OA19090A - Compositions comprising bacterial strains. - Google Patents

Abstract

The invention provides compositions comprising one or more bacterial strains for treating preventing diarrhea and/or constipation.

Description

There is a requirement for the potential effects of gut bacteria to be characterised so that new thérapies using gut bacteria can be developed.

US 2010/0247489 describes the use of minerai nutrients to treat digestive disorders. US‘789 proposes optionally also using numerous different généra of bacteria to prevent a wide variety of 5 abdominal symptoms but does not link any bacteria to any diseases or symptoms.

WO 2016/086206 suggests that bacteria in the order Clostridiales can be used to treat or prevent various conditions but does not link any bacteria to any diseases or symptoms.

-------WO 2012/142605 proposes that it may be possible to treat a number of different discasos with a combination of microorganisms. WO’605 suggests a large number of possible bacterial species that 10 could be eiiiplu^LÜ, but iIilil îj> nu IcaUiiiig hi WO’605 as lu Iww any uf il il ptupubcd lidLlciial species could be used to treat any of the diseases proposed.

WO 02/07741 suggests using various bacteria from the class Clostridia to treat gastro-intestinal disorders but does not link any bacteria to any diseases or symptoms. WO 2016/086209 suggests using a large number of possible bacteria in the order Clostridiales to decrease the sécrétion of pro15 inflammatory cytokines and/or increase the sécrétion of anti-inflammatory cytokines, but there is no teaching as to how any of the proposed bacterial species could be used to treat any of the diseases proposed.

SUMMARY OF THE INVENTION

The inventors hâve developed new thérapies for treating and preventing diarrhea and/or constipation. 20 In particular, the inventors hâve identified that bacterial strains from the genus Blautia can be effective for reducing diarrhea and/or constipation. As described in the examples, oral administration of compositions comprising Blautia hydrogenotrophica may reduce diarrhea and/or constipation in patients having irritable bowel syndrome (IBS). Therefore, in a first embodiment, the invention ΡΓθ¥ί468-&-&θ«ι·ρθ8ί·ΐίθΐί-6θ·ΐ&ρρί$Ηΐ^-3-ί3&ΐ·&ΡΪ&ί-8ΰ^,&ίη-οί-€ΐ6-@6ΐΐΰ8τ8·/Α»^ΰ!. for use-in-a-method-of25 treating or preventing diarrhea and/or constipation.

In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Blautia, for use in a method of treating or preventing diarrhea and/or constipation in a subject diagnosed with Crohn’s disease, ulcerative colitis, or, more preferably, IBS. In some embodiments, the subject diagnosed with IBS has IBS with both diarrhea and constipation. In some embodiments, 30 the subject diagnosed with IBS has IBS with diarrhea but without constipation or with only a small

19090 3 amount of constipation. In some embodiments, the subject diagnosed with IBS has IBS with constipation but without dianhea or with only a small amount of diarrhea. In preferred embodiments of the invention, the bacterial strain in the composition is of Blautia hydrogenotrophica. Closely related strains may also be used, such as bacterial strains that hâve a 16s 5 rRNA sequence that is at least 95%, 96%, 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 has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ΓΓ) T\TO'5 Mnst nrpfp!rAhl\7 fhp bartAvînl ctrnïrt in tliA γϊπ ïo
.siLalnJ.cpo.siicd..uiider ac.c.ession.numDcr.u.MVtoHD.u././-iÆ2.y4·..
10 In further embodiments of the invention, the bacterial strain in the composition is of Blautia
stercoris. Closely related strains may also be used, such as bacterial strains that hâve a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia stercoris. Preferably, the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 3. 15 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 further embodiments of the invention, the bacterial strain in the composition is of Blautia wexlerae. Closely related strains may also be used, such as bacterial strains that hâve a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA 20 sequence of a bacterial strain of Blautia wexlerae. Preferably, the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or 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, _____________In certain embodiments, the composition of the invention is for oral administration Oral
constipation. Also, oral administration is convenicnt for patients and practitioners and allows delivery to and ! or partial or total colonisation of the intestine. In certain embodiments, the composition of the invention comprises one or more pharmaceutically acceptable excipients or carriers. 30 In certain embodiments, the composition of the invention comprises a bacterial strain that has been lyophilised. Lyophilisation is an effective and convenient technique for preparing stable

	19090
	4
	compositions that allow delivery of bacteria, and is shown to provide effective compositions in the examples.
	In certain embodiments, the invention provides a food product comprising the composition as described above.
5	In certain embodiments, the invention provides a vaccine composition comprising the composition as described above.
	Additionally, the invention provides a method of treating or preventing diarrhea and/or constipation.
^uinpiiuiiig mmiimuieiiiig u uumpuLHLiun uumprmmg a BüCTCl'iai BÎl'ain Bt TI1C gémis ifluniiLl.
	In preferred embodiments, the invention provides a composition comprising a bacterial strain of the
10	genus Blautia, for use in a method of treating or preventing a disease associated with Enterobacteriaceae infection, such as E. coli infection. In certain embodiments, the compositions of the invention are for use in treating or preventing diarrhea, gastroenteritis, urinary tract infection or néonatal meningitis.
15	In fùrther preferred embodiments, the compositions of the invention are for use in reducing the level of Enterobacteriaceae in the gastrointestinal tract, preferably the level of E1. coli, in the treatment or prévention of IB S, Crohn’s disease, ulcerative colitis, fünctional dyspepsia, diarrhea, gastroenteritis, urinary tract infection or néonatal meningitis.
20	In particularly preferred embodiments, the compositions of the invention are for use a method of treating or preventing diarrhea associated with Enterobacteriaceae infection, such as E. coli infection, or are for use in reducing the level of Enterobacteriaceae in the gastrointestinal tract, preferably the level of E. coli, in the treatment or prévention of diarrhea.
	BRIEF DESCRIPTION OF DRAWINGS
.Figure jl: cnanges m panent symptoms during dosmg period (days 1-1b) ot Phase 1 climcal tnal.
	Figure 2: Changes in patient symptoms during washout period of Phase I clinical trial.
25	Figure 3: Hydrogen breath test Cmax results for day 1, day 2, day 15 and day 16 for IBS patients treated with Blautix (Figure 3a) and IBS patients treated with placebo (Figure 3b). Figure 3c is a graph comparing the percentage of Blautix treated patients with a réduction in hydrogen between the mean of days 15 and 16 and the mean of days 1 and 2 to the percentage of placebo treated patients with a réduction in hydrogen between these time points.

