species (e.g. less than 100, 99, 98, 97, 96, 95, 93, 90, 85, 80, 75 or 70 bacterial species) and/or less than 195 different strains (e.g. less than 193, 190, 187, 185, 183, 180, 175, 170, 165, 160, 150, 140 bacterial strains) in its microbiota. For example, in some embodiments, the composition is for use in treating a subject that has at least one bacterial genus (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 bacterial généra) fewer in its intestinal microbiota compared to a healthy subject or compared to a population of healthy subjects. In some embodiments, the treatment or prévention comprises a step of diagnosing a subject as having a reduced level of microbiota diversity and then if a reduced level of diversity is found to be present, the subject is then treated with a composition according to the invention.

In certain embodiments, the compositions are for use in subjects that exhibit, or are expected to exhibit, reduced stability of the microbiota. In some embodiments, the compositions are for use in subjects that exhibit, or are expected to exhibit, reduced stability in its microbiota, for example, when compared to a healthy subject, or a population of healthy subjects. In some embodiments, the treatment or prévention comprises a step of diagnosing a subject as having a reduced stability in its microbiota and then if reduced stability is found to be present, the subject is then treated with a composition according to the invention.

In certain embodiments, the subject is an infant. In certain embodiments, the subject is a child. In certain embodiments, the subject is an adult.

In certain embodiments, the subject is a healthy subject. For example, in some embodiments in which the composition is used for preventing a disease or disorder, the subject is a healthy subject, optionally one identified as being at risk of developing a disease or disorder characterised by a réduction in microbiota diversity.

In certain embodiments, the subject has previously received, is receiving, or will be receiving antibiotic treatment. Accordingly, in some embodiments, the treatment or prévention comprises administering the composition of the invention after, together with, or before antibiotic treatment. The composition of the invention and the one or more antibiotics may be for separate, simultaneous or sequential administration.

In some embodiments, the composition of the invention is for use in a method of increasing the microbiota diversity and/or inducing the stability of the microbiota in a subject having an increased level of hydrogen in their breath relative to a healthy subject. In some embodiments, the composition of the invention is for use in reducing the hydrogen level in the breath of a subject exhibiting or who is expected to exhibit a reduced level of diversity of its microbiota and/or reduced stability of the microbiota. The subject is preferably a subject diagnosed as having IBS, IBD, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases and/or one or more metabolic diseases/disorders. Treatment with a composition of the invention reduces the level of hydrogen detected in hydrogen breath tests. Accordingly, the hydrogen levels are preferably assessed using a hydrogen breath test. The hydrogen breath test is well known in the art and so the skilled person will know how to conduct such a test. In some embodiments, the subject is administered lactulose as the substrate for the test.

The hydrogen breath test is also a useful tool for monitoring the effectiveness or likely effectiveness of increasing the microbiota diversity and/or inducing the stability of the microbiota and of treatment or prévention using a composition of the invention. For example, a réduction in the level of hydrogen detected in a subject’s breath following treatment with a composition of the invention may indicate that the treatment is having an increasing, stabilising, therapeutic or preventative effect. Accordingly, in some embodiments the methods and uses of the invention further comprise monitoring the hydrogen level in a subject’s breath during and/or following treatment with a composition of the invention and thereby assessing the effectiveness or likely effectiveness of increasing, stabilising, treatment or prévention. For example, hydrogen levels may be monitored at one or more (e.g. 1, 2, 3, 4 or more than 4) times, for example, including before treatment, at the start of treatment, during treatment, at the end of treatment and/or following treatment, as desired. In some embodiments, the level of hydrogen in the subject’s breath at the end and/or following the dosing period (during which the composition is administered to the subject) is compared to the level at the start and/or before the dosing period and a réduction in the level indicates the effectiveness or likely effectiveness of the increasing, stabilising, treatment or prévention. For example, in embodiments in which the dosing period is 16 days, it may be désirable to take measurements at day 1 and day 16, or for example at day 1, day 2, day 15 and day 16. In some embodiments, multiple measurements are taken and the mean of those measurements obtained (for example, the mean of day 1 and day 2 and the mean of day 15 and day 16). In some embodiments, a réduction in at least 40 ppm in the hydrogen level Cmax indicates that the increasing, stabilising, treatment or prévention is effective or likely to be effective. In some embodiments, the hydrogen level in the subject’s breath is measured only once, for example, at the end of or following treatment, and the finding that the level is at or close to a predetermined level is indicative that the increasing stabilising, treatment or prévention is likely to hâve been effective. The hydrogen breath test is a standard assay and so predetermined levels are known in the art.

Treatment or prévention may refer to, for example, an alleviation of the severity of symptoms or a réduction in the frequency of exacerbations or the range of triggers that are a problem for the subject.

Bacteria in the microbiota may be detected in faeces from a subject, using standard techniques, such as the qPCR techniques used in the examples.

Modes of administration

Preferably, the compositions of the invention are to be administered to the gastrointestinal tract in order to enable delivery to and / or partial or total colonisation of the intestine with the bacterial strain of the invention. Generally, the compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.

In certain embodiments, the compositions of the invention may be administered as a foam, as a spray or a 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 a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.

In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopie gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jéjunum and other suitable access ports.

The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily. The examples demonstrate that daily administration provides successful delivery and clinical benefits.

In certain embodiments, the compositions of the invention are administered regularly, such as daily, every two days, or weekly, for 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 B. hydrogenotrophica administration may not resuit in permanent colonisation of the intestines, so regular administration for extended periods of time may provide greater therapeutic and/or prophylactic benefits.

In certain embodiments of the invention, treatment according to the invention is accompanied by assessment of the subject’s gut microbiota. Treatment may be repeated if delivery of and / or partial or total colonisation with the strain of the invention is not achieved such that efficacy is not observed, or treatment may be ceased if delivery and / or partial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may be administered to a prégnant animal, for example a mammal such as a human in order to prevent reduced levels of diversity in the microbiota and/or reduced stability of the microbiota developing in her child in utero and / or after it is born.

The compositions of the invention may be administered to a subject that has been diagnosed with reduced microbiota diversity relative to a healthy subject and/or reduced stability of the microbiota or a disease or disorder associated with reduced microbiota diversity relative to a healthy subject and/or reduced stability of the microbiota, or that has been identified as being at risk of reduced microbiota diversity relative to a healthy subject and/or reduced stability of the microbiota. The compositions may also be administered as a prophylactic measure to prevent the development of reduced microbiota diversity relative to a healthy subject and/or reduced stability ofthe microbiota in a healthy subject.

The compositions of the invention may be administered to a subject that has been identified as having an abnormal gut microbiota. For example, the subject may hâve reduced or absent colonisation by Blautia, and in particular Blautia hydrogenotrophica, Blautia stercoris or Blautia wexlerae.

The compositions ofthe invention may be administered as a food product, such as a nutritional supplément.

Generally, the compositions of the invention are for the treatment of humans, although they may be used to treat animais including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animais. If administered to animais, oral gavage may be used.

Compositions

Generally, the composition of the invention comprises bacteria. In preferred embodiments of the invention, the composition is formulated in freeze-dried form. For example, the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain ofthe invention.

Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [30-32], The examples demonstrate that lyophilisate compositions are particularly effective.

Alternatively, the composition ofthe invention may comprise a live, active bacterial culture.

