WO2022003057A1 - Compositions comprising bacterial strains - Google Patents
Compositions comprising bacterial strains Download PDFInfo
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- WO2022003057A1 WO2022003057A1 PCT/EP2021/068072 EP2021068072W WO2022003057A1 WO 2022003057 A1 WO2022003057 A1 WO 2022003057A1 EP 2021068072 W EP2021068072 W EP 2021068072W WO 2022003057 A1 WO2022003057 A1 WO 2022003057A1
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- Prior art keywords
- bacteroides
- ncimb
- composition
- bacterial strain
- compositions
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- This invention is in the field of bacterial strains and the use of such bacterial strains in the treatment of disease.
- the human intestine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1],
- the human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2],
- the successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions.
- the enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host.
- the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5],
- Bacteroidetes is composed of three classes of gram negative bacteria, including the Bacteroidia class which includes the genus Bacteroides.
- Bacteroides present in the gut are Bacteroides caccae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis, Bacteroides dorei, Bacteroides koreensis, Bacteroides kribbi and Bacteroides xylanisolvens [7],
- Bacteroides spp. play a role in the intestine by digesting complex molecules or producing nutrients for the host. Bacteroides spp. can secrete inflammatory neurotoxins, metalloproteinases and pro-inflammatory molecules, including lipopolysaccharides (LPS) [8], These metalloproteinases can disrupt the gut epithelium and facilitate the propagation of inflammatory neurotoxins and LPS throughout the body. Once the LPS is translocated from the gut to the systemic circulation it is recognised by TLR receptors.
- LPS lipopolysaccharides
- TLR receptors subsequently activate the systemic immune system, resulting in the release of pro-inflammatory cytokines [9,10].
- Certain bacterial strains have been proposed for use in the treatment of various diseases (see, for example, [11-14]).
- Lactobacillus and Bifidobacterium strains have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the gastrointestinal tract (see [15] and [16] for reviews).
- WO2016/102950 describes the efficacy of the Bacteroides species, BT2013, against gastrointestinal inflammatory disorders and the use of BT2013 as a therapeutic agent in preventing inflammatory, autoimmune and allergic disorders. The exact relationship between different diseases and different bacterial strains is poorly understood.
- Bacteroides species including Bacteroides fragilis, Bacteroides theta, Bacteroides thetaiotaomicron, Bacteroides vulgatus and Bacteroides faecalis for treating autism spectrum disorders, epilepsy and schizophrenia [22-27].
- WO02/07741 suggests that compositions comprising Clostridia in combination with other bacterial strains can be used to treat diseases associated with the presence of abnormal microflora in the gastrointestinal tract, including neurological syndromes and psychiatric disorders.
- WO2009/149149 suggests the use of bacteria from the genus Bacteroides in the treatment or prevention of immune-related disorders and in particular the use of Bacteroides fragilis to protect against experimental autoimmune encephalomyelitis.
- this document does not demonstrate the ability of other species of Bacteroides to treat experimental autoimmune encephalomyelitis or the ability of any other bacteria from the genus Bacteroides to treat inflammatory central nervous system disorders or neurodegenerative disease.
- US2008/254009 discloses the manipulation of the gut microbiome, either through the administration of antibiotics or the administration of probiotic mixtures comprising organisms from the Clostridium genus for treating neurological and gastrointestinal disorders.
- WO2017160711 discusses engineering bacteria, and the utilisation of bacteria, to produce GABA for the treatment of mental illnesses or diseases of the central nervous system. A number of bacteria are identified which may be able to inherently do so. Additionally, Table 10 of that document sets out a significantly longer list of 2219 bacterial species which are potentially capable of being engineered to produce GABA but do not appear to possess the ability to do so naturally. There is a need in the art to provide further treatments for treating central nervous system diseases or disorders. SUMMARY OF THE INVENTION The inventors have developed new therapies for treating central nervous system diseases or disorders, in particular those associated with inflammation and oxidative stress. The inventors have demonstrated that bacterial strains from the genus Bacteroides have immuno-inhibitory and anti-oxidant properties.
- compositions comprising bacterial strains from the genus Bacteroides can protect against reactive oxygen species and prevent inflammation and therefore have an antioxidant effect.
- the examples demonstrate the ability of organisms from the genus Bacteroides to induce the production of GABA in human cell lines.
- the invention provides compositions comprising bacteria from the genus Bacteroides for use in treating or preventing a central nervous system disease or disorder.
- the compositions are for use in treating or preventing an inflammatory central nervous system disease or disorder.
- the composition comprises a bacterial strain of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides koreensis, Bacteroides kribbi, Bacteroides thetaiotaomicron Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii or Bacteroides caccae.
- Bacteroides dorei Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides koreensis, Bacteroides kribbi, Bacteroides thetaiotaomicron Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii or Bacteroides caccae.
- the bacterial strain can be the strain deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601. Additionally, the invention provides methods of treating or preventing a central nervous system disease or disorder, comprising administering a composition comprising a bacterial strain of the genus Bacteroides. In preferred embodiments, the invention provides methods of treating or preventing an inflammatory central nervous system disease or disorder.
- the invention provides uses of a composition comprising a bacterial strain of the genus Bacteroides for the manufacture of a medicament for the treatment or prevention of a central nervous system disease or disorder.
- the central nervous system disease or disorder is an inflammatory central nervous system disease or disorder.
- the invention also provides compositions, methods and uses for treating or preventing a central nervous system disease or disorder mediated by dysfunction of the microbiota-gut-brain axis, comprising administering a composition comprising a bacterial strain of the genus Bacteroides.
- the central nervous system disease or disorder may be mediated by the microbiota-gut-brain axis.
- the composition is for use in a method of modulating the microbiota-gut-brain axis.
- the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodegenerative disease, a neurodevelopmental disorder, a neuropsychiatric condition or a brain injury.
- the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodegenerative disease.
- the neurodegenerative disease may be selected from the group consisting of Parkinson’s disease (including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism); Alzheimer’s disease (including Benson's syndrome); multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia and spinal muscular atrophy.
- the invention also provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating a brain injury.
- the brain injury can be the result of a stroke, a traumatic brain injury, an acquired brain injury, a trauma, a brain haemorrhage, a tumour, encephalitis, cerebral hypoxia and/or cerebral anoxia.
- the invention also provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating epilepsy.
- the invention also provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition.
- the neurodevelopmental disorder or neuropsychiatric condition may be selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post- traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment, and corticobasal degeneration.
- the composition of the invention can be for oral administration.
- Oral administration of the strains of the invention can be effective for treating central nervous system diseases and disorders, in particular those mediated by the microbiota-gut-brain axis and/or those associated with inflammation. Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or total colonisation of the intestine.
- the composition of the invention may comprise one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the composition of the invention has been lyophilised.
- the composition of the invention can also comprise a lyophilised bacteria strain of the genus Bacteroides. The bacterial strain may have been lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.
- the bacterial strain is viable and capable of partially or totally colonising the intestine.
- the invention further provides a food product comprising a composition comprising a bacterial strain of the genus Bacteroides as described above.
- the invention also provides a vaccine composition comprising a bacterial strain of the genus Bacteroides as described above.
- the inventors have identified and characterised Bacteroides bacterial strains that are particularly useful for therapy.
- the Bacteroides dorei species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders.
- bacterial strains from the species Bacteroides dorei are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders.
- the invention provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, NCIMB 43599 or NCIMB 43601, or a derivative thereof.
- the invention also provides compositions comprising such cells, or biologically pure cultures of such cells.
- the invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, NCIMB 43599 or NCIMB 43601, or a derivative thereof, for use in therapy, in particular for the diseases described herein.
- the Bacteroides ovatus species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders.
- the invention provides a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600 , or a derivative thereof.
- the invention also provides compositions comprising such cells, or biologically pure cultures of such cells.
- the invention also provides a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof, for use in therapy, in particular for the diseases described herein.
- the Bacteroides stercoris species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders.
- the invention provides a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof.
- the invention also provides compositions comprising such cells, or biologically pure cultures of such cells.
- the invention also provides a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof, for use in therapy, in particular for the diseases described herein.
- the Bacteroides xylanisolvens species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders.
- the invention provides a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof.
- the invention also provides compositions comprising such cells, or biologically pure cultures of such cells.
- the invention also provides a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof, for use in therapy, in particular for the diseases described herein.
- the Bacteroides koreensis species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders.
- bacterial strains from the species Bacteroides koreensis are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof.
- the invention also provides compositions comprising such cells, or biologically pure cultures of such cells.
- the invention also provides a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof, for use in therapy, in particular for the diseases described herein.
- the Bacteroides sp species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders.
- bacterial strains from the species Bacteroides sp are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides sp strain deposited under accession number NCIMB 43593 or NCIMB 43598, or a derivative thereof.
- the invention also provides compositions comprising such cells, or biologically pure cultures of such cells.
- the invention also provides a cell of the Bacteroides sp.strain deposited under accession number NCIMB 43593 or NCIMB 43598, or a derivative thereof, for use in therapy, in particular for the diseases described herein.
- Figure 1 Inhibition of IL-6 secretion in U373 cells by strains of Bacteroides.
- Figure 2 Secretion of IL-6 and IL-10 in peripheral blood mononuclear cells (PBMCs) after treatment with different strains of Bacteroides from an average of ten healthy human donors.
- Figure 3 Comparison of NF-kB activation after treatment with LPS, a-synuclein mutant A53T and different strains of Bacteroides .
- Figure 4 A) The anti-oxidant potential of different strains of Bacteroides; B) the DPPH radical scavenging activity of different strains of Bacteroides; C) the antioxidant concentration of different strains of Bacteroides.
- Figure 4 represents three biological replicates (mean ⁇ SEM).
- Indole production is expressed as concentration calculated from the standard curve.
- Figure 5 Antioxidant capacity of different strains of Bacteroides in SHSY-5Y cells.
- Figure 6 Effect of bacteria from the genus Bacteroides on intestinal ileum permeability
- Figure 7 Effect of bacteria from the genus Bacteroides on intestinal colon permeability
- Figure 8 Effect of bacteria from the genus Bacteroides on the gene expression of the tight junction protein TJP1 in (A) the ileum and (B) the colon.
- Figure 9 Effect of bacteria from the genus Bacteroides on the gene expression of the tight junction protein Occulin in (A) the ileum and (B) the colon.
- Figure 10 Effect of bacteria from the genus Bacteroides on the gene expression of Indoleamine 2,3 dioxygenase-1 (IDO1) (A) the ileum and (B) the colon.
- Figure 11 Effect of bacteria from the genus Bacteroides on the gene expression Tryptophan hydroxylase-1 (TPH-1) in (A) the ileum and (B) the colon.
- Figure 12 Effect of bacteria from the genus Bacteroides on GABA production (A) and the production of GABAergic neuron markers (B) DISCLOSURE OF THE INVENTION Bacterial strains
- the compositions of the invention comprise a bacterial strain of the genus Bacteroides.
- Bacteroides species are gram-negative, obligate anaerobic, non-spore-forming, rod shaped, bile resistant and can be motile or non-motile [28]. Bacteroides species are one of the major species present in the human microbiome.