Figure 4a: Hydrogen uncorrected and hydrogen corrected breath test paired data for day 1 and day for Blautix treated IBS patients; Figure 4b: graph comparing mean hydrogen uncorrected breath test results for day 1 and day 15 for the Blautix treatment group; Figure 4c: graph comparing mean hydrogen corrected breath test results for day 1 and day 15 for the Blautix treatment group,

Figure 5a: Hydrogen uncorrected and hydrogen corrected breath test paired data for day 1 and day for placebo treated IBS patients; Figure 5b: graph comparing mean hydrogen uncorrected breath test results for day 1 and day 15 for the placebo group; Figure 5c: graph comparing mean hydrogen ---------corrected breath test results for day 1 and day 15 for the placebo group.-Figure 6; CraplwLJutpàiiüg Hit mtan hydiugcn bicath test rcsulty lïdffl day 1 aiid day 15 for the......

Bautix treatment group (Verum) and placebo group (Figure 6a: uncorrected hvdrogen: Figure 6h· corrected hydrogen).

DISCLOSURE OF THE INVENTION

Bacterial strains

The compositions of the invention comprise a bacterial strain of the genus Blautia. The examples demonstrate that bacteria of this genus are useful for treating or preventing diarrhea and/or constipation. The preferred bacterial strains are of the species Blautia hydrogenotrophîca, Blautia stercoris and Blautia wexlerae. Other preferred bacterial strains for use in the invention are Blautia producta, Blautia coccoides and Blautia hansenii.

Examples of Blautia strains for use in the invention include Blautia hydrogenotrophîca, 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 [17], Blautia may be isolated from the human gut, although B. producta was isolated from a septicaemia sample.

Blautia hydrogenotrophîca (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 hydrogenotrophîca isi

S5a33 = DSM 10507 = JCM 14656. The GenBank accession number for the 16S rRNA geneί sequence of Blautia hydrogenotrophîca strain S5a36 is X95624.1 (disclosed herein as SEQ IDi

NO:5). This exemplary Blautia hydrogenotrophîca strain is described in [17] and [18], The S5a33j strain and the S5a36 strain correspond to two subclones of a strain isolated from a faecal sample of aί 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 ofSEQ ID N0:5.

The Blautia hydrogenotrophica bacterium deposited under accession number DSM 10507 and also 5 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) in January 1996 ------as “Ruminococcus hydrogenotrophicus” under accession number DSM 10507 and also under ac.cessiorL.num

MIS!

iliUI de Microbiologie CR de Clermont-Ferrand/Theix 63122 Saint Genès Champanelle, France.

Uwnerstup ot the deposits has passed to 4D Pharma Pic by way of assignaient.

The GenBank accession number for the 16S rRNA gene sequence of Blautia stercoris strain GAM61^T is HM626177 (disclosed herein as SEQ ID NO:1). An exemplary Blautia stercoris strain is described in [19]. The type strain of Blautia wexlerae is WAL 14507 = ATCC BAA-1564 = DSM 15 19850 [17]. 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 [17],

A preferred Blautia stercoris strain is the strain deposited under accession number NCIMB 42381, which is also referred to herein as strain 830. A 16S rRNA sequence for the 830 strain is provided in 20 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 is the strain dqiosited-iimlei-aeeesxttiii-imÎÏber-NClAIB 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 30 Blaqutia/Ruminococcus MRx()008” 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 treating or preventing diarrhea and/or constipation. In certain embodiments, the bacterial strain

19090 7 for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia hydrogenotrophica. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5. 5 In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia stercoris. Preferably, the bacterial strain for use in the invention has a 16s rRNA spquence that is at least °5% °6% 97% oooz, 00 5% m 00 oo/ Μηπτΐρτί cm τη
—,QT——tllC— idênt-lt-y—IS -to—SJzîîJ—1-U—pFcfeFahlvTthi11!—-
10 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 95%, 96%, 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 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or 15 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 treating or preventing diarrhea and/or 20 constipation. 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
-----22----10507/14294, NCIMB 42381 or NCIMB 42486 For exfimple^subsianiiaily theA^We-gonom^-may---------
be sequenced and a biotype strain for use in the invention may hâve at least 95%, 96%, 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 30 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 [20], Biotype strains may hâve sequences with at least 95%, 96%, 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 as 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 ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 and has the 16S rRNA sequence of SEQ ID NO:5.

------Altematively, strains that are biotypes of a bacterium deposited under accession number DSM .. .....' ~ y B 4.23-8-LoilN.C1m13^ 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 identîfy 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 20 restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example,[21]). Altematively, 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 25 biotypes of a bacterium deposited under accession nnmhsr 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 30 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 elicït

19090 9 comparable effects on diarrhea and/or constipation 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 5 and shown to be effective for treating disease. 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 described herein.
A dvnvalivL·. uf iLc sliam dcpusiivd under accession number DS1V1 1UDU//!4Zy4, JNCllVLti 42381 or 10 NCIMB 42486 may be a daughter strain inrogenv) or a strain cultur ed isubjcloned)from the nriuinnl
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 15 comparable effects on diarrhea and/or constipation 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 20 strain. References 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 characterîstics as the strains deposited under accession number DSM 10507/14294, and such cells are encompassed by the invention. References to cells ofthe Blautia stercoris strain deposited under accession number
------25-----NCIMB 42381 encompass any cells that hâve the same safety and-therap&utiG-eSÏGaGy-GharaeÎeri^-Îes-----------
as the strains deposited under accession number NCIMB 42381, and such cells are encompassed by the invention. References to cells of the Blautia wexlerae strain deposited under accession number