In some embodiments, the bacterial strain in the composition of the invention has not been inactivated, for example, has not been heat-inactivated. In some embodiments, the bacterial strain in the composition of the invention has not been killed, for example, has not been heatkilled. In some embodiments, the bacterial strain in the composition of the invention has not been attenuated, for example, has not been heat-attenuated. 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, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine. For example, in some embodiments, the bacterial strain in the composition of the invention is viable and capable of partially or totally colonising the intestine.

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

In preferred embodiments, the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from dégradation until delivery at the target location through, for example, rupturing with Chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [33] and [34].

The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Blautia are anaerobes. Other ingrédients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo. Altematively, the probiotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effective amount of a bacterial strain of the invention. A therapeutically effective amount of a bacterial strain is sufficient to exert a bénéficiai effect upon a subject. A therapeutically effective amount of a bacterial strain may be sufficient to resuit in delivery to and / or partial or total colonisation of the subject’s intestine.

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 10³to about 1 x 10¹¹ colony forming units (CFU); for example, from about 1 x 10⁷ to about 1 x 10¹⁰CFU; in another example from about 1 x 10⁷ to about 1 x 10¹¹ CFU; in another example from about 1 x 10⁸ to about 1 x 10¹⁰ CFU; in another example from about 1 x 10⁸ to about 1 x 10¹¹CFU; in another example from about 1 x 10⁶ to about 1 x 10¹⁰ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cells per day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cells per day.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 10⁶ to about 1 x 10¹¹ CFU/g, respect to the weight of the composition; for example, from about 1 x 10⁸ to about 1 x 10¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.

Typically, a probiotic, such as the composition of the invention, is optionally combined with at least one suitable prebiotic compound. A prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which 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 the présent invention includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans, arable gum modified and résistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molécule to which three glucose molécules are bonded.

The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [35], Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [36], Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol, sorbitol and the like. Examples of suitable diluents include éthanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, cysteine and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. A further example of a suitable carrier is saccharose. A further example of a preservative is cysteine.

The compositions of the invention may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect ofthe invention, such as in a nutritional supplément. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to 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- based product having a varying fat content. The milk-based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.

In certain embodiments, the compositions of the invention contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture or lyophilisate that is substantially free from other species of organism.

In certain embodiments, the compositions of the invention comprise one or more bacterial strains of the genus Blautia, for example, a Blautia hydrogenotrophica, and do not contain any other bacterial genus, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another genus. In certain embodiments, the compositions of the invention comprise a single species of Blautia, for example, a Blautia hydrogenotrophica, and do not contain any other bacterial species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another species. In certain embodiments, the compositions of the invention comprise a single strain of Blautia, for example, of Blautia hydrogenotrophica, and do not contain any other bacterial strains or species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another strain or species.

In some embodiments, the compositions of the invention comprise more than one bacterial strain or species. For example, in some embodiments, the compositions of the invention comprise more than one strain from within 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, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise less than 50 strains from within 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, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, ΙΙΟ, 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 from within the same species and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise more than one species from within 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, do not contain bacteria from any other genus. In some embodiments, the compositions of the invention comprise less than 50 species from within the same genus (e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3 species), and, optionally, do not contain bacteria from any other genus. In some embodiments, the compositions ofthe invention comprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 19, 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 species from within the same genus and, optionally, do not contain bacteria from any other genus. The invention comprises any combination of the foregoing.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises the Blautia bacterial strain, for example, a Blautia hydrogenotrophica bacterial strain as part of a microbial consortium. For example, in some embodiments, the Blautia bacterial strain is présent in combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from other généra with which it can live symbiotically in vivo in the intestine. For example, in some embodiments, the composition comprises a bacterial strain of Blautia hydrogenotrophica in combination with a bacterial strain from a different genus. In some embodiments, the microbial consortium comprises two or more bacterial strains obtained from a faeces sample of a single organism, e.g. a human. In some embodiments, the microbial consortium is not found together in nature. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms. In some embodiments, the two different organisms are from the same species, e.g. two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal.

In some embodiments, the composition of the invention additionally comprises a bacterial strain that has the same safety and therapeutic efficacy characteristics as the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294, but which is not the Blautia hydrogenotrophica strain deposited under accession number DSM 10507/14294, or which is not a Blautia hydrogenotrophica or which is not a Blautia.

In some embodiments in which the composition of the invention comprises more than one bacterial strain, species or genus, the individual bacterial strains, species or généra may be for separate, simultaneous or sequential administration. For example, the composition may comprise ail of the more than one bacterial strain, species or généra, or the bacterial strains, species or généra may be stored separately and be administered separately, simultaneously or sequentially. In some embodiments, the more than one bacterial strains, species or généra are stored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention is obtained from human adult faeces. In some embodiments in which the composition of the invention comprises more than one bacterial strain, ail of the bacterial strains are obtained from human adult faeces or if other bacterial strains are présent they are présent only in de minimis amounts. In some embodiments, the bacteria may hâve been cultured subséquent to being obtained from the human adult faeces and being used in a composition of the invention.

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

The compositions for use in accordance with the invention may or may not require marketing approval.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is spray dried. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is capable of partially or totally colonising the intestine. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable and capable of partially or totally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

The compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to increase the microbiota diversity in a subject and/or induce stability of the microbiota and/or treat a disorder associated with reduced microbiota diversity and/or reduced stability of the microbiota when administered to a subject in need thereof, microbiota diversity, for example, a disease or disorder such as IBS, IBD, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases or one or more metabolic diseases/disorders.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 χ 10³ to about 1 χ 10¹¹ colony forming units per gram with respect to a weight of the composition.

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

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

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol and sorbitol.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of éthanol, 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, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stéarate, magnésium stéarate, 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 esters of p-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4”C or about 25°C and the container is placed in an atmosphère having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

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

The composition of the présent invention may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a tablet or capsule. In some embodiments, the capsule is a gélatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.

Pharmaceutical formulations suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed Systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), émulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an enteric formulation, i.e. a gastrorésistant formulation (for example, résistant to gastric pH) that is suitable for delivery of the composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to dégradation under gastric conditions.

In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric-coated tablet or an enteric-coated capsule, or the like. The enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery. The formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric, for example, gastrorésistant without the need for an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule comprises a shell that does not contain any film forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [37 ]). In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsules are capsules which may, owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, hâve a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gélatine or starch. Gelatine-based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as, for example, orally or rectally, soft capsules can hâve various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional processes, such as, for example, by the Scherer process, the Accogel process or the droplet or blowing process.

Culturing methods

The bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [38-40],

The solid or liquid medium used for culture may be YCFA agar or YCFA medium. YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCI (0.09 g), (NH₄)₂SO₄ (0.09 g), MgSO₄ 7H₂O (0.009 g), CaCI₂ (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 pg), cobalamin (1 pg), p-aminobenzoic acid (3 pg), folie acid (5 pg), and pyridoxamine (15 pg).

Bacterial strains for use in vaccine compositions

The inventors hâve identified that the bacterial strains of the invention are useful for treating or preventing diseases or disorders associated with a level of microbiota diversity that is reduced relative to the microbiota diversity of a healthy subject (or relative to the microbiota diversity of a population of healthy subjects) and/or diseases or disorders that are associated with reduced stability of the microbiota compared to a healthy subject (or compared to a population of healthy subjects). This is likely to be a resuit of the effect that the bacterial strains of the invention hâve on the host immune System. Therefore, the compositions of the invention may also be useful for preventing such diseases or disorders when administered as vaccine compositions. In certain such embodiments, the bacterial strains of the invention are viable. In certain such embodiments, the bacterial strains of the invention are capable of partially or totally colonising the intestine. In certain such embodiments, the bacterial strains of the invention are viable and capable of partially or totally colonising the intestine. In other certain such embodiments, the bacterial strains of the invention may be killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration via injection, such as via subcutaneous injection.