- Bacteroides species for use in the invention include Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides salyersae, Bacteroides cellulosilyticus, Bacteroides intestinalis, Bacteroides koreensis, Bacteroides kribbi and Bacteroides xylanisolvens.
- the examples show that bacterial strains of the genus Bacteroides have immuno-inhibitory, GABAergic and anti-oxidant properties.
- the bacterial strains of the invention can be selected from the species: Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae.
- the composition can comprise a bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis or Bacteroides kribbi. Therefore, in preferred embodiments of the invention the bacterial strain in the composition is Bacteroides dorei.
- Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides dorei.
- 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 12.
- the sequence identity is to SEQ ID NO:5 or 12.
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 5 or 12.
- the bacterial strain in the composition is Bacteroides ovatus. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides ovatus.
- 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: 11.
- the sequence identity is to SEQ ID NO: 11.
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 11.
- the bacterial strain in the composition is Bacteroides stercoris. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides stercoris.
- 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:7.
- the sequence identity is to SEQ ID NO:7.
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:7.
- the bacterial strain in the composition is Bacteroides xylanisolvens. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides xylanisolvens.
- 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:8.
- the sequence identity is to SEQ ID NO:8.
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:8.
- the bacterial strain in the composition is Bacteroides koreensis.
- Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides koreensis.
- 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: 9.
- the sequence identity is to SEQ ID NO: 9 .
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:9.
- the bacterial strain in the composition is from the genus Bacteroides sp and has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:6 or 10.
- the sequence identity is to SEQ ID NO:6 or 10.
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:6 or 10.
- the compositions of the invention comprise bacterial strains of the species Bacteroides dorei.
- the bacterial strains are NCIMB 43595, NCIMB 43599 or NCIMB 43601.
- the compositions of the invention comprise bacterial strains of the species Bacteroides ovatus. In preferred embodiments, the bacterial strains are NCIMB 43600. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides koreensis. In preferred embodiments, the bacterial strains are NCIMB 43594. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides sp. In preferred embodiments, the bacterial strains are. NCIMB 43593 or NCIMB 43598.In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides stercoris. In preferred embodiments, the bacterial strain is NCIMB 43597.
- compositions of the invention comprise bacterial strains of the species Bacteroides xylanisolvens.
- the bacterial strain is NCIMB 43596.
- the bacterial strain is not of the species Bacteroides fragilis.
- the compositions of the invention may not contain bacterial strains from the species Bacteroides fragilis.
- compositions of the invention may not contain bacterial strains from the genus Clostridia. Examples of Bacteroides vulgatus strains are DSM 1447, DSM 28735, DSM 23289.
- GenBank accession number for the 16S rRNA gene sequence of Bacteroides vulgatus strain DSM 1447 is HQ012024 (disclosed herein as SEQ ID NO:1).
- An exemplary strain of Bacteroides dorei is DSM 17855.
- the GenBank accession number for the 16S rRNA gene sequence of Bacteroides dorei is DSM 17855 is NZ_DS995567.1 (disclosed herein as SEQ ID NO:2).
- Exemplary Bacteroides xylanisolvens strains include DSM 18836.
- DSM 19555 is an example of a Bacteroides stercoris and DSM2151 is an example of a Bacteroides fragilis strain.
- Bacteroides thetaiotaomicron strain BT2013 has been deposited under accession number NCIMB 42341 on 3rd December 2014 at the National Collections of Industrial, Food and Marine Bacteria (NCIMB) at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, UK, AB21 9YA. The deposit was made under the terms of the Budapest Treaty. The deposit was made by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). GT Biologics Ltd. Has subsequently changed its name to 4D Pharma Research Limited.
- BT2013 The genome sequence of BT2013 was provided as SEQ ID NO:1 in WO2016/102950, the contents of which is incorporated herein by reference (and referred to herein as SEQ ID NO:3).
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 42408, which is also referred to herein as strain 675.
- a 16S rRNA sequence for the 675 strain that was tested is provided in SEQ ID NO:4. The deposit was made under the terms of the Budapest Treaty. Strain 675 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroidales 675” on 13th May 2015 by 4D Pharma Research Ltd.
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43593. Also referred to herein as Ref 27. A 16S rRNA sequence for this strain is provided in SEQ ID NO:10. The deposit was made under the terms of the Budapest Treaty. NCIMB 43593 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides sp.” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland).
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43594. Also referred to herein as Ref 17.
- a 16S rRNA sequence for this strain is provided in SEQ ID NO:9. The deposit was made under the terms of the Budapest Treaty.
- NCIMB 43594 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland) as “Bacteroides koreensis” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland).
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43595. The deposit was made under the terms of the Budapest Treaty.
- NCIMB 43595 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides dorei” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland).
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43596. Also referred to herein as Ref 1.
- a 16S rRNA sequence for this strain is provided in SEQ ID NO:8. The deposit was made under the terms of the Budapest Treaty.
- NCIMB 43596 was deposited with the international depositary authority NCIMB, Ltd.
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43597. Also referred to herein as Ref 10.
- a 16S rRNA sequence for this strain is provided in SEQ ID NO:7. The deposit was made under the terms of the Budapest Treaty. NCIMB 43597 was deposited with the international depositary authority NCIMB, Ltd.
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43598. Also referred to herein as Ref 2.
- a 16S rRNA sequence for this strain is provided in SEQ ID NO:6. The deposit was made under the terms of the Budapest Treaty. NCIMB 43598was deposited with the international depositary authority NCIMB, Ltd.
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43599. Also referred to herein as Ref 20. A 16S rRNA sequence for this strain is provided in SEQ ID NO:5. The deposit was made under the terms of the Budapest Treaty. NCIMB 43599 was deposited with the international depositary authority NCIMB, Ltd.
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43600. Also referred to herein as Ref 7. A 16S rRNA sequence for this strain is provided in SEQ ID NO:11. The deposit was made under the terms of the Budapest Treaty. NCIMB 43600 was deposited with the international depositary authority NCIMB, Ltd.
- a preferred Bacteroides strain is the strain deposited under accession number NCIMB 43601. Also referred to herein as Ref 8. A 16S rRNA sequence for this strain is provided in SEQ ID NO:12. The deposit was made under the terms of the Budapest Treaty. NCIMB 43601 was deposited with the international depositary authority NCIMB, Ltd.
- the bacterial strain has a 16s rRNA sequence that is at least 98.65% sequence similarity to SEQ ID NO:5-12. Pairwise similarities between 16S rRNA gene sequences can be calculated based on robust global sequence alignment algorithms such as the EzTaxon server described in [29].
- Bacterial strains closely related to the strain tested in the examples are also expected to be effective for treating or preventing central nervous system diseases and disorders, and in particular inflammatory neurodegenerative disorders and can thus be used in the invention.
- 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 Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae.
- 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: 5-12.
- the sequence identity is to SEQ ID NO: 5-12.
- the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 5-12.
- Bacterial strains that are biotypes of the bacteria disclosed herein are also expected to be effective for treating or preventing central nervous system diseases and disorders, and in particular inflammatory neurodegenerative disorders.
- a biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.
- Strains that are biotypes of the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601.
- substantially the whole genome may be sequenced and a biotype strain for use in the invention may have 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).
- Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG) 5 , or REP or [30].
- Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601.
- the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g.
- Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of the genome.
- the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:3 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:3.
- sequence identity e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity
- the bacterial strain for use in the invention may have a genome with at least 90% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:33, or at least 90% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 30% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 30% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 98% sequence identity to SEQ
- strains that are biotypes of the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 , NCIMB 43601, NCIMB 42408 or NCIMB 42341 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting
- such techniques may be used to identify other Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides kribbi, Bacteroides koreensis or Bacteroides caccae strains.
- strains that are biotypes of the bacteria deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 and that are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and
- biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341.
- the examples demonstrate that bacteria from the genus Bacteroides are capable of inducing the production of GABA, which is useful in the treatment or prevention of central nervous system diseases or disorders, such as epilepsy.
- the bacterial strain for use in the invention can induce GABA production to a greater extent than media alone using the assay described in Example 11.
- bacterial strains from the genus Bacteroides or the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides kribbi, Bacteroides koreensis or Bacteroides caccae that can induce GABA production to a greater extent than media alone.
- strains that are biotypes of the bacteria deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 and that are suitable for use in the invention are strains which can induce the production of GABA to a greater extent than media.
- Bacteroides strains that are useful in the compositions and methods of the invention, such as biotypes of the bacteria deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341, may be identified using any appropriate method, including the assays described in the examples.
- strains for use in the invention may be identified by culturing a candidate strain with inflammatory neuroblastoma cells and then assessing cytokine levels and levels of inflammatory neuroprotection or inflammatory neuro- proliferation.
- bacterial strains that have similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 may be useful in the invention.
- a useful strain will have comparable immune modulatory activity to the NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 strain.
- a biotype strain will elicit comparable effects on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
- a biotype strain will be able to reduce IL-6 in neuroblastoma cells, increase IL-6 and IL-10 secretion in peripheral blood mononuclear cell (PBMCs) or have a high antioxidant capacity compared to compared to YCFA+ media.
- the Bacteroides dorei species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders.
- a particularly preferred strain of the invention is the Bacteroides dorei strain deposited under accession number NCIMB 43595. This is the exemplary strain Ref 25 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders.
- the invention provides a cell, such as an isolated cell, of the Bacteroides dorei strain deposited under accession number NCIMB 43595, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides dorei strain deposited under accession number NCIMB 43595
- the invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- a particularly preferred strain of the invention is the Bacteroides dorei strain deposited under accession number NCIMB 43599.
- This is the exemplary strain Ref 20 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides dorei strain deposited under accession number NCIMB 43599, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43599, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides dorei strain deposited under accession number NCIMB 43599.
- the invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43599, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- a particularly preferred strain of the invention is the Bacteroides dorei strain deposited under accession number NCIMB 43601. This is the exemplary strain Ref 8 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides dorei strain deposited under accession number NCIMB 43601, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43601, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides dorei strain deposited under accession number NCIMB 43601.
- the invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43601, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- a particularly preferred strain of the invention is the Bacteroides ovatus strain deposited under accession number NCIMB 43600.
- the invention provides a cell, such as an isolated cell, of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides ovatus strain deposited under accession number NCIMB 43600.
- the invention also provides a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- a particularly preferred strain of the invention is the Bacteroides koreensis strain deposited under accession number NCIMB 43594. This is the exemplary strain Ref 17 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides koreensis strain deposited under accession number NCIMB 43594.
- the invention also provides a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 435934 or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- the invention provides a composition comprising the species Bacteroides koreensis for use in therapy.
- the Bacteroides species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders.
- a particularly preferred strain of the invention is the Bacteroides strain deposited under accession number NCIMB 43593. This is the exemplary strain Ref 27 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides strain deposited under accession number NCIMB 43593, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides strain deposited under accession number NCIMB 43593, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides strain deposited under accession number NCIMB 43593.
- the invention also provides a cell of the Bacteroides strain deposited under accession number NCIMB 43593, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- a particularly preferred strain of the invention is the Bacteroides strain deposited under accession number NCIMB 43598. This is the exemplary strain Ref 2 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides strain deposited under accession number NCIMB 43598, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides strain deposited under accession number NCIMB 43598, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides strain deposited under accession number NCIMB 43598.