NCIMB 42486 encompass any cells that hâve the same safety and therapeutic efficacy characterîstics as the strains deposited under accession number NCIMB 42486, and such cells are encompassed by 30 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 preferred embodiments, the compositions of the invention are for use in treating diarrhea and/or constipation. In some embodiments the composition is for use in treating a patient having both diarrhea and constipation. In some embodiments the composition is for use in treating a patient 5 having diarrhea without constipation. In some embodiments the composition is for use in treating a patient having constipation without diarrhea. Although patients often exhibit either diarrhea or constipation, a single patient may exhibit both conditions at different times. Patients having IB S and/or inflammatory diseases of the intestine often exhibit diarrhea and/or constipation. Accordingly, the invention provides the composition of the invention for use in frmiing _a patient 1 u diagnosed witn or suspected ot having IBS or an inflammatory disease of the intestine. For example,

IB S pÎitien.ÎS-ixi.a.y.ËXperj.enca.a.ltema.t.i.ng.c.y&les-aiίΑΪΑ^(Μ^_ιη^-Β&η&ί·ίρΑί4Θΐί-ΘΓ-·»·η-Ι·Β8-ρ'&ΐ'ίθΐί^·ΐΐίη;4χ}characterised as an IBS patient with diarrhea or an IBS patient with constipation. In some embodiments, a composition of the invention may be used to treat a patient having IBS, a patient having IBS with diarrhea or a patient having IBS with constipation. For example, in some embodiments, the subject diagnosed with IBS has IBS with both diarrhea and constipation. In some embodiments, the subject diagnosed with IBS has IBS with diarrhea but without constipation or with only a small amount of constipation. In some embodiments, the subject diagnosed with IBS has IBS with constipation but without diarrhea or with only a small amount of diarrhea. Tn some embodiments, the inflammatory disease is of the small intestine, colon or rectum. Diarrhea is a symptom of Crohn’s disease and ulcerative colitis. Accordingly, a composition of the invention may be used to treat a patient having Crohn’s disease or ulcerative colitis.

As demonstrated in the examples, bacterial compositions of the invention may be effective for reducing diarrhea and/or constipation.

In preferred embodiments, the compositions of the invention are for use in treating or preventing diarrhea and/or constipation associated with Crohn’s disease, ulcerative colitis or, more preferably,

-25IBS—In pieferied embodiments^ the compositions of the invention are for use m treating or preventing diarrhea and/or constipation in a subject diagnosed with Crohn’s disease, ulcerative colitis or, more preferably, IBS. In preferred embodiments the compositions of the invention are for use in treating or preventing diarrhea and/or constipation in the treatment of Crohn’s disease, ulcerative 30 colitis or, more preferably, IBS, In some embodiments, the patient has abdominal pain and/or bloating in addition to diarrhea and/or constipation. In such embodiments, the patient may hâve any combination of two, three or ail four of these conditions. For example, the patient may hâve diarrhea, constipation, abdominal pain and bloating; diarrhea, constipation and abdominal pain;

diarrhea, constipation and bloating; diarrhée, abdominal pain and/or bloating without constipation;

constipation, abdominal pain and/or bloating without diarrhea.

In certain embodiments, the composition may be in the form of a bacterial culture. In some embodiments, the composition may preferabiy be a lyophilisate.

In some embodiments, the composition of the invention is for use in treating diarrhea and/or constipation 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 ot a subject having diarrhea and/or constipation.	The subject is preferabiy a
	10	—uuujuul umgnooea aa naving iu?j ana/pr an iiilldiiiiuatuiy UtseasE· embodiments, the inflammatory disease is of the small intestine.	ui ntu iuitjyLiiid. m yôftie colon or rectum. In some

embodiments, the subject has been diagnosed with Crohn’s disease, ulcerative colitis or, more preferabiy, IBS, for example one of the types of IBS described herein. Without being bound by theory, it is speculated that the increased hydrogen level arises from the mechanisms underlying the inflammation in the intestine. The examples show that treatment with a composition of the invention 15 reduces the level of hydrogen detected in hydrogen breath tests. Accordingly, the hydrogen levels are preferabiy 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 patient 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 20 of treatment or prévention using a composition of an invention. For example, a réduction in the level of hydrogen detected in a subject’s breath following treatment or prévention by a composition ofthe invention may indicate that the treatment is having a 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 or prévention with a 25 composition of the invention and thereby assessing the effectiveness or likely effectiveness of 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 30 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 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 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 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.

-------------In-C.enain..embodimcnts,.the.compQsitions..ol-thc-mvent-ion-ai-e-toi-use-in-Î)rcvent-ina-diaFi^:heaand/'or 10 constipation in a subject that is receiving or has received or is about to receive (for example, later the same day, the next day, or within 2, 3, 4, 5, 6, 7, 10, 14 or 21 days) antibiotic treatment or that is suffering from or has suffered from bacterial gastroenteritis. Antibiotic treatment and bacterial gastroenteritis are associated with changes in the gut microbiota that may précédé diarrhea and/or constipation and that may be prevented by the compositions of the invention. The compositions of 15 the invention may be administered simultaneously, separately or sequentially with an antibiotic treatment. For example, the compositions of the invention may be administered concurrently with an antibiotic treatment.

In preferred embodiments, treatment with compositions of the invention results in a réduction in diarrhea and/or constipation.

Treatment or prévention of diarrhea and/or constipation 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 patient.

In certain embodiments, the compositions of the invention are for use in treating or preventing a disease associated with Entoroèr/ctenfrcwe infection, such as E. coZz infection. In certain

gastroenteritis, urinary tract infection or néonatal meningitis. In such embodiments the Enterobacteriaceae may be a pathogenic strain.