General

The practice of the présent invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references [41] and [42-48], etc.

The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The term “about” in relation to a numerical value x is optional and means, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the définition of the invention.

Référencés to a percentage sequence identity between two nucléotide sequences means that, when aligned, that percentage of nucléotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [49], A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [50],

Unless specifically stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive).

MODES FOR CARRYING OUT THE INVENTION

Example 1 - Changes in patient microbiota after Blautia hydrogenotrophica treatment

Summary

The effect of Blautia hydrogenotrophica on the diversity and stability of patient microbiota was tested in healthy and IBS patients.

Methodology

Study Design

A Phase I clinical trial was conducted in which Blautia hydrogenotrophica (“Blautix”, strain deposited under accession number DSM 10507 and also under accession number DSM 14294) was administered to human patients having IBS or healthy human patients. Patients were administered Blautix during a dosing period (days 1-16) with the washout period being day 1923. Faecal samples were collected from IBS & healthy subjects, placebo or Blautix treated, at: baseline, day 1 (D1) prior to treatment; end of treatment day 16 (D-16); and at end of study (EOS), which was 2-4 weeks (wash-out) post-treatment.

16S Amplicon seguencing

A Qiagen DNeasy Blood & Tissue Kit was used following the manufacturées instructions, to extract microbial DNA from 0.2g of frozen faecal samples from IBS & Healthy subjects, placebo or Blautix treated, at: baseline, day 1 (D1) prior to treatment; end of treatment day 16 (D-16); and at end of study (EOS), which was 2-4 weeks (wash-out) post -treatment.

Préparation and sequencing of the 16S rRNA gene amplicons was carried out using the 16S Sequencing Library Préparation Nextera protocol developed by Illumina (San Diego, California, USA). 50 ng of each of the DNA faecal extracts was amplified using PCR and primers targeting the V3/V4 variable région of the 16S rRNA gene. The products were purified and forward and reverse barcodes were attached by a second round of adapter PCR. The resulting PCR products were purified, quantified and equimolar amounts of each amplicon were then pooled before being sent for sequencing to the commercial Supplier GATC Gmbh., on either the MiSeq (2x250 bp chemistry) or HiSeq (2 x 300 bp chemistry) platforms.

Data Analysis (Post-sequencinq)

The raw sequence data was merge trimmed using flash methodology. This filters out low quality reads. The USEARCH pipeline methodology (version 8.1.1861 J86Jinux64) was used to identify singletons and hide them from the OTU (Operational Taxonomie Unit) generating step. The UPARSE algorithm was used to cluster the sequences into OTUs. Chimeric sequences generated in the amplification step were removed using the UCHIME chimera removal algorithm with the Chimeraslayer reference database (downloaded: 9th September 2016). The USEARCH global alignment algorithm was then used to map ail reads, including singletons onto the remaining OTU sequences. In-house scripts were used to then group the sequences into OTUs as classified by the USEARCH global alignment algorithm. Individual sequences were grouped into OTUs to give microbiome compositional information (abundance and diversity).

Hiqh-level Data analysis

The Bray-Curtis dissimilarity matrix was generated for each sample pairing using the Vegan library in R 3.3.1. Dataset was visualised using Principal Coordinate analysis with the BrayCurtis dissimilarity matrix.

An in-house heatplot R function was used to generate a heatmap visualisation with hierarchical clustering based on the Bray-Curtis dissimilarity and ward linkage.

Shannon and Simpson diversity indexes were generated using the phyloseq library in R.

DESeq2 methodology was used to identify taxonomie variables that were significant for chosen comparisons.

Permutational MANOVA was performed on the dissimilarity matrix using the Adonis function in R.

Results

Samples from ail time points were pooled for both groups (71 IBS patients and 67 healthy contrais including both the Blautix treated and placebo groups). Analysis was performed using a distance measure generated on the full microbiome dataset. Figure 1 reports that the microbiota of IBS subjects is significantly different from that of healthy subjects.

Diversity analysis was carried out using Observed number of predicted Taxa (OTUs), Shannon diversity index and Simpson Diversity index. Both treatment groups showed an increase in diversity at Day 16 timepoint which was significant for the observed OTUs and showed a trend for the Simpson (Raw P-value: < 0.1) (Figure 2). This increase in diversity was not observed with patients treated with the placebo. A significant decrease in microbiota diversity was observed in the untreated IBS placebo group between End of study and Day 1.

Figure 3 reports that Blautix treatment increased the microbiota network connectivity of certain health-associated taxa. In healthy patients a substantial increase in inter-microbe connections was observed from Day 1 to Day 16 (after Blautix treatment), which suggests an increase in coopération and microbiota structure (Figure 3A). Connectivity is correlated with diversity and stability. After the wash period the network structure reverted to a network similar to that observed on Day 1. Blautix treatment was, therefore, able to increase interconnectivity in healthy patients but the effect was lost post wash out. In IBS patients the network remained similar in terms of connectivity between Day 1 and Day 16, but an increase in connectivity was observed by the end of the study suggesting an increased microbiota structure post washout period in Blautix-treated IBS patients (Figure 3B). The effect of Blautix on microbiome connectivity was, therefore, delayed in IBS patients compared to healthy patients.

Instability/change in the microbiota profiles were represented by Bray Curtis distances between time-points of the same subject. Bray-Curtis shows dissimilarity between species abundance profiles limited between 0-1 (0 = same; 1 = do not share any species). Treatment of IBS patients with Blautix reduced the magnitude of microbiota changes during the treatment (Fig. 4A) and after the treatment (Figures 4B-4C). This shows that Blautix increased the stability of the microbiota in IBS patients and that the change continues after the intervention. This increased stability was not observed when IBS patients were administered the placebo (Figures 4 A-C).

Figure 5 reports that there was a significant increase in microbiota diversity at the genus level for IBS patients treated with Blautix at Day 16 compared to Day 1. The diversity analysis was carried out using the Shannon diversity index applied to the Genus level (Raw p-value:0.04, Day 1 versus Day 16).

Figure 6A and Figure 6B show the changes in the mutual exclusion networks in healthy and IBS patients after Blautix treatment. In healthy individuals the mutual exclusion network becomes more dense and interconnected at Day 16, which is suggestive of increased compétition and inhibition. This effect was lost, however, by the end of the study as the network structure reverted back to the initial time point during the washout period (Figure 6A). In IBS patients the effect of Blautix on mutual exclusion connectivity was to increase the network diameter over the treatment period and the washout period. This was opposite to the effect seen in the healthy individuals where the network become denser. During the washout phase for the IBS patients, multiple independent interactions were observed that were not seen previously. Multiple independent interactions represent pairs of taxa that are interacting in a manner that is independent of the rest of the network, i.e. they do not hâve any interactions to the rest of the network.

Visualisation of microbiota shows that after Blautix treatment there was an increased network connectivity for certain health-associated taxa (Figure 7). The health associated taxa include Clostridium cluster IV, Bifidobacterium and Prevotella. Oscillibacter is also potentially a health associated généra. These health-associated taxa are implicated in the response to treatment.