- the invention also provides a cell of the Bacteroides strain deposited under accession number NCIMB 43598, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- the Bacteroides xylanisolvens species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders.
- a particularly preferred strain of the invention is the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596.
- This is the exemplary strain Ref 1 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596.
- the invention also provides a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- the Bacteroides stercoris species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders.
- a particularly preferred strain of the invention is the Bacteroides stercoris strain deposited under accession number NCIMB 43597.
- the invention provides a cell, such as an isolated cell, of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof.
- the invention also provides a composition comprising a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof.
- the invention also provides a biologically pure culture of the Bacteroides stercoris strain deposited under accession number NCIMB 43597.
- the invention also provides a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders.
- a derivative of the strain deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original.
- a derivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity.
- a derivative strain of the invention is therapeutically active.
- a derivative strain will have comparable immune modulatory activity to the original NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601strain.
- a derivative strain will elicit comparable effects on the central nervous system disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
- a derivative of the NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601 strain will generally be a biotype of the strain NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601.
- the bacterial strain may also be a strain that has the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601, and such cells are encompassed by the invention.
- the bacterial strains in the compositions of the invention are viable and/or live. They may be capable of partially or totally colonising the intestine.
- the bacterial strain for use in the invention is naturally-occurring.
- the bacterial strain may have been isolated from the mammalian digestive tract.
- the bacterial strain for use in the invention has not been not genetically engineered.
- the bacterial strain may not have been transformed with recombinant DNA.
- Therapeutic uses demonstrate that bacterial strains from the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae can decrease the secretion of the pro-inflammatory cytokine IL-6 in human glioblastoma astrocytoma cells.
- bacteria from the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides koreensis, Bacteroides kribbi and Bacteroides xylanisolvens have high antioxidant capacities and can protect neuroblastoma cells from oxidative damage. Therefore, the inventors have shown that bacteria from the genus Bacteroides are particularly effective in preventing or treating central nervous system diseases or disorders, in particular those associated with inflammation. Central nervous system diseases or disorders include neurodegenerative diseases, neurodevelopmental disorders, neuropsychiatric conditions and brain injuries.
- the invention provides compositions comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodegenerative disease.
- the neurodegenerative disease may be selected from the group consisting of Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia and spinal muscular atrophy.
- Parkinson’s disease including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arterioscler
- compositions of the invention may be useful for treating central nervous system diseases or disorders in adult patients and in particular inflammatory central nervous system diseases or disorders.
- the patients may be between 18 and 65 years old, for example between 40 to 65 years old; or they may be older than 40 years.
- the compositions of the invention may be for use in treating a disease associated with old age, for example, a disease diagnosed in a patient who is older than 50 years.
- the compositions of the invention may be effective at treating for treating central nervous system diseases or disorders, such as neurodegenerative diseases that occur in elderly patients, for example, the patients are 65 years or older.
- the inventors have found that bacterial strains of the genus Bacteroides can reduce the secretion of pro-inflammatory cytokines, such as IL-6.
- Bacteroides strains according to the invention can decrease the secretion of IL-6 in human glioblastoma astrocytoma cells. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prevention of central nervous system diseases or disorders, in particular those associated with inflammation. In some embodiments, the bacterial strains are useful in the treatment of central nervous system diseases or disorders characterised by the enhanced activation of IL-6, such as inflammatory central nervous system diseases or disorders. Therefore, the compositions of the invention may be useful for decreasing the levels of IL-6, particular in patients with a neurodegenerative disease.
- IL-10 is an anti-inflammatory cytokine that is synthesised in the CNS and can act to limit clinical symptoms of stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioural changes that occur during bacterial infections [32].
- the examples show that the bacterial strains of the invention can increase the per se production of IL-10.
- the compositions of the invention may be useful for treating or preventing central nervous system diseases or disorders, including stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioural changes that occur during bacterial infections.
- the bacterial strains may be useful for treating or preventing central nervous system diseases or disorders that are characterised by a reduction in the production of IL-10, such as inflammatory central nervous system diseases or disorders.
- the inventors have demonstrated that bacterial strains of the genus Bacteroides can decrease the gut permeability. Bacteria can modulate signalling of the microbiota-gut-brain axis by modulating the levels of gastrointestinal permeability. Abrogation of the gut epithelial barrier allows harmful substances to leave the intestine and is linked to the induction of inflammatory and autoimmune diseases. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prevention of central nervous system diseases or disorders characterised by increased gut permeability. The bacterial strains of the invention are also useful in the treatment or prevention of inflammatory central nervous system diseases or disorders characterised by increased gut permeability.
- compositions of the invention may be useful for decreasing gut permeability, particularly in patients with a neurodegenerative disease.
- the examples show that bacterial strains of the genus Bacteroides have potent antioxidant activity.
- bacterial strains from the genus Bacteroides produce high levels of the known antioxidant indole, have high radical scavenging activity (e.g. against DPPH radicals) and can reduce the production of reactive oxygen species (ROS).
- ROS reactive oxygen species
- the bacterial strains are useful in the treatment of central nervous system diseases or disorders characterised by oxidative stress, and in particular those associated with inflammation.
- the compositions of the invention may be useful for reducing the oxidative stress by increasing the production of antioxidants, particularly in patients with a neurodegenerative disease.
- the bacterial strains of the invention useful in the treatment of central nervous system diseases or disorders characterised by increased ROS levels, and in particular those associated with inflammation.
- the compositions of the invention may be useful for reducing the production of ROS, particularly in patients with a neurodegenerative disease.
- the compositions of the invention may be for use in reducing or preventing the loss of dopaminergic cells in the substantia nigra.
- the compositions of the invention may be used for reducing or preventing the degeneration of dopaminergic inflammatory neurons in the substantia nigra pars compacta.
- compositions of the invention may be used for reducing or preventing the degeneration of dopaminergic inflammatory neurons in the substantia nigra pars compacta and the consequent loss of their projecting nerve fibres in the striatum.
- the compositions of the invention may be used for reducing or preventing loss of nigrostriatal dopaminergic inflammatory neurons.
- the compositions of the invention may be for use in treating a central nervous system disease or disorder mediated or characterised by the accumulation of protein, in particular mis-folded protein, such as an inflammatory central nervous system disease or disorder.
- the compositions of the invention are for use in treating a central nervous system disease or disorder associated with grey matter inflammatory neuronal loss, such as an inflammatory central nervous system disease or disorder.
- compositions of the invention are for treating a central nervous system disease or disorder that is not associated with white matter lesions, such as an inflammatory central nervous system disease or disorder.
- the compositions of the invention may be for use in treating a central nervous system disease or disorder associated with permanent symptoms, such as an inflammatory central nervous system disease or disorder. Permanent symptoms are present on clinical examination and expected to last throughout the person’s life.
- Permanent neurological symptoms include numbness, hyperaesthesia (increased sensitivity), paralysis, localised weakness, dysarthria (difficulty with speech), aphasia (inability to speak), dysphagia (difficulty in swallowing), visual impairment, difficulty in walking, lack of co- ordination, tremor, seizures, lethargy, dementia, delirium and coma.
- the neuroprotective properties of the compositions of the invention mean that the compositions may be particularly effective for preventing or delaying onset or progression of a central nervous system disease or disorder, such as an inflammatory central nervous system disease or disorder.
- the compositions of the invention are for use in delaying onset or progression of a central nervous system disease or disorder, and in particular an inflammatory central nervous system disease or disorder.
- bacteria from the species Bacteroides stercoris have anti- inflammatory and anti-oxidant properties. These properties make bacteria strains from the species Bacteroides stercoris particularly effective at treating or preventing an inflammatory disease, an autoimmune disease and cancer.
- the invention provides a method of treating or preventing an inflammatory disease, an autoimmune disease and cancer, comprising administering a composition comprising a bacterial strain of the species Bacteroides stercoris to a subject.
- the invention provides a use of a composition comprising a bacterial strain of the species Bacteroides stercoris for the manufacture of a medicament for the treatment or prevention of an inflammatory disease, an autoimmune disease and cancer.
- the invention provides a composition comprising a bacterial strain of the species Bacteroides stercoris for use in a method of treating or preventing an inflammatory disease.
- the inflammatory disease can be selected from the group consisting of: adult-onset Still's disease, amyloid A amyloidosis, polymyalgia rheumatica, remitting seronegative symmetrical synovitis with pitting edema, Behcet's disease, uveitis, graft-versus-host diseases, and tumor necrosis factor receptor-associated periodic syndrome, Systemic sclerosis, ANCA-associated vasculitis, Takayasu arteritis, osteoarthritis, osteoporosis, multiple sclerosis, asthma, atopic dermatitis, sciatica, Polymyalgia rheumatica, chronic glomerulonephritis, inflammatory bowel disease and cardiovascular disease (such as pulmonary arterial hypertension, atherosclerosis and Non-ST elevation myocardial infarction).
- adult-onset Still's disease amyloid A amyloidosis
- polymyalgia rheumatica remitting sero
- compositions comprising a bacterial strain of the species Bacteroides stercoris for use in a method of treating or preventing an autoimmune disease, wherein the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, systemic sclerosis, polymyositis, vasculitis syndrome including giant cell arteritis, Takayasu arteritis, cryoglobulinemia, myeloperoxidase-antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis and rheumatoid vasculitis; organ-specific, rheumatoid arthritis, juvenile idiopathic arthritis; systemic onset juvenile idiopathic arthritis, Graves ophthalmopathy, Relapsing polychondritiis, ankylosing spondylitis, Type II diabetes, obesity, Crohn's disease, relapsing polychondritis, Castleman’s disease, Neuromyelitis optica (
- compositions comprising a bacterial strain of the species Bacteroides stercoris for use in a method of treating or preventing an cancer, wherein the cancer is selected from the group consisting of: multiple myeloma, prostate cancer, metastatic renal cell carcinoma, metastatic kidney cancer, non-small cell lung cancer, colorectal cancer,;andpancreatic cancer, T-cell acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukaemia, multiple myeloma, colorectal cancer and myeloma (such as multiple myeloma).
- the cancer is selected from the group consisting of: multiple myeloma, prostate cancer, metastatic renal cell carcinoma, metastatic kidney cancer, non-small cell lung cancer, colorectal cancer,;andpancreatic cancer, T-cell acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukaemia, multiple myeloma, colorectal cancer
- compositions according to the invention may also be for use in a method of treating or preventing an inflammatory disease, an autoimmune disease or cancer associated with oxidative stress.
- the inflammatory disease, autoimmune disease or cancer associated with oxidative stress is selected from gastrointestinal diseases; (such as peptic ulcers) gastrointestinal cancers; inflammatory bowel disease; cardiovascular diseases; (such as atherosclerosis), ischemia; hypertension; cardiomyopathy; cardiac hypertrophy and congestive heart failure; renal diseases, such as glomerulonephritis and tubulointerstitial nephritis, chronic renal failure, proteinuria, uremia; inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD); ocular diseases (such as cataracts and retinal diseases) and joint diseases (such as rheumatoid arthritis and rheumatism).
- COPD chronic obstructive pulmonary disease
- compositions comprising bacterial strains of the genus Bacteroides.