In certain embodiments, the compositions of the invention are for use in treating or preventing a disease associated with increased levels of Enterobacteriaceae, such as E. coli. In certain 30 embodiments, the compositions of the invention are for use in treating or preventing diarrhea, gastroenteritis, urinary tract infection or néonatal meningitis. In such embodiments, the Enterobacteriaceae may be a commensal or non-pathogenic strain.

In preferred embodiments, the compositions of the invention are for use in reducing the level of Enterobacteriaceae in the gastrointestinal tract, preferably the level of E. coli, in the treatment or prévention of a disease associated with increased levels οΐ Enterobacteriaceae, such as IB S, Crohn’s disease, ulcerative colitis, functional dyspepsia, diarrhea, gastroenteritis, urinary tract infection or 5 néonatal meningitis. Enterobacteriaceae and in particular E. coli are known to be potential triggers for, or known to exacerbate, Crohn’s disease and ulcerative colitis [22-24], so the effect shown in the examples for the compositions of the invention may be bénéficiai in the treatment of these conditions.

In certain embodiments, the compositions ofthe invention, are ior use in treating or preventing IBS. Crohn’s disease, ulcerative colitis, functional dyspepsia, diarrhea, gastroenteritis, urinary tract inrection or néonatal meningitis by reducing the level of Enterobacteriaceae in the gastrointestinal tract.

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 20 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.

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 suitabie access ports.

The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. hi certain embodiments, the compositions of the 30 invention are to be administered daily. The examples demonstrate that administration provides successful colonisation and clinical benefîts in treatment of diarrhea and/or constipation.

19090 14 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 BH administration may not resuit in permanent colonisation of the intestines, so regular administration for extended 5 periods of time may provide greater therapeutic benefits. In some embodiments the compositions of the invention are administered for 7 days, 14 days, 16 days, 21 days or 28 days or no more than 7 days, 14 days, 16 days, 21 days or 28 days. For example, in qnmff pmbndÎmpntc tbp rnmnncifînnQ n-ftliA îmrAntïnn qyp QrbnîniotAtwl fnr IA rlcK/o
111 bbl 141111 L111UUU.1111L111S U1 L11U 111VCHLiUllj, UCdLlllClll dLLUlUlliy LU U1C HlvCllllüll IS aLCUllipUillcU Dy 10 assessment of the patient’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, 15 for example a mammal such as a human in order to prevent diarrhea and/or constipation developing in her child in utero and / or after it is bom. The compositions of the invention may be administered to a patient that has been diagnosed with diarrhea and/or constipation or a disease or condition associated with diarrhea and/or constipation, or that has been identified as being at risk of diarrhea and/or constipation. The compositions may also 20 be administered as a prophylactic measure to prevent the development of diarrhea and/or constipation in a healthy patient. The compositions of the invention may be administered to a patient that has been identified as having an abnormal gut microbiota. For example, the patient may hâve reduced or absent colonisation by
Blautia, and in particular Blautia hydrogenotrophica, Blautia stercoris or Blautia wexlerae.
25 The compositions of the 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 încluding 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 30 of animais. If administered to animais, oral gavage may be used.

15 In some embodiments, the subject to whom the composition is to be administered is an adult human. In some embodiments, the subject to whom the composition is to be administered is an infant human. Compositions Generally, the composition of the invention comprises bacteria. In preferred embodiments of the 5 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 of the invention.
Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of
--------------------„---------------------------------------------------------------------—------------------------------.------------------------------------------------------------------------------------------ ... ----------------------------------------------------~--------------------.___________________________________________.
bacieria is a wcii-csiaonsnea proceaure ano relevant guiaance is avanaoie in, ror exampie, rererences 10 Γ25-271. Lvonhilisate compositions mav he nartic.nlarlv effective Tn nreferre.d embo.diments the
compositions of the invention comprises lyophilised bacteria and is for the treatment of diarrhea and/or constipation associated with IB S. Alternatively, the composition of the invention may comprise a live, active bacterial culture. The examples demonstrate that cultures of the bacteria of the invention are therapeutically effective. 15 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 heat-killed. 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 20 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	—
------25----composition ofthe mvcnuün is viable and capable ofpartially or Îotallycolonising the intesTinù. ------------ 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 30 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, selfaggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be usefül for preparing compositions of the invention is available in, for example, references [28-29].

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-deliveiy-...and./„or-pamal.jor.ÎO.ÎaLcolomsaiion ancLsurvi.vaL4»--wvo^A-ltemati-vely,4-he-pr-0bÎ0ti& 10 composition of the invention may be administered orally as a food or nutritional product, such as milK or wliey based iermented dairy product, or as a pharmaceutical product.

The composition may be formulated as a probiotic.

A composition ofthe invention includes a therapeutically effective amount of a bacterial strain ofthe invention. A therapeutically effective amount of a bacterial strain is sufficient to exert a bénéficiai 15 effect upon a patient. A therapeutically effective amount of a bacterial strain may be sufficient to resuit in delivery to and / or partial or total colonisation of the patient’s intestine.

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 10³ to about 1 x 10¹¹ colony forming units (CFU); for example, from about 1 x 10⁷ to about 1 x 1O^{10 *} CFU; in another example from about 1 x 10⁶ to about 1 x 10¹⁰ CFU; in another example from about 1 x 10⁷ 20 to about 1 x 10¹¹ CFU; in another example from about 1 x 10⁸ to about 1 x 1O¹⁰ CFU; in another example from about 1 x 10^s to about 1 x 10¹¹ 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.

4i+eei+aîfl-&mbodtmenfetr+'he-eomÎ>osi'tioiÎ-coiTtérrrr

10⁶to about 1 x 10¹¹ CFU/g, respect to the weight ofthe composition; for example, from about 1 x

10^s to about 1 x 1O¹⁰ CFU/g. The dose may be, for example, 1 g, 3 g, 5g, and 10g.