Example 2 - Protective effect in models of neurodevelopmental disorders

The BTBR mouse model

The BTBR mouse model uses inbred, genetically modified mice that display a robust autisticlike phenotype. Déficits in social behaviours, increased répétitive behaviours and increased anxiety-related behaviours hâve been reported in this strain [51]. Due to this robust behavioural phenotype, the BTBR mouse is an idéal animal model to assess the efficacy of novel therapeutic agents for the treatment of autistic-related behaviours. Alleviation of such symptoms by a live biotherapeutic can also be indicative of efficacy of the biotherapeutic in the treatment of other psychiatrie or neurological diseases.

Mice

Male BTBR mice were bred in house. The animais were housed in a température- and humiditycontrolled room on a 12hr dark cycle (lights on from 7:00-19:00 hr). Ail experiments were conducted in accordance with the European Directive 2010/63/EEC, the requirements of S.l. No 543 of 2012, and approved by the Animal Expérimentation Ethics Committee of University College Cork.

Strain

Blautia hydrogenotrophica bacterium deposited under accession number DSM 10507 and also under accession number DSM 14294.

Biotherapeutic was provided in glycerol stock. Live biotherapeutics were grown in the facility in anaérobie conditions.

Live biotherapeutic administration

Dosing with Blautia hydrogenotrophica commenced when the mice were 8 weeks old. These mice were treated once daily for 3 weeks via oral gavage.

Administration schedule

The vehicle for oral administration is PBS. Daily oral administration occurs via oral gavage.

Fecal collection

Fresh fecal samples were collected from individual mice before and after administration of Blautia hydrogenotrophica. At least 20 mg of fresh faeces were placed in a microcentrifuge tube, placed immediately on ice and then stored at -80°C.

Results

The effect of Blautix treatment on microbiota between timepoints (D14, D32) is shown in Figure 8. Significant temporal variation in the microbiota profiles was observed (p-value = 0.001) between the before treatment (D14) and after treatment (D32) study timepoints.

Differential analysis using DESeq2 yielded 25 significant (adjusted p-value< 0.05) differentially 15 abundant taxa for the Blautix treatment between the D14 and D32 Autism study timepoints. The taxa are listed in Table 1 below.

Lowest	Iog2 fold change	st error	adjusted p-value
OTUID	Level	Classification
2440650	Genus	Clostridium XlVa	19.706	3.008	6.9E-10
307526	Species	Bacteroides acidifaciens	11.275	0.912	5.0E-33
39008	Species	Bacteroides acidifaciens	10.501	1.345	1.0E-13
277773	Species	Alistipes finegoldii	9.954	0.906	2.8E-26
1105465	Genus	Barnesiella	9.255	0.923	2.8E-22
943687	Family	Porphyromonadaceae	9.200	0.850	1.1E-25
47662	Species	Barnesiella intestinihominis	8.844	0.988	7.0E-18
181003	Genus	Alistipes	8.370	2.069	4.2E-04

Barnesiella

1282905	Species	intestinihominis	7.373	1.004	2.8E-12
1370810	Species	Barnesiella intestinihominis	6.633	1.986	0.006
1203483	Species	Bacteroides acidifaciens	6.599	1.584	2.7E-04
74179	Species	Alistipes massiliensis	6.318	1.899	0.006
1640334	Species	Barnesiella intestinihominis	6.258	2.066	0.013
76239	Family	Lachnospiraceae	6.202	1.229	4.6E-06
308030	Species	Barnesiella intestinihominis	6.196	1.451	1.8E-04
1156020	Family	Erysipelotrichaceae	5.827	1.607	0.002
712755	Species	Barnesiella intestinihominis	5.614	1.749	0.008
11297	Family	Porphyromonadaceae	5.450	1.021	1.0E-06
2218722	Genus	Clostridium IV	3.983	1.017	0.001
594012	Species	Clostridium lactatifermentans	2.900	0.952	0.013
453043	Species	Eubacterium ventriosum	-3.675	1.260	0.018
451019	Species	Barnesiella intestinihominis	-4.055	1.540	0.041
466087	Species	Akkermansia muciniphila	-6.727	0.876	2.5E-13
2153421	Genus	Blautia XlVa	-8.051	2.577	0.010
866478	Species	Barnesiella intestinihominis	-8.961	0.846	9.3E-25

Table 1. Significant differentially abundant taxa between D14 and D32 time points in the autism study. A positive fold change is interpreted as increased at D32 when compared to D14.

Summary

In a mouse model of autism in which animais were administered Blautix, a significant variation on their microbiome was observed, including a substantial net increase in bacterial diversity.

Example 3 -Effect in models of cérébral ischemia

Summary

The protective effect of Blautia hydrogenotrophica was tested in mouse models of cérébral ischemia. To this end three groups of 5-17 mice were tested. Only normally behaving animais were included in the study. The first dosing day was Day -14. One group received freeze dried bacteria daily from the first dosing day until termination. The control groups received either vehicle or lyobuffer.

On Day 1, ail mice were anesthetized. A midline incision was created in the ventral side ofthe neck to expose the right and left common carotid-arteries. A cérébral ischemia-reperfusion l/R model was then induced by bilateral common carotid artery occlusion (BCCAO) using vascular clips for 15 minutes. At the end of each occlusion, the clips were removed.

Strain

Administration Schedule

No. of Animais	Treatment	Dose Level (mg/kg)	Dose Volumes (ml/kg or ml/animal)
12	PBS (négative control)	n/a	10
17	Freeze-dried Powder	7.8 mg in 100 pl	100 μΙ per animal
13	Freeze-dried Bacteria	15.6 mg in 100μΙ (bacteria dose = 2x10⁸)	100 μΙ per animal

Study design

Days -14 to 14: Daily dose of PBS control (lyobuffer), freeze dried powder control (vehicle) or freeze dried bacteria (Blautix).

Day 1 : Cérébral ischemia-reperfusion l/R model induced by surgery.

Day 14: Half of the mice in each group were terminated.

Day 14 to 28: Daily dose of PBS control (lyobuffer), freeze dried powder control (vehicle) or freeze dried bacteria (Blautix) for the remaining mice in each group.

Day 28: Termination of remaining mice.

Faecal pellets were collected at three time points: Day -14, Day 14 and Day 28. Each take was carried out in a stérile environment (fully aseptie = cleaned between animais), with every mouse being taken out of the cage and placed separately into a new stérile box for individual pellet harvesting. As many pellets as possible were collected in order to reach a minimum of 80mg and preferably 100mg of material per mouse.

Results

No signifïcant différences in microbiota profiles between the Blautix treatment, Vehicle and Lyobuffer groups were detected at D-14 (p-value= 0.177) before administration of Blautix (see Figure 9A). However, signifïcant différences were observed in microbiota profiles between the different treatment groups at Day 14 (see Figure 9B) with a p-value of 0.011 observed. The inventors further assessed the temporal variation in the microbiota of the Blautix treated group and found a signifïcant différence (see Figure 9C) with a p-value of 0.002 observed.

Differential analysis using DESeq2 yielded signifïcant (adjusted p-value <0.05) differentially abundant taxa for the Vehicle, Lyobuffer and Blautix treatment between timepoints in the Stroke study, as shown in Table 2, demonstrating a longer term impact on bacterial diversity imparted by Blautix. The taxa for the Blautix treatment are listed in Table 3, Table 4 and Table 5.

	D-14->D14	D14->D28	D-14->D28
Vehicle	4	0	2
Lyobuffer	17	2	0
/Blautix	7	14	12

Table 2. Signifïcant differential y abundant taxa between time points in the Stroke study.