- the inventors have also shown that treatment with bacterial strains from the genus Bacteroides can reduce the activation of proinflammatory molecules, such as NF ⁇ B and IL-6, by LPS and mutant ⁇ -synuclein A53T. This decrease is particularly effective in human glioblastoma astrocytoma cells.
- the compositions of the invention therefore, may be particularly effective at preventing or delaying the onset or progression of neurodegenerative diseases.
- the compositions of the invention are for use in delaying the onset or progression of neurodegenerative diseases.
- compositions of the invention can be measured relative to the disease state or disorder level observed in the patient before treatment, a healthy individual or an individual with a more severe form of the neurodegenerative disease or disorder.
- Chronic inflammation induced by IL-6 can ultimately lead to cell death.
- Bacterial strains of the invention can be used to reduce or prevent neuronal cell death. Therefore, in preferred embodiments, the compositions of the invention are for use in reducing or preventing neuron death, in particular, in the treatment of neurodegenerative diseases.
- compositions of the invention can be measured relative to a healthy individual, an individual with a more severe form of the neurodegenerative disease or to the rate of neuron cell death observed in the patient before treatment with the compositions of the invention.
- administration of LPS can replicate some characteristics of Parkinson’s disease, including extensive activation of microglia and selective loss of dopaminergic neurons in the nigrostriatal system [33].
- the inventors have demonstrated that bacterial strains of the genus Bacteroides can decrease the secretion of the pro-inflammatory cytokine IL-6 after induction with LPS. Therefore, in preferred embodiments, compositions comprising bacterial strains from the genus Bacteroides may be particularly effective at treating environmental Parkinson’s.
- IL-6 can act as an anti-inflammatory regulator by stimulating potent anti-inflammatory cytokines such as IL-10 [34, 35].
- potent anti-inflammatory cytokines such as IL-10 [34, 35].
- the examples show that bacterial strains of the genus Bacteroides have anti- inflammatory activity and can induce the per se production of IL-6 and IL-10 in peripheral blood mononuclear cells. Therefore, the compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are the result of inflammation.
- bacterial strains of the genus Bacteroides are useful in the treatment of neurodegenerative diseases characterised by the reduced production of IL-6.
- IL-10 limits inflammation in the brain via three major pathways: (1) reducing the synthesis of proinflammatory cytokines, (2) suppressing cytokine receptor expression, and (3) inhibiting receptor activation [Error! Bookmark not defined.]. Therefore, in certain embodiments the composition of the invention may be useful for treating or preventing inflammation in the brain. IL-10 promotes the survival of neurons and all glial cells in the brain by blocking the effects of proapoptotic cytokines and by promoting expression of cell survival signals [Error! Bookmark not defined.]. Therefore, in certain embodiments the invention can increase the survival of neurons in the brain. In other embodiments, the compositions of the invention can increase the survival of glial cells in the brain.
- compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are the result of neuronal or glial cell death in the brain.
- the cytokines IL-6 and IL-10 are known to share the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signalling pathway [34,35].
- Activation of the JAK/STAT3 signalling pathway by IL-10 promotes cell survival by inhibiting both ligand- and mitochondrial-induced apoptotic pathways [Error! Bookmark not defined.].
- the JAK/STAT3 pathway is a common mediator of astrocyte reactivity which is a hallmark of neurodegenerative diseases, such as Alzheimer's and Huntington's diseases [36].
- the bacterial strains of the invention can activate the JAK/STAT3 pathway.
- the compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are mediated by the STAT-3 signalling pathway.
- the bacterial strains of the invention can promote astrocyte reactivity. Therefore, in certain embodiments, the composition of the invention may be useful for treating or preventing neurodegenerative diseases, such as Alzheimer's and Huntington's disease that are the result of astrocyte reactivity.
- the examples demonstrate that compositions of the invention can decrease the activation of the NF- ⁇ B promoter which activates cytokine production, for example the cytokines IL-1 ⁇ , IL-1 ⁇ , IL-18, TNF ⁇ and IL-6.
- compositions of the invention may be effective at treating Parkinson’s disease by inhibiting the ability of TLR4 signalling to activate the NF- ⁇ B promoter.
- Oxidative damage is implicated in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease and stroke (brain ischemia/reperfusion injury) [38].
- compositions of the invention may be particularly effective at treating neurodegenerative diseases by reducing the oxidative damage by free radicals.
- the compositions of the invention can therefore be effective for treating neurodegenerative diseases with a pathogenesis of oxidative damage, including Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease and stroke (brain ischemia/reperfusion injury).
- Tau associated neurodegenerative diseases are neurodegenerative diseases associated with the pathological aggregation of tau protein in neurofibrillary or gliofibrillary tangles in the human brain.
- Alzheimer’s disease is an example of a tauopathology.
- Synucleinopathies also called ⁇ -Synucleinopathies
- Parkinson’s disease is an example of a synucleinopathology. There is clinical and pathological overlap between these two pathologies. Parkinson's disease patients frequently have dementia and Alzheimer's disease patients often manifest parkinsonism [39].
- LBD Lewy body disease
- compositions of the invention may be useful for treating or preventing tauopathies.
- the compositions of the invention may be useful for treating or preventing Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson- Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism.
- the compositions of the invention may be useful for treating or preventing Alzheimer’s disease, including Benson's syndrome.
- the compositions of the invention may be useful for treating or preventing dementia; including Lewy body; vascular and frontotemporal dementia.
- Parkinson’s disease Parkinson’s disease is a common neurodegenerative disease neuropathologically characterised by degeneration of heterogeneous populations of neural cells (dopamine-producing cells).
- the clinical diagnosis of Parkinson’s disease requires bradykinesia and at least one of the following core symptoms: resting tremor; muscle rigidity and postural reflex impairment.
- Other signs and symptoms that may be present or develop during the progression of the disease are autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature.
- Parkinson’s disease is a neurodegenerative disease that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing Parkinson’s disease in a subject.
- the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing Parkinson’s disease.
- Compositions comprising a bacterial strain of the genus Bacteroides may improve motor and cognitive functions in models of Parkinson’s disease.
- Treatment with Bacteroides strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of Parkinson’s disease.
- the composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens.
- compositions using Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating Parkinson’s disease and are thus preferred.
- the compositions of the invention may prevent, reduce or alleviate one or more of the symptoms of Parkinson’s disease in a subject.
- compositions of the invention may prevent, reduce or alleviate one or more core symptoms, such as bradykinesia, resting tremor; muscle rigidity and/or postural reflex impairment in a subject.
- the compositions of the invention may prevent, reduce or alleviate one or more symptoms associated with Parkinson’s disease progression selected from autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature.
- the compositions of the invention may prevent, reduce or alleviate depressive symptoms comorbid with Parkinson’s disease.
- compositions of the invention may improve verbal memory, executive functions, attention, working memory, verbal fluency and/or anxiety.
- compositions of the invention may prevent, reduce or alleviate cognitive dysfunctions comorbid with Parkinson’s disease.
- the compositions of the invention may prevent, reduce or alleviate Parkinson’s disease progression.
- the compositions of the invention may prevent, reduce or alleviate late motor complications, late motor fluctuations and/or neuronal loss.
- the compositions of the invention may improve symptoms of Parkinson’s disease dementia (PDD).
- PDD Parkinson’s disease dementia
- Symptoms of PDD include changes in memory, concentration and judgment, trouble interpreting visual information, muffled speech, visual hallucinations, delusions, especially paranoid ideas, depression, irritability, anxiety and sleep disturbances, such as excessive daytime drowsiness and rapid eye movement (REM) sleep disorder.
- the compositions of the invention improve the symptoms associated the PDD according to a symptomatic or diagnostic test and/or scale.
- the test or scale is selected from the Hopkins Verbal Learning Test – Revised (HVLT-R); the Delis-Kaplan Executive Function System (D-KEFS) Color-Word Interference Test; the Hamilton Depression Rating Scale (HAM-D 17; depression); the Hamilton Anxiety Rating Scale (HAM-A; anxiety) and the Unified Parkinson’s Disease Rating Scale (UPDRS; PD symptom severity).
- the compositions of the invention may prevent, reduce or alleviate impairment of executive function, attention and/or working memory.
- the compositions of the invention may improve dopaminergic neurotransmission, or prevent, reduce or alleviate impaired dopaminergic neurotransmission.
- the compositions of the invention may improve the symptoms of Parkinson’s disease.
- the test for assessing symptomatic improvement of motor function in Parkinson’s disease may be the Unified Parkinson’s Disease Rating Scale.
- UPDRS II considers the activity of daily life and UPDRS III considers motor-examination.
- the compositions of the invention may improve the Clinical Global Impression – Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders.
- the compositions of the invention may display a positive effect on global social and occupational impairment of the subject with Parkinson’s disease.
- Neurocognitive disorder is a heterogeneous class of psychiatric diseases.
- the most common neurocognitive disorder is Alzheimer’s disease, followed by vascular dementias or mixed forms of the two.
- Other forms of neurodegenerative disease e.g. Lewy body disease, frontotemporal dementia, Parkinson’s dementia, Creutzfeldt-Jakob disease, Huntington’s disease, and Wernicke-Korsakoff syndrome
- Alzheimer’s disease and dementia are also characterised by neuronal loss, so the neuroprotective effects shown in the examples for the compositions of the invention indicate that they may be useful for treating or preventing these conditions.
- the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing Alzheimer’s disease.
- the composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens.
- the composition for use in preventing or treating Alzheimer’s disease does not comprise Bacteroides fragilis.
- the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing dementia.
- the composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens.
- compositions using Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating dementia, and thus bacterial strains from these species are preferred.
- the symptomatic criteria for dementia under DSM-5 are evidence of significant cognitive decline from a previous level of performance in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition.
- the cognitive deficits must interfere with independence in everyday activities.
- the cognitive deficits do not occur exclusively in the context of a delirium and are not better explained by another mental disorder (for example MDD or schizophrenia).
- subjects with neurodegenerative disease display behavioural and psychiatric symptoms including agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances. Neurodegenerative diseases may develop or persist due to dysfunction of the microbiota-gut-brain axis.
- the compositions of the invention are for use in treating or preventing neurodegenerative diseases in a subject.
- the neurodegenerative disease is Alzheimer’s disease.
- the neurodegenerative disease is selected from vascular dementias; mixed form Alzheimer’s disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson’s dementia; Creutzfeldt-Jakob disease; Huntington’s disease; and Wernicke-Korsakoff syndrome.
- the compositions of the invention may prevent, reduce or alleviate one or more of the symptoms of neurodegenerative diseases in a subject.
- compositions of the invention may prevent, reduce or alleviate the occurrence of cognitive decline in a subject and/or improve the level of performance of a subject with neurodegenerative disease or disorder in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual- motor and social cognition.
- the compositions of the invention may prevent, reduce or alleviate the occurrence of one or more behavioural and psychiatric symptoms associated with neurodegenerative disorders selected from agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances.
- the compositions of the invention may prevent, reduce or alleviate symptomatic disease by intervention in the suspected pathogenic mechanisms at a preclinical stage.
- compositions of the invention may improve disease modification, by slowing or arrest the symptom progression.
- the slowing or arresting of symptom progression correlates with evidence in delaying the underlying neuropathological process.