Typically, a probiotîc, 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 30 upper digestive tract. Known prebiotics include commercial products such as inulin and transgalactooligosaccharides.

In. certain embodiments, the probiotic composition of the present 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, isomalt5 oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), betaglucans, 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 inchiding a saccharose molécule to which 10 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 [30]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [31]. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, 15 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 20 of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, freeflow 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 25 provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, m i umiiÎj i fau i«wa w cjm wj wfey nu ιπ^Μΐι»ίίυιΐί»·Ηΐιιιΐίϊΐιι preservative is selected from sodium benzoate, sorbic acid 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 of the 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

19090 18 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 5 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.
	_
10 In some embodiments, the compositions of the invention comprise one or more bacterial strains of
me genus mautia ana ao nor contain bacteria îroni any orner genus, or wnicli comprise only de minimis or biologically irrelevant amounts of bacteria from another genus. 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 15 minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organisai. In some embodiments, such compositions may be a 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 20 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
25 Blautia, for example, of Blautia hydrogenotrophica, and do not contain anv 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 30 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. Tn 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, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,250, 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 5 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 gémis (o g 1^ tb^n sn 45 4η ?5_; 30, 05, 20, 1U 16, 12, 10, 8, 7, 6, 5, 4 or 3 species), and, optionally, do not contain bacteria from any other genus.

Tn som^emhQdiaaen,to_r4h^CQmpasUiawfeaf4k^inwarien-gemp}iiee-l-T1 20, 1 15, 1-10,

1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 3150 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 as part of a microbial consortium. For example, in some embodiments, the Blautia bacterial strain is present in combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from other 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 djfferetrt-eFgafflsms-are-fromrthe-sanTe-^eciesT^Tg-Iwn-dTfferHrirhnmmTg—1 lÎBjrêjnis t Î a ns 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.

iïi some embodiments, the composition of the invention additionally comprises a bacterial strain that 30 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 5 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.

------hi some embodiments, the bacterial strain for use in the invention is obtained from human adult bacterial strain, ail of the bacterial strains are obtained from human adult faeces or if other bacterial strains are present they are present only in de minimis amounts. 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—Ln^eertairuembediments^ftie^inverrtion^r-evÎdes^tite-ahovg^^phaHnaceüÎieal coinposîtioii, 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.

2l

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 of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the 5 bacterial strain is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is diarrhea and/or constipation, such as diarrhea and/or constipation associated with Crohn’s disease, ulcerative colitis or, more preferably, IB S.

________In certain embodiments, the invention provides the above pharmaceutical composition, wherein the ainuunl uf the bacterial sliain is ûuiii abuiiL 1 a ÏO¹ tu aboli! 1 χ 10¹¹ uolouy fbmilllg llïllïy per gfâtif 10 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, 15 subcutaneous, 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 20 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-lactosc, corn sweelener, acacia/tragacanth, sodium alginatc, carboxymethyl cellulose, polyeihy 1 enc glycoJ, sodium oleatc, sodium slearate, magnésium stéarate, sodium 25 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 p30 hydroxybenzoic acid.

19090 22 In certain embodiments, there is provided the pharmaceutical composition of the invention, wherein the composition does not comprise any minerais, or more specifically, does not comprise any metals with an atomic number greater than 33, for example, wherein the composition does not comprise any minerais from the group consisting of sélénium, molybdenum, tungsten, sélénium compounds, 5 molybdenum compounds and tungsten compounds. 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
Λ11C11 Ulv wvdllpuiSlllvdÎ 14 Ali/lvd ±ai ci avalvil vuiilaiiivi at abuaL 4“C· ui ubuuL diiu U1C CUIlUlInef lü 10 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. 15 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”). 20 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.
rnuimttoouueui lormuiaiions suiiaisie toi· oral admmistiauuii induite suliil plnjjs wnlui
25 microparticulates, semi-solid and liquid (including multiple phases or dispersed Systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquide (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 gastro-résistant 30 formulation (for example, résistant to gastric pH) that is suitabie for delivery ofthe composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful

19090 23 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 enteric5 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 acïd-insohihle polymer
UL JÜIUC CmDOaimUlU, Tllv vlllvllv io uib.juioivcillj' l/iiLl/IIC-, Ibi cAolliplt!, 10 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 15 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 [32 ]). 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, 20 owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, présent in the capsule shell, hâve a certain elasticity and sofitness. Soft capsules can be produced, for example, on the basis of gélatine or starch. Gelatine-based soft capsules are commercially available from varions 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,
25 round, oval, oblong or torpedo-shaped. Soft capsules can be produced hy conventional
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 présent invention can be cultured using standard microbiology techniques as detailed in, for example, references [33-35], 30 The solid or liquid medium used for culture may for example 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), NaCl (0.09 g),

(NH4)₂SO₄ (0.09 g), MgSO₄ · 7H₂O (0.009 g), CaCl₂ (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 ftg)·

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 [36-43], etc.

The term “comprising” encompasses “including” as well as “consisting” e.s. a composition èomprising' A may consisr exclusively or λ or may melude something additional e.g. X !- V.

tu i he terni a boni m relation ro a numencai value x is opuonai and means, îor example, 1U%.

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.

References to a percentage sequence identity between two nucléotide sequences means that, when 15 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. [44], 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 Smith20 Waterman homology search algorithm is disclosed in ref. [45].

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 combîned, omitted or performed in an alternative order, if appropriate.

Vafiôïïs embodiments of the invention are described Hefêin.Tt will be âppreciafédThat the featurcs 25 specified in each embodiment may be combîned with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combîned with each other (except when they are mutually exclusive).