OTUJD	Lowest	Iog2 fold change	st error	adjusted p-value
Level	Classification
321825	Family	Ruminococcaceae	1.647	0.470	0.027
74771	Species	Alistipes massiliensis	1.530	0.442	0.027
567799	Genus	Alistipes	-1.215	0.308	0.008
77091	Genus	Clostridium	-1.634	0.489	0.036
472737	Family	Lachnospiraceae	-2.585	0.667	0.008
615246	Family	Lachnospiraceae	-3.003	0.711	0.007
166882	Family	Lachnospiraceae	-5.547	1.406	0.008

Table 3. Significant differentially abundant taxa between D-14 and D14 timepoints for the Blautix treatment in the Stroke study. A positive fold change is interpreted as increased at D14 when compared to D-14.

OTUJD	Lowest	Iog2
fold	adjusted p-value
Level	Classification	change	st error
1101936	Order	Clostridiales	3.275	0.709	0.001
218505	Species	Roseburia faecis	2.568	0.630	0.002
948888	Genus	Barnesiella	2.499	0.575	0.001
612631	Genus	Clostridium XlVa	2.473	0.723	0.011
201398	Phylum	Bacteroidetes	2.045	0.605	0.011
1370810	Species	Barnesiella intestinihominis	1.878	0.579	0.016
770554	Species	Alistipes putredinis	1.868	0.626	0.033
558330	Genus	Prevotella	1.795	0.453	0.002

943687	Family	Porphyromonadaceae Barnesiella	1.586	0.546	0.039
308030	Species	intestinihominis	1.324	0.361	0.005
176124	Phylum	Bacteroidetes	1.163	0.294	0.002
565518	Species	Oscillospira guilliermondii	-1.571	0.488	0.016
544582	Species	Flavonifractor plautii	-1.599	0.569	0.050
25678	Species	Mucispirillum schaedleri	-2.751	0.640	0.001

Table 4. Significant differentially abundant taxa between D14 and D28 timepoints for the Blautix treatment in the Stroke study. A positive fold change is interpreted as increased at D28 when compared to D14.

OTU_ID	Lowest Level	Classification	Iog2 fold change	st error	adjusted p-value
688867	Genus	Clostridium XlVa	8.296	1.136	3.8E-11
612631	Genus	Clostridium XlVa	7.814	1.348	3.1E-07
560658	Family	Lachnospiraceae	5.241	1.243	0.001
929749	Species	Eubacterium ruminantium	3.190	0.829	0.003
518034	Species	Desulfovibrio fairfieldensis	3.098	0.982	0.024
74771	Species	Alistipes massiliensis	2.548	0.714	0.007
23310	Species	Odoribacter laneus	1.621	0.475	0.011
117624	Order	Clostridiales	-1.748	0.612	0.049
411272	Genus	Clostridium XlVa	-2.923	1.019	0.049
39008	Species	Bacteroides acidifaciens	-2.953	0.816	0.007
331352	Genus	Clostridium XlVa	-3.969	0.626	1.6E-08

77091 Genus Clostridium -4.247 1.426 0.039

Table 5. Significant differentially abundant taxa between D-14 and D28 timepoints for the Blautix treatment in the Stroke study. A positive fold change is interpreted as increased at D28 when compared to D-14.

Differential analysis using DESeq2 yielded significant (adjusted p-value <0.05) differentially 5 abundant taxa for the Blautix treatment vs. Vehicle as well as Blautix treatment vs. Lyobuffer for the Stroke study timepoints, as shown in Table 6. The taxa are listed in Table 7, Table 8 and Table 9.

	D-14	D14	D28
Blautix vs. Vehicle	0	10	0
Blautix vs. Lyobuffer	2	13	0

Table 6. Significant differential y abundant taxa for the Blautix treatment in the Stroke study.

OTUJD	Lowest Level	Classification	Iog2 fold change	st error	adjusted p-value
25678	Species	Mucispirillum schaedleri	2.604	0.688	0.014
3119687	Family	Lachnospiraceae	2.445	0.642	0.014
321825	Family	Ruminococcaceae	2.174	0.564	0.014
627	Genus	Clostridium XlVa	1.915	0.601	0.043
308030	Species	Barnesiella intestinihominis	-1.324	0.419	0.043
1370810	Species	Barnesiella intestinihominis	-1.540	0.425	0.019
187271	Species	Ruminococcus flavefaciens	-3.475	1.065	0.042

277773	Species	Alistipes finegoldii	-3.751	1.178	0.043
940566	Species	Staphylococcus lentus	-5.228	1.519	0.026
930972	Genus	Staphylococcus	-5.418	1.536	0.023

Table 7. Significant differentially abundant taxa for the Blautix treatment vs. Vehicle at D14 in the Stroke study.

OTUJD	Lowest Level	Iog2
Classification	fold	adjusted p-value
change	st error
1161472	Family	Lachnospiraceae	6.511	1.403	0.001
392940	Kingdom	Bacteria	-5.169	1.346	0.022

Table 8. Significant differentially abundant taxa for the Blautix treatment vs. Lyobuffer at D-14 in the Stroke study

OTUJD	Lowest Level	Classification	Iog2 fold change	st error	adjusted p-value
25678	Species	Mucispirillum schaedleri	2.704	0.753	0.012
1379349	Genus	Clostridium XlVa	2.517	0.771	0.027
742656	Species	Oscillibacter valericigenes	1.738	0.459	0.009
558330	Genus	P revote lia	-1.634	0.406	0.006
1370810	Species	Barnesiella intestinihominis	-1.780	0.390	0.001
712755	Species	Barnesiella intestinihominis	-1.827	0.464	0.006
47662	Species	Barnesiella intestinihominis	-2.109	0.606	0.014
1640334	Species	Barnesiella intestinihominis	-2.260	0.693	0.027

1105465	Genus	Barnesiella	-2.306	0.627	0.010
161658	Family	Lachnospiraceae	-2.565	0.816	0.037
277773	Species	Alistipes finegoldii	-3.619	1.034	0.014
187271	Species	Ruminococcus flavefaciens	-3.924	1.057	0.010
459041	Species	Lactobacillus johnsonii	-4.029	0.981	0.006

Table 9. Significant differentially abundant taxa for the Blautix treatment vs. Lyobuffer at D14 in the Stroke study

Summary

Blautix effects a significant increase in microbiota diversity throughout the period of the study in a mouse model of stroke, when compared to lyobuffer or vehicle control.

Example 4 - Protective effect in models of neuroinflammatory conditions

Experimental Autoimmune Encephalomyelitis (EAE) is a mouse model of CNS inflammation that mirrors many aspects of the human disease MS and EAE is the most commonly used experimental model for human MS. EAE is also used more generally as a model for CNSspecific autoimmune disorders [52] and for other spécifie conditions, including acute disseminated encephalomyelitis. EAE is induced using immunisation with myelin peptides and adjuvants to elicit an immune and inflammatory response that closely corresponds to the mechanisms underlying many autoimmune and inflammatory disorders of the CNS, and in particular MS. Many thérapies showing efficacy in EAE hâve also shown efficacy in treatment of MS in human patients [52], Most importantly, EAE reproduces key features of MS, including inflammation, demyelination, axonal loss and gliosis. The effects of demyelination are mainly restricted to the spinal cord in EAE, with little alteration of the brain stem and the cerebellum. In EAE the CD4+ T cells are the dominant cell population found in the CNS.