- the compositions of the invention may improve symptoms of neurodegenerative disorders comprising enhanced cognitive and functional improvement, for example, the compositions of the invention may improve the behavioural and psychiatric symptoms of dementia (BPSD).
- BPSD behavioural and psychiatric symptoms of dementia
- the compositions of the invention may improve the ability of a subject with neurodegenerative disease to undertake everyday activities.
- the compositions of the invention may improve both cognition and functioning in a subject with Alzheimer’s disease.
- the composition of the invention improve the cognitive endpoint in a subject with Alzheimer’s disease and/or the functional endpoint in a subject with Alzheimer’s disease.
- compositions of the invention may improve the overall clinical response (the global endpoint) in a subject with Alzheimer’s disease.
- the compositions of the invention may improve the symptoms of neurodegenerative disorders according to a symptomatic or diagnostic test.
- the tests for assessing symptomatic improvement of Alzheimer’s disease (and other neurodegenerative disorders) are selected from objective cognitive, activities of daily living, global assessment of change, health related quality of life tests and tests assessing behavioural and psychiatric symptoms of neurodegenerative disorders.
- the objective cognitive tests for assessment of symptomatic improvement use the Alzheimer’s disease may be the Assessment Scale cognitive subscale (ADAS-cog) and the classic ADAS scale. Symptomatic improvement of cognition is assessed using the neurophysiological Test Battery for Use in Alzheimer’s Disease (NTB).
- NTB neurophysiological Test Battery for Use in Alzheimer’s Disease
- the global assessment of change test uses the Clinical Global Impression – Global Improvement (CGI- I) scale for assessing psychiatric and neurological disorders.
- CGI- I Clinical Global Impression – Global Improvement
- the global scale can be the Clinician's Interview Based Impression of Change plus (CIBIC-plus) or the Alzheimer’s Disease Cooperative Study Unit Clinician’s Global Impression of Change (ADCS-CGIC).
- the health related quality of life measures are the Alzheimer’s Disease-Related QOL (ADRQL) and the QOL- Alzheimer’s Disease (QOL-AD).
- the tests assessing behavioural and psychiatric symptoms of neurodegenerative disorders can be selected from the Behavioural pathology in Alzheimer’s Disease Rating Scale (BEHAVE-AD); the Behavioural Rating Scale for Dementia (BRSD); the neuropsychiatric Inventory (NPI); and the Cohen- Mansfield Agitation Inventory (CMAI).
- the compositions of the invention may be particularly effective at preventing, reducing or alleviating neurodegenerative diseases and disorders when used in combination with another therapy for treating neurodegenerative disorders.
- Such therapies include acetylcholinesterase inhibitors including donepezil (AriceptTM), galantamine (RazadyneTM) and rivastigmine (ExelonTM), and memantine.
- MS Multiple Sclerosis Multiple sclerosis
- MS is a demyelinating disease in which the myelin sheath surrounding neurons in the brain and spinal cord are damaged.
- the exact underlying causes of MS are unknown, but are thought to vary between individuals. Certain forms of MS are hereditary. Environmental factors are also thought to contribute to MS. In some individuals, a combination of both genetic and environmental factors may trigger the onset of MS.
- the compositions of the invention may prevent, reduce or alleviate one or more symptoms of MS in a subject.
- compositions of the invention may prevent, reduce or alleviate fatigue, resting tremor, muscle weakness, muscle spasms, muscle stiffness, paraesthesia and/or ataxia in a subject.
- compositions of the invention may prevent, reduce or alleviate one or more symptoms associated with MS progression selected from the list consisting of autonomic disturbances, constipation, micturition disturbances, sexual functioning, dysphagia, dysarthria, syncope, vertigo and/or dizziness, sleep disturbances, and disturbances in the sense of smell or sense of temperature.
- the compositions of the invention may prevent, reduce or alleviate one or more ocular symptoms associated with MS.
- the ocular symptom can be selected from the list consisting of loss of vision, eye pain, colour blindness, double vision and/or involuntary eye movements in a subject.
- the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing MS.
- the composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens.
- compositions using Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating MS and thus bacterial strains from these species are preferred.
- the composition for use in a method of preventing or treating MS does not comprise bacteria strains of the genus Bacteroides which have a 16s rRNA sequence that is at least 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO:4.
- the compositions of the invention may prevent, reduce or alleviate dizziness, vertigo, neuropathic pain, musculoskeletal pain, cognitive dysfunction, bowel incontinence, dysphagia, dysarthria, or any combination thereof.
- the compositions of the invention may prevent, reduce or alleviate depressive symptoms or anxiety in a patient diagnosed with MS. The improvement of symptoms can be determined using the 2017 McDonald criteria for diagnosing MS.
- Treatment with the compositions of the invention may result in a reduction in MS incidence or MS severity.
- the compositions of the invention may be for use in reducing relapse incidence or relapse severity.
- Treatment with the compositions of the invention may prevent a decline in motor function or results in improved motor function associated with MS.
- the compositions of the invention are therefore for use in preventing a decline in motor function or for use in improving motor function in the treatment of MS.
- Treatment with the compositions of the invention may prevent the development of paralysis in MS. Therefore, the compositions of the invention are for use in preventing paralysis in the treatment of MS.
- compositions of the invention are for use in preventing multiple sclerosis in a patient that has been identified as at risk of multiple sclerosis, or that has been diagnosed with early-stage multiple sclerosis or “relapsing-remitting” multiple sclerosis.
- the compositions of the invention may be useful for preventing the development of MS.
- the compositions of the invention may be useful for preventing the progression of MS.
- the compositions of the invention are for use in a patient identified as having a genetic predisposition to MS, such as major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2 or HLA- DR4.
- MHC major histocompatibility complex
- HLA human leukocyte antigen
- HLA human leukocyte antigen
- compositions of the invention may be particularly useful for reducing symptoms associated with MS.
- Treatment or prevention of MS may refer to, for example, an alleviation of the severity of symptoms or a reduction in the frequency of exacerbations or the range of triggers that are a problem for the patient.
- the compositions of the invention slow or stop progression of the disease.
- the compositions of the invention are for use in treating relapsing-remitting MS.
- compositions of the invention are for use in treating progressive MS, such as secondary progressive MS (SPMS), which develops over time following diagnosis of RRMS, primary progressive MS (PPMS) which exhibits gradual continuous neurologic deterioration and progressive relapsing MS (PRMS), which is similar to PPMS but with overlapping relapses.
- progressive MS such as secondary progressive MS (SPMS)
- SPMS secondary progressive MS
- PPMS primary progressive MS
- PRMS progressive relapsing MS
- the compositions of the invention are for use in treating one or more symptoms of MS selected from the group consisting of: fatigue, vision problems, numbness, tingling, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain, problems with thinking, learning and planning, depression and anxiety, sexual problems, bladder problems, bowel problems, speech and swallowing difficulties.
- the-gut-brain axis includes the central nervous system, neuroendocrine and neuroimmune systems, including the hypothalamus- pituitary-adrenal (HPA) axis, sympathetic and parasympathetic arms of the autonomic nervous system (ANS), including the enteric nervous system (ENS) and the vagus nerve, and the gut microbiota.
- HPA hypothalamus- pituitary-adrenal
- ANS autonomic nervous system
- ENS enteric nervous system
- vagus nerve the gut microbiota.
- the compositions of the invention are useful for modulating the microbiota-gut-brain axis to reduce the cell death associated with central nervous system diseases or disorders, such as inflammatory central nervous system diseases or disorders. Accordingly, the compositions of the invention may be used for treating or preventing neurodegenerative disease sand disorders, in particular those associated with dysfunction of the microbiota-gut-brain axis.
- IL-6 signalling and alterations in the compositions of the microbiota play key roles in the pathogenesis of depression.
- the intravenous administration of an IL-6 receptor antibody (MR16-1) resulted in the induction of depression in a rat model, but intracerebroventricular injection of MR16-1 did not induce any anti-depressant effects.
- the intravenous administration of MR16-1 lead to a decrease in the number of Firmicutes and the Firmicutes/Bacteroidetes ratio. It was suggested that MR16-1 may show antidepressant-like effects by normalizing abnormalities in Firmicutes (or Firmicutes/Bacteroidetes) [43].
- compositions comprising bacteria from the genus Bacteroides to decrease the secretion of IL-6 in human glioblastoma astrocytomas, but to increase IL-6 secretion in PMBCs. Therefore, the compositions of the invention may modulate the microbiota-gut-brain axis by increasing the levels of IL-6 in the periphery but not in the brain. Therefore, the compositions of the invention may be useful for treating or preventing depression by modulating the microbiota-gut- brain-axis.
- compositions of the invention may be useful for treating or preventing a disease or condition selected from the group consisting of: Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia; including Lewy body; vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration.
- Parkinson’s disease including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydro
- compositions of the invention may be useful for treating or preventing epilepsy.
- the compositions of the invention may be useful for treating or preventing chronic disease, treating or preventing disease in patients that have not responded to other therapies (such as treatment with Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, and/or anticholinergics), and/or treating or preventing the tissue damage and symptoms associated with dysfunction of the microbiota-gut-brain axis.
- therapies such as treatment with Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, and/or anticholinergics
- the compositions of the invention may be useful for modulating the CNS.
- compositions of the invention may modulate the autonomic nervous system (ANS), enteric nervous system (ENS), the hypothalamic, pituitary, adrenal (HPA) axis, neuroendocrine, dopaminergic and/or neuroimmune pathway.
- the signalling of the microbiota-gut-brain-axis is modulated by neural systems.
- the compositions of the invention may modulate signalling in neural systems, in the central nervous system in sensory neurons, and/or in motor neurons.
- the compositions of the invention may modulate the signalling in the ANS, for example in the parasympathetic or sympathetic ANS nervous system.
- the compositions of the invention may modulate the signalling in the vagus nerve or ENS.
- compositions of the invention may also module the levels of commensal metabolites and/or the gastrointestinal permeability of a subject.
- the signalling of ANS and ENS neurons may respond directly to luminal contents of the gastrointestinal tract or indirectly to neurochemicals produced by luminal bacteria.
- the signalling of ANS and ENS neurons may respond to neurochemicals produced by luminal bacteria or enteroendocrine cells.
- the neurons of the ENS may activate vagal afferents that influence the functions of the CNS.
- the compositions of the invention may regulate the activity of enterochromaffin cells.
- the microbiota-gut-brain axis is modulated by a number of different physiological systems.
- the microbiota-gut-brain axis is modulated by a number of signalling molecules. Alterations in the levels of these signalling molecules results in central nervous system diseases and disorders, such as inflammatory central nervous system diseases and disorders.
- the signalling of the microbiota-gut-brain axis is modulated by levels of neurochemical factors, neuropeptides and neurotransmitters. Dysregulation of the productions of these metabolites can lead to central nervous system diseases and disorders, such as Parkinson’s disease.
- compositions of the invention modulates levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain preferred embodiments, the compositions of the invention directly alter CNS biochemistry. - Indole The inventors have further demonstrated that bacterial strains from the genus Bacteroides can produce indole, which can attenuate inflammation and oxidative stress.
- bacteria from the genus Bacteroides and in particular bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae are useful in treating or preventing central nervous system diseases or disorders that are associated with inflammation and oxidative stress.