MODES FOR CARRYING OUT THE INVENTION

Example 1 - Changes in patient symptoms during Phase I clinical trial

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 5 administered to human patients having irritable bowel syndrome (IBS). Patients were administered

Blautix during a dosing period (days 1-16) with the washout period being day 19-23. Blautix was found to be both safe and well tolerated. Four symptoms were monitored, of which two were diarrhca and constipation. The study recorded whether patients experienced an improvement in, no

compared with those obtained using patients administered a placebo. Symptoms were monitored at three time points: day 1, day 15/16 and at the end of the study. The results are shown in Figures 1 and 2.

When the patients’ reported symptoms at day 16 were compared to the baseline from day 1, 82% of

IBS patients receiving Blautix reported an improvement in symptoms (Figure 1). Improvement of 15 symptoms, two of which are diarrhea and constipation, supports the use of Blautix for treating or preventing diarrhea and/or constipation.

50% of patients receiving placebo reported an improvement in symptoms (Figure 1). High placebo response rates are an established phenomenon in IBS clinical studies, Xifaxan was recently approved to treat IBS based on much smaller improvements over placebo (see:

http://www.accessdata.fda.gov/spl/data/5ab6fceb-4d22-4480-81fc-8bc28cl6770d/5ab6fceb~4d224480-81fc-8bc28cl6770d.xml ).

A worsening of symptoms at the study completion (day 19-23) compared to symptoms présent upon dosing completion (day 16) is expected based on the teaching presented here. This worsening of -------symptoms was seen in the Phase I clinical trial: 41% of IBS patients reported worsening of 25----------.sympttmts-fcdlvwing-cessÎi-tion-ui' Blautix dcwing-fFiguiu 2). l'Iie wuiseniiig uf syniptoiiis, two of which are diarrhea and constipation, following cessation of Blautix dosing therefore also supports the use of Blautix in treating or preventing diarrhea and/or constipation.

Example 2 — Hydrogen breath test results

Breath hydrogen levels are a biomarker of Blautix activity MoA involves metabolism of 30 endogenous H₂ to produce acetate. Human subjects were administered lactulose and hydrogen (H₂) levels (Cmax) were sampled at four time points: day 1, day 2, day 15 and day 16. Hydrogen uncorrected results were converted to hydrogen corrected results.

Some patients were excluded from the analysis. There were three reasons why a subject was not included in the hydrogen breath test analysis: 1) They produced a CMAX hydrogen breath test resuit of <20 on one of the four sampling days and were therefore deemed to hâve not responded to the test;

2) They were methane producers (taken as producing more methane than hydrogen in the breath test), this affects the hydrogen response; and/or 3) there was an aberrant value obtained in the hydrogen breath test (232 ppm). Subjects 3.12 (Blautix), 3.24 (Blautix), 4.07 (Blautix) were excluded as non-responders. Subjects 3.03 (Blautix) and 3,08 (Placebo) were excluded as methane producers (4.07, mentioned as excluded above, was also a methane producer). Subject 4.09 (Placebo) was excluded due to an aberrant value.

The results of the corrected hydrogen analysis from the end of the dosing period (day 15/16) were compared to those from the baseline (day 1/2). 10 out of 12 patients (83%) receiving Blautix had a réduction in hydrogen levels over this period (Figure 3a and 3c). In contrast, 3 out of 6 (50%) patients receiving placebo had reduced hydrogen levels (Figure 3b and 3c). These percentages are similar to the percentages of patients exhibiting an improvement in symptoms following Blautix 15 treatment or administration of placebo.

Figure 4 shows the uncorrected and corrected hydrogen results for the Blautix (Verum) treatment group together with a statistical analysis of the results. The mean values for both uncorrected and corrected H₂ were found to differ between day 1 and day 15. After 13.5 days of treatment, a statistically significant (p < 0.05) decrease of H₂ in Cmax breath test was detected after lactulose 20 stimulation. In contrast, for the placebo group, the mean was found to be équivalent for day 1 and day 15 (p > 0.05) (Figure 5). Thus, the mean for the treatment group (also referred to as the VERUM group), decreases between day 1 and day 15 whereas the mean for the placebo group is équivalent between day 1 and day 15 for both the uncorrected hydrogen results and the corrected hydrogen results (Figure 6).

Fxample 3 - Stahiïity testing

A composition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25”C or 4°C and the container is placed in an atmosphère having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% ofthe bacterial 30 strain shall remain as measured in colony forming units determined by standard protocols.

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

I fl....... ... - .............-ifaJ—gagjtg.a.a.g.g.a-ag.aag.t.aXc±—cggXaJLgtaa—g^agggaa.q.a—aa-a-faga-egg-t---421 acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca

4SI 00 3 1-1-1-3.^0-00.1^027=3.^.00-03.0.0=0=^3.0.3.0^00,^.3.0.3.0013^-0.1=-==.1000^0^---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 N0:2 (Blautia wexlerae strain WAL 14507 16S ribosomal RNA gene, partial sequence EF036467)

1 oa-a-g^eg-aao gggaattant ttattgaaac ttcggtcqnt ttpafetetaat tetaeftqac.Li 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 ggaggaaeac 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 agatggaqca aatcccaaaa ataacqtccc aqttcqqact gtagtctqca acccgactac

1261 acaaaactaa aatcactaat aato.gnnnat cagaatgr.cg r.ggtgaat-ac gt 1-nrrgggf

1321 uLLLjLauaua eugueuyLua uaucaLyyya youayLJduy CGeyauqLëa ynjüÆôtàad

1381 tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggt

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

TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTACGACAGAACCTT

CGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACA

GTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTATGAGAT

GGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGA

ACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAA

CCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGG

TACCTGACTAAGAAGCCCCGGCTAACTÀCGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTT

ACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGG aaactgtttttcttgagtgccggagaggtaagcggaattcctagtgtagcggtgaaatgcgtagatattaggaggaa caccagtggcgaaggcggcttactggacggtaactgacgttgaggctcgaaagcgtggggagcaaacaggattagat accctggtagtccacgccgtaaacgatgaatactaggtgttggggagcaaagctcttcggtgccgcagcaaacgcaa taagtattccacctggggagtacgttcgcaagaatgaaactcaaaggaattgacggggacccgcacaagcggtggag catgtggtttattcgaagcaacgcgaagaaccttaccaagtcttgacatcgatctgaccggttcgtaatggaacctt tccttcgggacagagaagacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaa

CGAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGGAGG

AAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGG

AAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGA

AGCTGGAATCGCTAGTTÏATCGCGAATCAGAATGTCGCGGTGAATACGTTCCGGGGTCTTGTACACÆCCGCCCGTCAC

ACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAACTG

GGGTGAAGTCTAGGGGGT

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

TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTAT

ACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGAGCTGCATGGCTCAGTGTGAAAAACTC

CGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCG

GCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG

CACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGG

GAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAG

CGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCT

GTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTA

GATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGC

AAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGGTG

CCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCC

GCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCGCCTGACCGA

TCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTT

GGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCA

GGGATAAGCTGGAGGAAGGGGGGGATGAGGTGAAATCATCATGCCCGTTATGATTTGGGCTACACACGTGCTaCAAT ““UUL-U i ΑΑΑι^ΑΑΑυυίϊΑΑΐζ,ι^ίιΗΐΞίΑΐ L-ij 1 ΙιυΑΙίυΛΛΗ,Ι UL-OArtHAAlMAOIj i i i IjTAljTU'iUU

AACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTTGTA

CACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACTGCAAAGAAGGAGCTGCCGAA

SEQ ID N0:5 (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

----------y-4-1- - l-l-c c ca-c c L-g—g g g ag t a c gt - tcgc aa ga a t-ga aac t c a aa g gaa ttg acg -gggac c cgc a

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

30 1441 gactgataac tggggtga REFERENCES [1] Spor et al. (2011) Nat Rev Microbiol. 9(4):279-90. [2] Eckburg ci a/. (2005) Science. 10;308(5728): 1635-8. [3] Tap et al. (2009) Environ Microbiol, 11(10):2574-84 [4] Macpherson et al. (2001) Microbes Infect. 3(12): 1021-35 [5] Macpherson et al. (2002) CellMol Life Sci. 59(12):2088-96.
[6] Mazmanian étal. (2005) Cell 15;122(1):1Ü7-18.
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[8] Scanlan et al. (2006) J Clin Microbiol. 44(11):3980-8.
L9J &l lll. (2U1UJ JiyZ/ÎJMJM JJZJWÎZ U!J. lB(12J!2UJ4-42. [10] Machiels et al. (2013) Gut. 63(8):1275-83. [11] Lopetuso et al. (2013), Gut Pathogens, 5: 23 [12] WO 2013/050792 [13] WO 03/046580 [14] WO 2013/008039 [15] WO 2014/167338 [16] Lee and Lee (2014) World JGastroenterol. 20(27): 8886-8897. [17] LiueieZ. (2008) Int JSyst Evol Microbiol 58,1896-1902. [18] Bemalier et al. (1996) Arch. Microbiol. 166 (3), 176-183. [19] ParkeiaZ. (2012) Int JSyst Evol Microbiol. 62(Pt 4):776-9. [20] Masco et al. (2003) Systematic and Applied Microbiology, 26:557-563. [21] Srûtkovâ et al. (2011) J. Microbiol. Methods, 87(1):10-6. [22] Darfeuille-Michaud et al. (2004) Gastroenterology 127(2):412-21. [23] Strus étal. (2015) Cent Eur .J ImmunolAC>{AyA2(}-?>(}. [24] Petersen et al. (2015) Scand J Gastroenterol.',. [25] Miyamoto-Shinohara et al. (2008) J. Gen. Appl. Microbiol., 54, 9-24.
[26] Cryopreservation and Freeze-Drying Protocols, ed. by Day and McLellan, Humana Press.
[27] Leslie et al. (1995) Appl. Environ. Microbiol. 61, 3592 3597. [28] Mitropoulou et al. (2013) JNutr Metab. (2013) 716861. [29] Kailasapathy et al. (2002) Curr Issues Intest Microbiol. 3(2):39-48. [30] Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller [31] Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) [32] US 2016/0067188 [33] Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press. [34] Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academie Press [35] Strobel (2009) Methods Mol Biol. 581:247-61.

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31
[36] Gennaro (2000) Remington: The Science and Practice ofPharmacy. 20th édition, ISBN: 0683306472. [37] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academie Press). [38] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academie Press, Inc.) [39] Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds, 1986, Blackwell Scientific Publications) [40] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd édition (Cold Spring Harbor Laboratory Press). [41] Handbook ofSurface and Colloïdal Chemistrv (Rirrli k R fri CRG Press 1007)
[43] PCR (Introduction to Biotechniques Sériés), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag) \MAJSun:eat±.r£itojoals inJHohsculaicBicda^tEAA^uwViell.eLal eds 1V.&7 1 SnpplementAû
[45] Smith & Waterman (1981)Tdv. Appl. Math. 2: 482-489.