Methodology

Blautia hydrogenotrophica (“Blautix”, strain deposited under accession number DSM 10507 and also under accession number DSM 14294) was used as a freeze-dried powder and reconstituted as required.

adult female C57BL/6J mice were used.

On Day 0 and Day 7, animais were administered with an émulsion containing MOG35-55 and complété Freund’s adjuvant (CFA) supplemented with Mycobacterium Tuberculosis H37Ra by subcutaneous injections under gas (isoflurane) anaesthesia. On Day 0, two subcutaneous injections were performed in the flanks; one in each of the lower quadrant of the back. On Day 7, two subcutaneous injections were performed in the flanks, one in each of the upper quadrant of the back.

On Day 0 and Day 2, animais were administered with pertussis toxin (PTx) in phosphate buffered saline (PBS) by intra-peritoneal injections. On Day 0, PTx administration was performed after MOG injections.

Treatments with Blautix or Controls were administered from Day -14 according to the following schedule:

Day 0: MOG/CFA, once, SC

Day 0: PTx, once, IP

Day 2, PTx, once, IP

Day 7: MOG/CFA, once, SC

Treatments were administered within 15 minutes of their préparation. Blautix was administered at a dose of 2 x 10⁸; 10Opl/mouse.

From Day 0 until the end of the experiment, animais were scored daily for clinical signs of EAE, including paresis and paralysis ofthe tail and/or limbs.

On Day -14, Day -1 and Day 34, faecal pellets were collected from each animal, immediately snap-frozen and stored at -80°C.

Results

The effect of Blautix treatment on microbiota between timepoints (D-14, D-1, D34) for the MS model is shown in Figure 10. Significant temporal variation in the microbiota profiles was observed (p-value = 0.001 ) for the study timepoints.

Differential analysis using DESeq2 yielded significant (adjusted p-value< 0.05) differentially abundant taxa for the Blautix treatment between study timepoints, as shown in Table 10. The taxa are listed in Table 11, Table 12 and Table 13.

	D-14->D-1	D-1->D34	D-14->D34
MS (Blautix)	42	30	58

Table 10. Significant differentially abundant taxa between timepoints in the MS study.

OTUJD	Lowest	Iog2 fold	adjusted p-value
Level	Classification	change	st error
1105465	Genus	Barnesiella	8.076	0.702	2.2E-28
48633	Genus	Clostridium XlVa	7.304	0.825	7.0E-17
490405	Species	Turicibacter sanguinis	6.824	0.778	1.0E-16
491106	Species	Flavonifractor plautii	5.116	0.923	4.3E-07
43241	Genus	Clostridium XlVa	5.041	0.739	2.2E-10
948888	Genus	Barnesiella	4.649	0.605	4.3E-13
		Barnesiella
47662	Species	intestinihominis	4.276	0.501	5.1E-16
1288839	Family	Lachnospiraceae	4.117	1.170	0.003
11297	Family	Porphyromonadaceae	4.081	0.600	2.2E-10
198591	Family	Lachnospiraceae	3.757	0.788	2.3E-05
49543	Family	Lachnospiraceae	3.275	0.897	0.002
1009304	Species	Oscillospira guilliermondii	3.140	1.043	0.015
930464	Species	Insolitispirillum peregrinum	2.804	1.033	0.029
1793164	Genus	Parasutterella	2.720	0.576	2.6E-05
1260915	Kingdom	Bacteria	2.678	0.804	0.006
36112	Species	Clostridium leptum	2.584	0.887	0.018
181003	Genus	Alistipes	2.581	0.555	3.3E-05
149837	Family	Lachnospiraceae	2.434	0.678	0.002
1056232	Genus	Clostridium XlVa	2.308	0.856	0.030
770554	Species	Alistipes putredinis	2.223	0.556	0.001
1176501	Family	Lachnospiraceae	2.079	0.758	0.028
33530	Species	Acetatifactor mûris	1.965	0.569	0.004
43033	Genus	Alistipes	1.788	0.379	2.6E-05
576748	Family	Ruminococcaceae	1.740	0.603	0.019
50759	Species	Oscillospira guilliermondii	1.570	0.409	0.001
		Pseudoflavonifractor
592877	Species	capillosus	1.512	0.418	0.002
		Barnesiella
712755	Species	intestinihominis	1.509	0.502	0.015
		Barnesiella
375558	Species	intestinihominis	1.505	0.554	0.029

307526	Species	Bacteroides acidifaciens	1.499	0.492	0.014
74641	Species	Bacteroides acidifaciens	1.418	0.532	0.032
943687	Family	Porphyromonadaceae	1.162	0.397	0.018
791734	Genus	Clostridium XlVa	-1.064	0.377	0.023
19031	Species	Anaerotruncus colihominis	-1.391	0.516	0.030
74179	Species	Alistipes massiliensis	-1.810	0.305	4.7E-08
		Anaeroplasma
211238	Species	abactoclasticum	-2.662	0.859	0.012
76239	Family	Lachnospiraceae	-2.721	0.668	4.2E-04
743544	Genus	Clostridium XlVa	-3.014	0.672	7.0E-05
993522	Genus	Clostridium XlVa	-3.394	0.708	2.2E-05
76325	Genus	Lactobacillus	-3.621	0.575	5.2E-09
209309	Family	Lachnospiraceae	-3.857	1.295	0.016
567799	Genus	Alistipes	-5.435	0.634	4.3E-16
77091	Genus	Clostridium	-6.877	1.048	1.0E-09

Table 11. Significant differentially abundant taxa between D-14 and D-1 timepoints in the MS study. A positive fold change is interpreted as increased at D-1 when compared to D-14.

OTUJD	Lowest	Iog2
fold	adjusted p-value
Level	Classification	change	st error
1370810	Species	Barnesiella intestinihominis	4.794	1.196	0.001
		Parasutterella
1684470	Species	excrementihominis	4.434	1.167	0.002
1070245	Species	Eubacterium plexicaudatum	3.870	0.961	0.001
518034	Species	Desulfovibrio fairfieldensis	3.867	0.962	0.001
1482481	Species	Clostridium disporicum	3.228	1.112	0.029
567799	Genus	Alistipes	3.218	0.864	0.002
1404432	Species	Bacteroides acidifaciens	2.978	0.835	0.004
1067514	Genus	Barnesiella	2.967	0.921	0.011
76325	Genus	Lactobacillus	2.893	0.683	0.001
307526	Species	Bacteroides acidifaciens	2.218	0.351	1.8E-08
1288839	Family	Lachnospiraceae	2.084	0.746	0.035

866478	Species	Barnesiella intestinihominis	1.936	0.647	0.022
23133	Family	Ruminococcaceae	1.840	0.544	0.007
472737	Family	Lachnospiraceae	1.697	0.524	0.011
842401	Order	Clostridiales	1.601	0.535	0.022
39008	Species	Bacteroides acidifaciens	1.494	0.390	0.002
74179	Species	Alistipes massiliensis	1.426	0.328	3.9E-04
277773	Species	Alistipes finegoldii	1.323	0.461	0.029
76234	Family	Lachnospiraceae	-1.183	0.333	0.004
948888	Genus	Barnesiella	-1.453	0.520	0.035
150155	Family	Lachnospiraceae	-1.609	0.421	0.002
783115	Family	Desulfovibrionaceae	-2.262	0.608	0.002
773427	Species	Anaerotruncus colihominis	-2.443	0.661	0.003
201157	Family	Lachnospiraceae	-2.587	0.754	0.006
596894	Genus	Clostridium XlVa	-2.616	0.909	0.029
43033	Genus	Alistipes	-3.236	0.718	2.2E-04
1793164	Genus	Parasutterella	-3.758	0.632	1.4E-07
49543	Family	Lachnospiraceae	-4.849	0.920	5.5E-06
490405	Species	Turicibacter sanguinis	-5.152	0.704	2.5E-11
48282	Family	Lachnospiraceae	-5.460	0.666	4.7E-14

Table 12. Significant differentially abundant taxa between D-1 and D34 timepoints in the MS study. A positive fold change is interpreted as increased at D34 when compared to D-1.