- the bacterial strain is from the species Bacteroides dorei, Bacteroides koreensis, Bacteroides sp or Bacteroides ovatus, for example NCIMB 43598 (Ref 2), NCIMB 43601 (Ref 8), NCIMB 43599 (Ref 20) or NCIMB 43593 (Ref 27).
- Oxidative stress has been implicated in the development of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, epileptic seizures and stroke [44]. Both the production of reactive oxygen species and autophagy have been linked to the onset and development of neurological disorders. Due to their high antioxidant activity indole compounds have been suggested to act as neuroprotectants [45].
- compositions of the invention can modulate the level of indole.
- compositions of the invention can increase the level of indole.
- GABA ⁇ -aminobutyric acid
- the compositions of the invention may be able to modulate the levels of GABA.
- GABA is an inhibitory neurotransmitter that reduces neuronal excitability.
- the compositions of the invention are effective at increasing the levels of GABA.
- the compositions of the invention may alter GABAergic neurotransmission.
- compositions of the invention may modulate the level of GABA transcription in different regions of the central nervous system.
- the commensal derived GABA crosses the blood-brain barrier and affects neurotransmission directly.
- the compositions of the invention may lead to a reduction of GABA in the hippocampus, amygdala and/or locus coeruleus.
- the compositions of the invention may lead to an increase of GABA in cortical regions.
- the levels of neuroactive molecules, such as GABA are linked to the pathophysiology of central nervous system diseases such as dementia, Alzheimer’s disease, Huntington’s disease and epilepsy.
- the compositions of the invention may be used for treating or preventing a disease mediated by GABA.
- compositions of the invention are for use in a method of treating epilepsy.
- Compositions which modulate the levels of GABA have been found to be useful in the treatment of epilepsy.
- the claimed compositions can increase the production of GABA. Consequently, in some embodiments, the compositions of the invention are for use in the treatment of epilepsy.
- the treatment reduces the frequency and/or the intensity of epileptic seizures in a subject. The frequency and/or intensity of epileptic seizures can vary greatly in different subjects.
- the treatment comprises reducing the frequency and/or intensity of seizures by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.
- Modes for measuring the reduction in frequency and/or intensity of epileptic seizures are known in the art.
- the intensity of a seizure may be assessed using the Chalfont Seizure Severity Scale.
- the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is awake. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is asleep.
- the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is awake but not whilst the subject is asleep, or whilst the subject is asleep but not whilst the subject is awake.
- the treatment comprises eliminating epileptic seizures.
- the frequency of epileptic seizures is reduced to once a day or less, once every two days or less, once every week or less, once every two weeks or less, once a month or less, once every three months or less, once every six months or less, once a year or less, once every two years or less, or once every three years or less.
- the treatment results in the subject experiencing an epileptic seizure free period of at least a day, at least two days, at least a week, at least a month, at least three months, at least six months, at least a year, at least two years, or at least three years.
- the compositions of the invention are particularly effective at treating epilepsy when used in combination with another therapy for treating epilepsy.
- the other therapy for use in combination with the composition of the invention is selected from one or more of: Acetazolamide, Brivaracetam, Carbamazepine, Clobazam, Clonazepam, Eslicarbazepine acetate, Ethosuximide, Everolimus, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Oxcarbazepine, Perampanel, Phenobarbital, Phenytoin, Piracetam, Pregabalin, Primidone, Rufinamide, Sodium valproate, Stiripentol, Tiagabine, Topiramate, Valproic acid, Vigabatrin, and Zonisamide.
- the treatment results in the subject being able to reduce the dose or stop taking one or more other anti-epileptic drugs without seeing a deterioration in symptoms.
- the dose of a GABA-mediator anti-epileptic drug may be reduced or the GABA- mediator anti-epileptic drug may no longer be taken.
- the GABA-mediator anti- epileptic drug is selected from valproic acid, sodium valproate and pregabalin.
- - Immune response The signalling of the microbiota-gut-brain axis is modulated by alterations in the immune response and inflammatory factors and markers.
- the compositions of the invention may modulate the immune response.
- compositions of the invention may modulate the systemic levels of circulating neuroimmune signalling molecules, pro-inflammatory cytokine production and/or inflammation.
- the compositions of the invention may modulate the inflammatory state.
- the compositions of the invention may decrease IL-6 production and secretion and/or decrease the activation of the NF ⁇ B promoter.
- the compositions of the invention are able to modulate the activation of IL-6 production by the potent pro-inflammatory endotoxin LPS. Therefore, the compositions of the invention are able to modulate the activation of the NF ⁇ B promoter by LPS and ⁇ -synuclein mutant proteins such as A53T.
- compositions of the invention are for use in reducing IL-6 levels and/or NF ⁇ B levels in the treatment of a central nervous system disease and disorder, such as inflammatory central nervous system diseases and disorders.
- the signalling of the microbiota-gut-brain axis is modulated by levels of gastrointestinal permeability.
- the compositions of the invention may alter the integrity of the gastrointestinal tract epithelium, modulate the permeability of the gastrointestinal tract and/or modulate gastrointestinal tract motility.
- the compositions of the invention may modulate the barrier function and integrity of the gastrointestinal tract.
- compositions of the invention may modulate the translocation of commensal metabolites and inflammatory signalling molecules into the bloodstream from the gastrointestinal tract lumen.
- the signalling of the microbiota-gut-brain axis is modulated by the composition of the microbiome in the gastrointestinal tract.
- the compositions of the invention may modulate the microbiome composition of the gastrointestinal tract.
- the compositions of the invention may prevent microbiome dysbiosis and associated increases in toxic metabolites (e.g. LPS).
- the compositions of the invention may modulate the levels of Clostridium in the gastrointestinal tract.
- the compositions of the invention may reduce the level of Clostridium in the gastrointestinal tract.
- the compositions of the invention may reduce the levels of Campylobacter jejuni.
- compositions of the invention may modulate the proliferation of harmful anaerobic bacteria and the production of inflammatory neurotoxins produced by these bacteria.
- the compositions of the invention may modulate the microbiome levels of Lactobacillus and/or Bifidobacterium.
- the compositions of the invention modulate the microbiome levels of Sutterella, Prevotella, Ruminococcus genera and/or the Alcaligenaceae family.
- the compositions of the invention may increase the level of Lactobacillus plantarum and/or Saccharomyces boulardii. Brain injury
- the examples demonstrate that the compositions of the invention have antioxidant activity and have neuroprotective properties.
- the compositions of the invention are for use in treating a brain injury.
- the brain injury can be for example a traumatic brain injury, an acquired brain injury, a brain injury resulting from trauma, a brain injury resulting from a tumour, a brain injury resulting from a stroke, a brain injury resulting from a brain haemorrhage, a brain injury resulting from encephalitis, a brain injury resulting from cerebral hypoxia or a brain injury resulting from cerebral anoxia.
- the compositions of the invention may be for use in treating stroke. The effects shown in the examples are particularly relevant to the treatment of stroke.
- Stroke occurs when blood flow to at least a part of the brain is interrupted. Without an adequate supply of blood to provide oxygen and nutrients to the brain tissue and to remove waste products from the brain tissue, brain cells rapidly begin to die.
- the symptoms of stroke are dependent on the region of the brain which is affected by the inadequate blood flow. Symptoms include paralysis, numbness or weakness of the muscles, loss of balance, dizziness, sudden severe headaches, speech impairment, loss of memory, loss of reasoning ability, sudden confusion, vision impairment, coma or even death.
- a stroke is also referred to as a brain attack or a cerebrovascular accident (CVA).
- the symptoms of stroke may be brief if adequate blood flow is restored within a short period of time.
- the stroke is cerebral ischemia.
- Cerebral ischemia results when there is insufficient blood flow to the tissues of the brain to meet metabolic demand.
- the cerebral ischemia may be focal cerebral ischemia, i.e. confined to a specific region of the brain or the cerebral ischemia may be global cerebral ischemia, i.e. encompassing a wide area of the brain tissue.
- Focal cerebral ischemia commonly occurs when a cerebral vessel has become blocked, either partially or completely, reducing the flow of blood to a specific region of the brain.
- the focal cerebral ischemia is ischemic stroke.
- the ischemic stroke is thrombotic, i.e. caused by a thrombus or blood clot, which develops in a cerebral vessel and restricts or blocks blood flow.
- the ischemic stroke is a thrombotic stroke.
- the ischemic stroke is embolic, i.e. caused by an embolus, or an unattached mass that travels through the bloodstream and restricts or blocks blood flow at a site distant from its point of origin.
- the ischemic stroke is an embolic stroke.
- Global cerebral ischemia commonly occurs when blood flow to the brain as a whole is blocked or reduced. The global cerebral ischemia can be caused by hypoperfusion, i.e.
- the subject diagnosed with brain injury may have suffered from cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered focal cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered an ischemic stroke. In some embodiments, the subject diagnosed with brain injury has suffered a thrombotic stroke. In some embodiments, the subject diagnosed with brain injury has suffered an embolic stroke. In some embodiments, the subject diagnosed with brain injury has suffered global cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered hypoperfusion. In some embodiments, the subject diagnosed with brain injury has suffered a cardiac arrest. In some embodiments, the compositions of the invention are for use in treating a brain injury caused by a cerebral ischemia.
- compositions of the invention may be for use in treating a brain injury caused by a focal cerebral ischemia.
- the compositions of the invention may be for use in treating a brain injury caused by a ischemic stroke.
- the compositions of the invention may be for use in treating a brain injury caused by a thrombotic stroke.
- the compositions of the invention may be for use in treating a brain injury caused by a embolic stroke.
- the compositions of the invention may be for use in treating a brain injury caused by a global cerebral ischemia.
- the compositions of the invention may be for use in treating a brain injury caused by a hypoperfusion.
- the stroke can be a hemorrhagic stroke.
- Hemorrhagic stroke is caused by bleeding into or around the brain resulting in swelling, pressure and damage to the cells and tissues of the brain. Hemorrhagic stroke is commonly a result of a weakened blood vessel that ruptures and bleeds into the surrounding brain.
- the hemorrhagic stroke is an intracerebral hemorrhage, i.e. caused by bleeding within the brain tissue itself.
- the intracerebral hemorrhage can be caused by an intraparenchymal haemorrhage or an intraventricular hemorrhage.
- the hemorrhagic stroke can be a subarachnoid hemorrhage i.e. bleeding that occurs outside of the brain tissue but still within the skull.
- the hemorrhagic stroke may be a result of cerebral amyloid angiopathy.
- the hemorrhagic stroke is a result of a brain aneurysm.
- the hemorrhagic stroke is a result of cerebral arteriovenous malformation (AVM).
- AVM cerebral arteriovenous malformation
- the subject diagnosed with brain injury has suffered hemorrhagic stroke.
- the subject diagnosed with brain injury may have suffered from an intracerebral haemorrhage or an intraparenchymal hemorrhage.
- the subject diagnosed with brain injury has suffered an intraventricular hemorrhage.
- the subject diagnosed with brain injury has suffered a subarachnoid hemorrhage.
- the subject diagnosed with brain injury has suffered cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with brain injury has suffered a brain aneurysm. In some embodiments, the subject diagnosed with brain injury has suffered cerebral AVM. In some embodiments, the compositions of the invention are for use in treating hemorrhagic stroke. In some embodiments, the compositions of the invention are for use in treating an intracerebral hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraparenchymal hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraventricular hemorrhage.