I

Claims (25)

1. 'CLAIMS

   1. A composition comprising a bacterial strain of the genus Blautia, for use in a method of treating or preventing diarrhea and/or constipatiplL
2. 2. A composition comprising a bacterial strain of the genus Blautia, for use in a method of treating or preventing diarrhea and/or constipation, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID N0:5 or which has the 16s rRNA sequence of SEQ ID N0:5.
3. 3. A composition comprising a bacterial strain ofthe species Blautia hydrogenotrophica, for

   --------use in a method of treating or preventing diarrhea and/or constipation.----------------------------
4. 4. The composition of claims 1-3, wherein Lire diarrhea and/or constipation is associated⁼ T© with IB S and/or an inflammatory disease of the intestine, optionally the inflammatory disease is

   --------------------.
5. 5. The composition of any preceding claim, wherein the diarrhea and/or constipation is associated with IB S, Crohn’s disease or ulcerative colitis.
6. 6. The composition of any one of claims 1 to 5, wherein the composition is for use:

   ^5 (a) in treating or preventing diarrhea and/or constipation in a subject diagnosed with

   IBS, Crohn’s disease or ulcerative colitis; and/or (b) in a method of treating diarrhea wherein the subject has diarrhea without constipation; and/or

   20 (c) in a method of treating constipation wherein the subject has constipation without diarrhea.
7. 7. The composition of any preceding claim, wherein the subject:

   (a) additionally has abdominal pain and/or bloating; and/or

   25 (b) has an increased level of hydrogen in their breath relative to a healthy subject.
8. 8. The composition of any preceding claim, wherein the use further comprises:

   (a) reducing the hydrogen level in the breath of the subject; and/or (b) monitoring the hydrogen level in the subject’s breath during and/or following the ' treatment or prévention and thereby assessing the likcly cffectiveness of treatment j;q or prévention. ;
9. 9. A composition comprising a bacterial strain of the genus Blautia, for use in a method of reducing the level of Enterobacteriaceae in the gastrointestinal tract in the treatment or prévention of diarrhea.
10. 10. A composition comprising a bacterial strain of the genus Blautia, for use in a method of 35 reducing the level of Enterobacteriaceae in the gastrointestinal tract in the treatment or prévention of diarrhea, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5 or which has the 16s rRNA ' _? sequence of SEQ ID NO:5.

    ? , ' 33 ’
11. 11. A composition comprising a bacterial strain of the species Blautia hydrogenotrophica, for use in a method of reducing the level of Enterobacteriaceae in the gastrointestinal tract in the treatment or prévention of diarrhea _{t v}
12. 12. A composition comprising a bacterial strain of the genus Blautia, for use in a method of

    5 treating or preventing diarrhea associated with Enterobacteriaceae infection.
13. 13. A composition comprising a bacterial strain of the genus Blautia, for use in a method of treating or preventing diarrhea associated with Enterobacteriaceae infection
14. 14. , wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID N0:5 or which has the 16s rRNA sequence of ¹⁰----SEQ ID N0:5. —:-------------------:-----------------------------------------................. 15 · Α-ί.ηι·ηρηf 1 nn-r.nmprτ·ίΐηg-a-haetemrl-straitwvf-the-spee-icSi-BlMutia-hydregenel^ephiea-ïoiV use in a method of treating or preventing diarrhea associated with Enterobacteriaceae infection.

    16. The composition of claims 10-15, wherein the Enterobacteriaceae is E. coli.
15. 15 17. The composition of claims 1, 10 and 13, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia stercoris or Blautia wexlerae.
16. 18. The composition of claim 1, wherein the composition comprises a bacterial strain of the species Blautia hydrogenotrophica, Blautia stercoris or Blautia wexlerae for use in a method of

    20 treating or preventing diarrhea and/or constipation in a subject diagnosed with IB S.
17. 19. A composition comprising at least 10⁶ colony forming units (CFUs) of a lyophilized bacteria strain of Blautia having a 16S rRNA gene sequence with at least 90% identity to SEQ ID NO:5 and a pharmaceutically acceptable excipient, diluent, or carrier.
18. 20. The composition of claim 18, wherein the bacterial strain has:

    25 (a) a 16s rRNA sequence that is at least 97%, 98%, 99%, 99.5% or 99.9% identical to

    SEQ ID NO:5 or which has the 16s rRNA sequence of SEQ ID NO:5, or wherein ~ the bacterial strain is Blautia hydrogenotrophica', or (b) a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Blautia stercoris or _m Blautia wexlerae: or wherein the bacterial strain is Blautia stercoris or Blautia wexlerae.
19. 21. The composition of any preceding claim, wherein:

    (a) the composition is for oral administration; and/or (b) the composition comprises one or more pharmaceutically acceptable excipients or

    35 carriers, and/or .

    (c) the bacterial strain is lyophilised; and/or (d) the bacterial strain is viable.
20. 22. The composition of any preceding claim, wherein the composition comprises a single * strain of the genus Blautia or the Blautia bacterial strain as part of a microbial consortium.

    ' 5* .
21. 23. ' A food product or a vaccine composition comprising the composition of any preceding claim, for the use of any preceding claim.
22. 24. The composition of claims 19-22, wherein thç.composition comprises:

    (a) a preservative, optionally whereînthe preservative comprises cysteine; and/or

    5 (b) at least l><10⁷, at least 1 χ 10⁸ or at least IxlO⁹ CFUs of the bacteria of Blautia, optionally wherein the composition comprises at least 1 x 10⁹ CFUs of the bacteria of Blautia', and/or (c) a pharmaceuticaîly acceptable excipient, wherein the pharmaceuticaîly acceptable excipient is selected from the group consisting of starch, gelatin, glucose, ,,__________________ anhydrous lactose, free-flow lactose, beta-lactose, corn swcetener, acacia,

    10 ^{v 1} tragacanth, sodîuih hl^lflillü, UUTbuXyindliyl udlduaC, polyethylene glyeol, sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, sodium acetate, and sodium chloride; and/or (d) the pharmaceuticaîly acceptable carrier, wherein the pharmaceuticaîly acceptable carrier is selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol, sorbitol, and saccharose; and/or
23. 25. The composition of claims 19-23, whereînthe composition is a solid enteric formulation.
24. 26. The composition of claim 24, wherein the enteric formulation is formulated as one or more tablets or capsules, optionally wherein the enteric formulation is formulated as one or more

    20 enteric-coated tablets.
25. 27. The composition of claims 19-25, wherein the composition comprises;

    (a) the pharmaceuticaîly acceptable carriers magnésium stéarate and mannitol; and the preservative cysteine; and/or (b) at least one of a preservative, an antioxidant, and a stabilizer.