OTU_ID	Lowest	Iog2 fold change	st error	adjusted p-value
Level	Classification
1105465	Genus	Barnesiella	7.221	0.754	2.1E-19
48633	Genus	Clostridium XlVa	6.734	0.959	8.7E-11
1288839	Family	Lachnospiraceae	5.820	0.811	3.5E-11
518034	Species	Desulfovibrio fairfieldensis Parasutterella	5.459	0.999	8.3E-07
1684470	Species	excrementihominis	5.289	1.408	0.001
1482481	Species	Clostridium disporicum	4.947	1.295	0.001
1370810	Species	Barnesiella intestinihominis	4.734	1.263	0.001

1070245	Species	Eubacterium plexicaudatum	4.620	0.794	1.3E-07
1067514	Genus	Barnesiella	4.544	1.103	2.8E-04
1575843	Species	Clostridium ruminantium	4.393	1.743	0.040
43241	Genus	Clostridium XlVa	4.284	0.817	2.4E-06
47662	Species	Barnesiella intestinihominis	4.273	0.478	3.8E-17
		Lachnospiracea incertae
1728285	Genus	sedis	4.204	1.221	0.003
11297	Family	Porphyromonadaceae	3.921	0.577	3.4E-10
307526	Species	Bacteroides acidifaciens	3.539	0.534	9.9E-10
198591	Family	Lachnospiraceae	3.273	0.762	1.4E-04
236126	Species	Oscillospira guilliermondii	3.175	1.086	0.015
930464	Species	Insolitispirillum peregrinum	3.152	0.848	0.001
948888	Genus	Barnesiella	3.040	0.629	1.6E-05
491106	Species	Flavonifractor plautii	3.039	1.170	0.034
563211	Family	Lachnospiraceae	2.564	0.965	0.030
149837	Family	Lachnospiraceae	2.562	0.890	0.016
770554	Species	Alistipes putredinis	2.520	0.455	6.2E-07
1260915	Kingdom	Bacteria	2.505	0.677	0.001
36112	Species	Clostridium leptum	2.483	0.916	0.026
1056232	Genus	Clostridium XlVa	2.319	0.664	0.002
39008	Species	Bacteroides acidifaciens	2.107	0.274	9.9E-13
23133	Family	Ruminococcaceae	2.049	0.614	0.004
74641	Species	Bacteroides acidifaciens	2.002	0.457	1.0E-04
712755	Species	Barnesiella intestinihominis	1.977	0.470	2.0E-04
277773	Species	Alistipes finegoldii	1.881	0.388	1.6E-05
1404432	Species	Bacteroides acidifaciens	1.805	0.563	0.006
1176501	Family	Lachnospiraceae	1.654	0.630	0.032
544582	Species	Flavonifractor plautii	1.418	0.540	0.032
76234	Family	Lachnospiraceae	-0.991	0.389	0.038
80190	Family	Lachnospiraceae	-1.113	0.348	0.006
182471	Order	Clostridiales	-1.315	0.472	0.021
494032	Species	Clostridium oroticum	-1.502	0.580	0.034
2367602	Order	Clostridiales	-1.518	0.472	0.006
74771	Species	Alistipes massiliensis	-1.617	0.408	0.001
172154	Genus	Clostridium XlVa	-1.628	0.442	0.001

993522	Genus	Clostridium XlVa	-1.799	0.594	0.011
791734	Genus	Clostridium XlVa	-1.842	0.387	2.2E-05
150155	Family	Lachnospiraceae	-1.859	0.532	0.002
743544	Genus	Clostridium XlVa	-2.196	0.558	0.001
567799	Genus	Alistipes	-2.378	0.513	3.9E-05
96345	Genus	Clostridium XlVa	-2.528	0.667	0.001
19031	Species	Anaerotruncus colihominis	-2.575	0.610	1.9E-04
201157	Family	Lachnospiraceae	-2.615	0.866	0.011
578360	Family	Lachnospiraceae	-2.870	0.586	1.4E-05
76239	Family	Lachnospiraceae	-3.325	0.631	2.3E-06
1165458	Family	Lachnospiraceae	-3.346	0.754	8.1E-05
773427	Species	Anaerotruncus colihominis	-3.475	0.776	7.0E-05
209309	Family	Lachnospiraceae	-3.639	1.042	0.002
320120	Genus	Clostridium XlVa	-3.670	0.811	5.9E-05
1628488	Species	Vallitalea guaymasensis	-4.144	1.538	0.027
48282	Family	Lachnospiraceae	-4.653	1.012	4.5E-05
77091	Genus	Clostridium	-7.493	1.192	7.9E-09

Table 13. Significant differentially abundant taxa between D-14 and D34 timepoints in the MS study. A positive fold change is interpreted as increased at D34 when compared to D-14.

Summary

Blautix effects a significant increase in microbiota diversity and results in significant temporal 5 variation during treatment in an animal model for multiple sclerosis.

The invention has been described above by way of example only and it will be understood that further modifications may be made which fall within the scope ofthe claims.

Sequences

SEQ ID NO:1 (Blautia stercoris strain GAM6-1 16S ribosomal RNA gene, partial sequence HM626177) tgcaagtcga gcgaagcgct tacgacagaa ccttcggggg aagatgtaag ggactgagcg 61 gcggacgggt gagtaacgcg tgggtaacct gcctcataca gggggataac agttggaaac 121 ggctgctaat accgcataag cgcacggtat cgcatgatac agtgtgaaaa actccggtgg 181 tatgagatgg acccgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg

241 atcagtagcc ggcctgagag ggtgaacggc cacattggga ctgagacacg gcccagactc

301 ctacgggagg cagcagtggg gaatattgca caatggggga aaccctgatg cagcgacgcc

361 gcgtgaagga agaagtatct cggtatgtaa acttctatca gcagggaaga aaatgacggt

421 acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca

481 agcgttatcc ggatttactg ggtgtaaagg gagcgtagac ggaagagcaa gtctgatgtg

541 aaaggctggg gcttaacccc aggactgcat tggaaactgt ttttcttgag tgccggagag

601 gtaagcggaa ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa caccagtggc

661 gaaggcggct tactggacgg taactgacgt tgaggctcga aagcgtgggg agcaaacagg

721 attagatacc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg ggagcaaagc

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 te

SEQ ID NO:2 (Blautia wexlerae strain WAL 14507 16S ribosomal RNA gene, partial sequence EF036467) caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg 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 gggggcaagc

481 gttatccgga tttactgggt gtaaagggag cgtagacggt gtggcaagtc tgatgtgaaa

541 ggcatgggct caacctgtgg aetgeattgg aaactgtcat acttgagtgc cggaggggta

601 ageggaatte ctagtgtagc ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa

661 ggcggcttac tggacggtaa ctgacgttga ggctcgaaag cgtggggagc aaacaggatt

721 agataccctg gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt ggcaaagcca