- the compositions of the invention are for use in treating a subarachnoid hemorrhage. In some embodiments, the compositions of the invention are for use in treating a cerebral amyloid angiopathy. In some embodiments, the compositions of the invention are for use in treating a brain aneurysm. In some embodiments, the compositions of the invention are for use in treating cerebral AVM.
- Restoration of adequate blood flow to the brain after a period of interruption, though effective in alleviating the symptoms associated with stroke, can paradoxically result in further damage to the brain tissue. During the period of interruption, the affected tissue suffers from a lack of oxygen and nutrients, and the sudden restoration of blood flow can result in inflammation and oxidative damage through the induction of oxidative stress.
- reperfusion injury This is known as reperfusion injury, and is well documented not only following stroke, but also following a heart attack or other tissue damage when blood supply returns to the tissue after a period of ischemia or lack of oxygen.
- the subject diagnosed with brain injury has suffered from reperfusion injury as a result of stroke.
- the compositions of the invention are for use in treating reperfusion injury as a result of stroke.
- a transient ischemic attack (TIA) often referred to as a mini-stroke, is a recognised warning sign for a more serious stroke. Subjects who have suffered one or more TIAs are therefore at greater risk of stroke.
- the subject diagnosed with brain injury has suffered a TIA.
- the compositions of the invention are for use in treating a TIA. In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has suffered a TIA.
- High blood pressure, high blood cholesterol, a familial history of stroke, heart disease, diabetes, brain aneurysms, arteriovenous malformations, sickle cell disease, vasculitis, bleeding disorders, use of nonsteroidal anti-inflammatory drugs (NSAIDs), smoking tobacco, drinking large amounts of alcohol, illegal drug use, obesity, lack of physical activity and an unhealthy diet are all considered to be risk factors for stroke.
- NSAIDs nonsteroidal anti-inflammatory drugs
- smoking tobacco drinking large amounts of alcohol
- illegal drug use obesity
- lack of physical activity and an unhealthy diet are all considered to be risk factors for stroke.
- lowering blood pressure has been conclusively shown to prevent both ischemic and hemorrhagic strokes [46, 47].
- the compositions of the invention are for use in treating brain injury in a subject who has at least one risk factor for stroke.
- the subject has two risk factors for stroke.
- the subject has three risk factors for stroke.
- the subject has four risk factors for stroke.
- the subject has more than four risk factors for stroke.
- the subject has high blood pressure.
- the subject has high blood cholesterol.
- the subject has a familial history of stroke.
- the subject has heart disease.
- the subject has diabetes.
- the subject has a brain aneurysm.
- the subject has arteriovenous malformations.
- the subject has vasculitis.
- the subject has sickle cell disease. In some embodiments the subject has a bleeding disorder. In some embodiments the subject has a history of use of nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments the subject smokes tobacco. In some embodiments the subject drinks large amounts of alcohol. In some embodiments the subject uses illegal drugs. In some embodiments the subject is obese. In some embodiments the subject is overweight. In some embodiments the subject has a lack of physical activity. In some embodiments the subject has an unhealthy diet.
- NSAIDs nonsteroidal anti-inflammatory drugs
- the subject smokes tobacco. In some embodiments the subject drinks large amounts of alcohol. In some embodiments the subject uses illegal drugs. In some embodiments the subject is obese. In some embodiments the subject is overweight. In some embodiments the subject has a lack of physical activity. In some embodiments the subject has an unhealthy diet.
- compositions of the invention may be for use in reducing the potential damage caused by a potential brain injury, preferably a stroke.
- the compositions may reduce the damage caused when they are administered before the potential brain injury occurs, in particular when administered to a patient identified as at risk of a brain injury.
- the compositions of the invention treat brain injury by reducing motoric damage.
- the compositions of the invention treat brain injury by improving motor function.
- the compositions of the invention treat brain injury by improving muscle strength.
- the compositions of the invention treat brain injury by improving memory.
- the compositions of the invention treat brain injury by improving social recognition.
- the compositions of the invention treat brain injury by improving inflammatory neurological function.
- Treatment of brain injury may refer to, for example, an alleviation of the severity of symptoms. Treatment of brain injury may also refer to reducing the inflammatory neurological impairments following stroke.
- Compositions of the invention for use in treating stroke may be provided to the subject in advance of the onset of stroke, for example in a patient identified as being at risk of stroke. Compositions of the invention for use in treating stroke may be provided after a stroke has occurred, for example, during recovery. Compositions of the invention for use in treating stroke may be provided during the acute phase of recovery (i.e. up to one week after stroke). Compositions of the invention for use in treating stroke may be provided during the subacute phase of recovery (i.e. from one week up to three months after stroke).
- compositions of the invention for use in treating stroke may be provided during the chronic phase of recovery (from three months after stroke).
- the compositions of the invention are for use in combination with a secondary active agent.
- the compositions of the invention are for use in combination with aspirin or tissue plasminogen activator (tPA).
- Other secondary agents include other antiplatelets (such as clopidogrel), anticoagulants (such as heparins, warfarin, apixaban, dabigatran, edoxaban or rivaroxaban), antihypertensives (such as diuretics, ACE inhibitors, calcium channel blockers, beta- blockers or alpha-blockers) or statins.
- compositions of the invention may improve the patient’s response to the secondary active agent.
- the compositions of the invention reduce the effect of ischemia on tissues.
- the compositions of the invention reduce the amount of damage to tissues caused by ischemia.
- the tissues damaged by ischemia are the cerebral tissues.
- the compositions of the invention reduce necrosis or the number of necrotic cells.
- the compositions of the invention reduce apoptosis or the number of apoptotic cells.
- the compositions of the invention reduce the number of necrotic and apoptotic cells.
- the compositions of the invention prevent cell death by necrosis and/or apoptosis.
- the compositions of the invention prevent cell death by necrosis and/or apoptosis caused by ischemia. In certain embodiments, the compositions of the invention improve the recovery of the tissue damaged by ischemia. In certain embodiments, the compositions of the invention improve the speed of clearance of necrotic cells and/or apoptotic cells. In certain embodiments, the compositions of the invention improve the efficacy of the clearance of necrotic cells and/or apoptotic cells. In certain embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues. In certain embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues damaged by ischemia.
- the compositions of the invention improve the overall histology of the tissue (for example upon a biopsy).
- 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.
- the compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.
- the bacteria may have colonised some or all of the gastrointestinal tract and / or such colonisation may be transient or permanent.
- the “total colonisation of the intestine” means that bacteria have colonised all parts of the intestine (i.e.
- total colonisation means that the bacteria engraft permanently in the some or all parts of the intestine.
- partial colonisation of the intestine means that bacteria have colonised some but not all parts of the intestine.
- partial colonisation means that the bacteria engraft transiently in some or all parts of the intestine. The transience of engraftment can be determined by assessing (e.g. in a fecal sample) the abundance of the bacterial strain of the invention periodically (e.g. daily) following the end of a dosing interval to determine the washout period, i.e.
- the washout period is 14 days or less, 12 days or less, 10 days or less, 7 days or less, 4 days or less, 3 days or less, 2 days or less or 1 day or less.
- the bacteria of the present invention engraft transiently in the large intestine.
- the compositions of the invention may be administered as a tablet, a foam, as a spray or a gel.
- 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.
- compositions of the invention may be administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
- a tube such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum 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.
- the compositions of the invention may be administered daily, weekly or monthly (either once or several times).
- 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.
- 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.
- the compositions of the invention are administered for 16 days. Treatment according to the invention may be accompanied by 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.
- the composition of the invention may be administered to a pregnant animal, for example a mammal such as a human in order to prevent a central nervous system disease or disorder, such as a neurodegenerative disease, a neurodevelopmental disorder, a neuropsychiatric condition or a brain injury developing in her child in utero and / or after it is born.
- compositions of the invention may be administered to a patient that has been diagnosed with a central nervous system disease or disorder, or that has been identified as being at risk of a central nervous system disease or disorder.
- the compositions may also be administered as a prophylactic measure to prevent the development of a central nervous system disease or disorder in a healthy patient.
- the compositions of the invention may be administered to a patient that has been diagnosed with a neurodegenerative disease, or that has been identified as being at risk of a neurodegenerative disease.
- the compositions may also be administered as a prophylactic measure to prevent the development of neurodegenerative disease in a healthy patient.
- compositions disclosed herein may be administered to a patient that has been diagnosed with a central nervous system disorder or condition, in particular a central nervous system disorder or condition mediated by the microbiota-gut-brain axis, or that has been identified as being at risk of a central nervous system disorder or condition, in particular central nervous system disorder or condition mediated by the microbiota-gut-brain axis.
- the compositions may also be administered as a prophylactic measure to prevent the development of central nervous system disorders or conditions, in particular central nervous system disorders or conditions mediated by the microbiota-gut-brain axis 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.
- the patient may have reduced or absent colonisation by Bacteroides, and in particular Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae.
- the compositions of the invention may be administered as a food product, such as a nutritional supplement.
- compositions of the invention are for the prevention or treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits.
- the compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used.
- the subject to whom the composition is to be administered is an adult human.
- the subject to whom the composition is to be administered is an infant human.
- the compositions of the invention comprise bacteria. The inventors have identified the surprising ability of bacteria from the genus Bacteroides to treat central nervous system diseases or disorders, such as inflammatory central nervous system diseases or disorders.
- compositions of the invention therefore does not comprise inactivated bacteria of the species Bacteroides, in particular heat-inactivated bacteria of the species Bacteroides.
- the invention provides compositions which are formulated to prevent the bacteria from being degraded or absorbed in the upper digestive tract and being unable to exert their effect.
- the compositions may comprises oxygen scavengers and/or prebiotic substrates, such as vitamin C and non-digestible carbohydrates.
- the composition can be enterically formulated to ensure that the bacteria are not degraded on route to the small intestine.
- the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine.
- Encapsulation protects the composition from degradation 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.
- the composition is formulated in freeze-dried form.
- the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain of the invention.
- 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 [50-52]. The examples demonstrate that lyophilised compositions are particularly effective.
- 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.
- the bacterial strain in the composition of the invention may not have been inactivated, for example, may not have been heat-inactivated.
- the bacterial strain in the composition of the invention may not have been killed, for example, not been heat-killed.
- the bacterial strain in the composition of the invention may not have been attenuated, for example, not been heat-attenuated.
- the bacterial strain in the composition of the invention may not have been killed, inactivated and/or attenuated.
- the bacterial strain in the composition of the invention is live.
- the bacterial strain in the composition of the invention is viable.
- the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine.
- the bacterial strain in the composition of the invention may be viable and capable of partially or totally colonising the intestine.
- the bacterial strain in the composition of the invention may be live and capable of partially or totally colonising the intestine.
- the bacterial strain in the composition of the invention may be live and viable.
- the bacterial strain in the composition of the invention may be live, viable and capable of partially or totally colonising the intestine.
- the composition can comprises a mixture of live bacterial strains and bacterial strains that have been killed.
- the invention provides compositions which are formulated to prevent the bacteria from being degraded or absorbed in the upper digestive tract and being unable to exert their effect.