781 ttcggtgccg tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg caagaatgaa

841 actcaaagga attgacgggg acccgcacaa gcggtggagc atgtggttta attcgaagca

901 acgcgaagaa ccttaccaag tcttgacatc cgcctgaccg atccttaacc ggatctttcc

961 ttcgggacag gcgagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg

1021 gttaagtccc gcaacgagcg caacccctat cctcagtagc cagcatttaa ggtgggcact

1081 ctggggagac tgccagggat aacctggagg aaggcgggga tgacgtcaaa tcatcatgcc

1141 ccttatgatt tgggctacac acgtgctaca atggcgtaaa caaagggaag cgagattgtg

1201 agatggagca aatcccaaaa ataacgtccc agttcggact gtagtctgca acccgactac

1261 acgaagctgg aatcgctagt aatcgcggat cagaatgccg cggtgaatac gttcccgggt

1321 cttgtacaca ccgcccgtca caccatggga gtcagtaacg cccgaagtca gtgacctaac

1381 tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggt

SEQ ID NO:3 (consensus 16S rRNA sequence for Blautia stercoris strain 830)

TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGC GCTTACGACAGAACCTTCGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAA CGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTGGAAACGGCTGCTAATACCGCA TAAG CG CACAGTATCG CATG ATACAGTGTG AAAAACTCCG GTG GTATG AG ATG G ACCCG C GTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCT GAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC AGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGA AGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAG CCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGA TTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCT TAACCCCAG G ACTG CATTG G AAACTGTTTTTCTTG AGTG CCG G AG AG GTAAG CGG AATT CC TAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACT G G ACG GTAACTG ACGTTG AG G CTCG AAAG CGTG GGG AG CAAACAG G ATTAG ATACCCTGG TAGTCCACG CCGTAAACG ATG AATACTAG GTGTTG G G G AG CAAAG CTCTTCG GTG CCG CA GCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATT GACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTATTCGAAGCAACGCGAAGAACCTT ACCAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAACCTTTCCTTCGGGACAGAGAAG ACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCC AGGGATAACCTGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGG CTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGCCCGCGAGGGGGAGCAAAT CCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAAT

CG CTAGTAATCG CG AATCAG AAT GTCG CGGT G AAT ACGTT CCCGGGT CTT GT ACACACCG CCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGA GCTGCCGAAGGCGGGATTGATAACTGGGGTGAAGTCTAGGGGGT

SEQ ID NO:4 (consensus 16S rRNA sequence for Blautia wexlerae strain MRX008)

TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGT GGGTAACCTGCCTTATACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGC GCACAGAGCTGCATGGCTCAGTGTGAAAAACTCCGGTGGTATAAGATGGACCCGCGTTGG ATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCGGCCTGAGAG GGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGG GGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATC TCG GTATGTAAACTTCTATCAGCAG GG AAG AT AGT G ACG GT ACCT G ACT AAG AAG CCCCG GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACT GGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCT GTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTG TAG CG GTG AAATG CGTAG ATATTAGG AG G AACACCAGTG G CG AAGGCG G CTTACTG G ACG GTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC CACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGGTGCCGTCGCAA ACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACG GGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC AAGTCTTGACATCCGCCTGACCGATCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACA GGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCAGGG ATAACCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACA CACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAA AATAACGTCCCAGTTCG G ACTGTAGTCTGCAACCCG ACT ACACG AAG CT G G AAT CGCT AGT AATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCA CACCATG GG AGTCAGTAACG CCCG AAGTCAGTG ACCTAACT G CAAAG AAG G AGCT G CCG A A

SEQ ID NO:5 (Blautia hydrogenotrophica strain S5a36 16S ribosomal RNA gene, partial sequence - X95624.1) gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagcgatag agaacggaga 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 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

Claims

1. A composition comprising a bacterial strain of the genus Blautia, for use in a method of increasing the microbiota diversity and/or inducing stability of the microbiota of a subject.

2. The composition for use according to claim 1, for use in a method of treating or preventing a disease or disorder in a subject, wherein the disease or disorder is associated with a level of microbiota diversity that is reduced relative to the microbiota diversity of a healthy subject, and wherein the treatment or prévention comprises increasing the microbiota diversity in the subject.

3. The composition for use according to claim 2, wherein the subject has a reduced microbiota diversity relative to a healthy subject, optionally wherein the subject has less than 99 different bacterial species and/or less than 190 different bacterial strains in its microbiota.

4. The composition for use according to claim 1, for use in a method of treating or preventing a disease or disorder in a subject, wherein the disease or disorder is associated with reduced stability of the microbiota compared to a healthy subject, and wherein the treatment or prévention comprises inducing stability of the microbiota in the subject.

5. The composition for use according to any one of claims 2 to 4, wherein the subject has reduced stability of its microbiota compared to a healthy subject.

6. The composition for use of any preceding claim, wherein the increase in microbiota diversity and/or induction of stability of microbiota is for:

(a) non-acetogenic bacteria; or (b) acetogenic and non-acetogenic bacteria.

7. The composition for use of any one of claims 1 to 6, wherein the composition is for use in treating or preventing (a) IBS;

(b) IBD;

(c) obesity;

(d) type 2 diabètes;

(e) one or more infectious diseases, for example, a viral, bacterial orfungal disease;

(f) one or more allergie diseases, for example, asthma;

(g) one or more autoimmune diseases, for example, rheumatoid arthritis or multiple sclerosis; or (h) one or more metabolic diseases/disorders, for example, diabètes or obesity.

8. A composition comprising a bacterial strain of the genus Blautia, for use in a method of treatment or prévention of IBS, IBD, obesity, type 2 diabètes, one or more infectious diseases, one or more allergie diseases, one or more autoimmune diseases or one or more metabolic diseases/disorders.

9. The composition for use of any one of the preceding claims, wherein the subject is:

(a) a frail elderly subject; or (b) an infant who has been delivered by Caesarean section.

10. The composition for use of any one of the preceding claims, wherein the microbiota diversity is increased and/or the stability of the microbiota is induced in the intestine and/or the distal gut of the subject.

11. The composition for use of claim 1, wherein the composition comprises a bacterial strain of the species Blautia hydrogenotrophica, for use in a method of increasing the microbiota diversity and/or inducing the stability ofthe microbiota in a subject diagnosed with IBS.

12. The composition for use of any of claims 1-10, wherein the bacterial strain has a 16s rRNA sequence that is at least 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia hydrogenotrophica, or the bacterial strain is Blautia hydrogenotrophica, optionally wherein the bacteria strain is the Blautia hydrogenotrophica bacterium deposited under accession number DSM 14294.

13. The composition for use of any preceding claim, wherein the composition is for oral administration and/or the composition comprises one or more pharmaceutically acceptable excipients or carriers.

14. The composition for use of any preceding claim, wherein the bacterial strain is:

(a) lyophilised; and/or (b) viable; and/or (c) capable of partially or totally colonising the intestine.

15. The composition for use of any preceding claim, which comprises:

(a) a single strain of Blautia; and/or (b) Blautia and does not comprise bacteria from any other genus or comprises such other bacteria only in de minimis amounts; and/or (c) Blautia hydrogenotrophica and does not comprise bacteria from any other species or comprises such other bacteria only in de minimis amounts.

16. The composition for use of any one of claims 1-14, which comprises Blautia as part of a microbial consortium.

17. A food product or vaccine comprising the composition of any preceding claim, for the use of any preceding claim.