- the compositions may comprises oxygen scavengers and/or prebiotic substrates, such as vitamin C and non-digestible carbohydrates.
- the composition can be enterically formulated. This ensures that the bacteria are not degraded on the way to the small intestine.
- Encapsulation protects the composition from degradation 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.
- compositions of the invention may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Bacteroides are anaerobes.
- 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 beneficial effect upon a patient. A therapeutically effective amount of a bacterial strain may be sufficient to result in delivery to and / or partial or total colonisation of the patient’s intestine.
- a suitable daily dose of the bacteria may be from about 1 x 10 3 to about 1 x 10 11 colony forming units (CFU); for example, from about 1 x 10 7 to about 1 x 10 10 CFU; in another example from about 1 x 10 6 to about 1 x 10 10 CFU; in another example from about 1 x 10 7 to about 1 x 10 11 CFU; in another example from about 1 x 108 to about 1 x 1010 CFU; in another example from about 1 x 10 8 to about 1 x 10 11 CFU.
- the dose of the bacteria is at least 109 cells per day, such as at least 1010, at least 1011, or at least 10 12 cells per day.
- a dose of the composition may comprise the bacterial strain from about 1 x 106to about 1 x 10 11 colony forming units (CFU) /g, respect to the weight of the composition.
- the dose may be suitable for an adult human.
- the composition may comprise the bacterial strain from about 1 x 10 3 to about 1 x 10 11 CFU/g; for example, from about 1 x 10 7 to about 1 x 10 10 CFU/g; in another example from about 1 x 10 6 to about 1 x 10 10 CFU/g; in another example from about 1 x 10 7 to about 1 x 10 11 CFU/g; in another example from about 1 x 10 8 to about 1 x 10 10 CFU/g; in another example from about 1 x 10 8 to about 1 x 10 11 CFU/g, from about 1 x 108 to about 1 x 10 10 CFU/g.
- the dose may be, for example, 1g, 3g, 5g, and 10g.
- the composition may be formulated as a probiotic.
- a probiotic is defined by the FAO/WHO as a live microorganism that, when administered in adequate amounts, confers a health benefit on the host.
- a probiotic such as a composition of the invention, is optionally combined with at least one suitable prebiotic compound.
- the probiotic composition of the present invention may include 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.
- Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides.
- 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.
- Potential prebiotics can have a number of beneficial physiological properties including: resistance to digestion in the upper sections of the alimentary tract, can be fermented by the intestinal microbiota, have a beneficial effect on the host’s health, can selectively stimulate the growth of the probiotic and is stable in various food or feed processing conditions.
- the 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 resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers.
- FOS fructo- oligosaccharides
- FOS short-chain fructo-oligosaccharides
- inulin isomalt-oligosaccharides
- pectins pectins
- xylo-oligosaccharides or XOS
- chitosan-oligosaccharides or COS
- beta-glucans arable gum modified and
- 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 molecule to which three glucose molecules are bonded.
- the prebiotic can stimulate the growth and activity of bacteria from the genus Bacteroides in the gastrointestinal tract. This allows the bacteria to exert their beneficial effect on the host of treating central nervous system diseases or disorders, such as inflammatory central nervous system diseases or disorders.
- Other prebiotic compounds may be included as oxygen scavengers and to improve the delivery and/or partial or total colonisation and survival in vivo.
- 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 compositions of the invention may be used in combination with another therapeutic compound for treating or preventing central nervous system diseases and disorders, such as inflammatory central nervous system diseases or disorders.
- the compositions of the invention may be administered with nutritional supplements that modulate neuroprotection or neuroproliferation.
- the nutritional supplements comprise or consist of nutritional vitamins.
- the vitamins can be vitamin B6, magnesium, dimethylglycine (vitamin B16) and vitamin C.
- the compositions of the invention may be administered in combination with another probiotic.
- compositions of the invention are for use in enhancing the effect of a second agent on a central nervous system disease and disorder, such as inflammatory central nervous system diseases or disorders.
- the immune modulatory effects of the compositions of the invention may make the brain more susceptible to conventional therapies such as Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, or anticholinergics, which are exemplary secondary agents to be administered in combination (sequentially or contemporaneously) with the compositions of the invention.
- the compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [55].
- Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [56].
- suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
- suitable diluents include ethanol, 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).
- binders examples 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.
- suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
- Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition.
- preservatives examples include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
- compositions of the invention may be formulated as a food product.
- a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement.
- 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.
- the composition of the invention is formulated as a milk-based product.
- 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.
- milk beverages such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.
- compositions of the invention may comprise one or more bacterial strains of the genus Bacteroides and do not contain bacteria from any other genera, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another genera.
- the invention provides a composition comprising one or more bacterial strains of the genus Bacteroides, which does not contain bacteria from any other genera or which comprises only de minimis or biologically irrelevant amounts of bacteria from another genera, for use in therapy.
- compositions of the invention may comprise one or more bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola and do not contain bacteria from any other species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another species.
- the invention provides a composition comprising one or more bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola, which does not contain bacteria from any other species or which comprises only de minimis or biologically irrelevant amounts of bacteria from another species, for use in therapy.
- the composition does not comprise bacteria of the species Clostridium and/or does not comprise bacteria of the species Bacteroides fragilis.
- the composition does not comprise Bacteroides thetaiotaomicron. In some embodiments, the composition does not comprise bacteria of the phylum Actinobacteria and/or does not comprise bacteria of the phylum Tenericutes, or comprises only de minimis or biologically irrelevant amounts of bacteria from the phylum Actinobacteria and/or the phylum Tenericutes for use in therapy.
- the compositions of the invention may contain a single bacterial species and do not contain any other bacterial species. In certain embodiments, the compositions of the invention contain a single bacterial strain and do not contain any other bacterial strains.
- compositions of the invention may comprise a bacterial strain only of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola.
- Such compositions may comprise only de 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 organism. In some embodiments, such compositions may be a lyophilisate that is substantially free from other species of organism.
- the invention also provides a composition comprising a single bacterial strain of the genus Bacteroides, which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy.
- the invention also provides a composition comprising a single bacterial strain of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy.
- compositions of the invention may comprise more than one bacterial strain.
- 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.
- 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.
- the compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1- 18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains from within the same species and, optionally, do not contain bacteria from any other species.
- the invention comprises any combination of the foregoing.
- the composition may comprise a microbial consortium.
- the composition may comprises a Bacteroides strain as part of a microbial consortium.
- the Bacteroides strain is present in combination with one or more (e.g.
- the composition comprises a bacterial strain of the genus Bacteroides in combination with a bacterial strain from a different genus.
- the composition comprises a bacterial strain of Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola in combination with a bacterial strain from the genus Bacteroides or the composition comprises a bacterial strain of Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron.
- the microbial consortium comprises two or more bacterial strains obtained from a faeces sample of a single organism, e.g. a human.
- the microbial consortium may not found together in nature.
- the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms.
- the microbial consortium can comprises bacteria from two different organisms which are from the same species, e.g. two different humans. For example, the two different organisms are an infant human and an adult human.
- the microbial consortium can comprises bacteria from two different organisms, which are not from the same species, such as a human and a non-human mammal.
- the composition comprises the Bacteroides bacterial strain as part of a microbial consortium which contains fewer than 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 bacterial species.
- the bacterial species may be from the genus Bacteroides and/or from other genera with which it can live symbiotically in vivo in the intestine.
- the composition is not a faecal sample, such as a human faecal sample.
- the composition may be formulated as part of a microbial consortium which contains fewer than 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, 20, 30, 40 or 50 bacterial strains.
- the bacterial strains may be from the genus Bacteroides and/or from other genera with which it can live symbiotically in vivo in the intestine.
- the microbial consortium contains fewer than 30 bacterial strains.
- the microbial consortium contains fewer than 20 bacterial strains. In some embodiments, the microbial consortium contains fewer than 10 bacterial strains.
- the composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43600, but which is not NCIMB 43600 or which is not a Bacteroides ovatus.
- the composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as NCIMB 43595, NCIMB 43599 or NCIMB 43601, but which is not NCIMB 43595, NCIMB 43599 or NCIMB 43601, or which is not a Bacteroides dorei.
- the composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43597, but which is not NCIMB 43597, or which is not a Bacteroides stercoris.
- the composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43596, but which is not NCIMB 43596, or which is not a Bacteroides xylanisolvens.
- the composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43594, but which is not NCIMB 43594 which is not a Bacteroides koreensis.
- the composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43593 or NCIMB 43598, but which is not NCIMB 43593 or NCIMB 43598, or which is not a Bacteroides sp.
- the composition of the invention comprises more than one bacterial strain, species or genus
- the individual bacterial strains, species or genera may be for separate, simultaneous or sequential administration.
- the composition may comprise all of the more than one bacterial strain, species or genera, or the bacterial strains, species or genera may be stored separately and be administered separately, simultaneously or sequentially.
- the more than one bacterial strains, species or genera are stored separately but are mixed together prior to use.
- 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, all 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 have been cultured subsequent to being obtained from the human adult faeces and being used in a composition of the invention. In some embodiments, the bacterial strain for use in the invention is obtained from human infant faeces.
- the composition of the invention comprises more than one bacterial strain
- all of the bacterial strains are obtained from human infant faeces or if other bacterial strains are present they are present only in de minimis amounts.
- the bacteria may have been cultured subsequent to being obtained from the human infant faeces and being used in a composition of the invention
- the one or more Bacteroides bacterial strains is/are the only therapeutically active agent(s) in a composition of the invention.
- 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.
- the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides a lyophilised pharmaceutical composition comprising the bacterial strains as discussed earlier. 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.
- 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.
- the lyophilised bacterial strain is reconstituted prior to administration.
- the reconstitution is by use of a diluent described herein.
- the compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.
- the invention provides a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat an inflammatory neurodegenerative disorder when administered to a subject in need thereof.
- the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent an inflammatory neurodegenerative disorder.
- the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 ⁇ 103 to about 1 ⁇ 1011 colony forming units per gram with respect to a weight of the composition.
- 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.
- the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.
- the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.
- the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.
- 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 stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
- the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.
- 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.
- the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.
- 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 atmosphere 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.
- the composition of the invention is provided in a sealed container comprising a composition as described herein.
- the sealed container is a sachet or bottle.
- the composition of the invention is provided in a syringe comprising a composition as described herein.
- the composition of the present invention may, in some embodiments, be provided as a pharmaceutical formulation.
- the composition may be provided as a tablet or capsule.
- the capsule is a gelatine capsule (“gel-cap”).
- the capsule can be a hard or a soft capsule.
- 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, have a certain elasticity and softness.
- Soft capsules can be produced, for example, on the basis of gelatine 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 have 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.
- compositions of the invention are administered orally.
- Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract.
- 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), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
- the pharmaceutical formulation is an enteric formulation, i.e.
- a gastro-resistant formulation for example, resistant to gastric pH
- Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to degradation under gastric conditions.
- the enteric formulation comprises an enteric coating.
- the formulation is an enteric-coated dosage form.
- 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.
- the enteric formulation is intrinsically enteric, for example, gastro-resistant without the need for an enteric coating.
- the formulation is an enteric formulation that does not comprise an enteric coating.
- the formulation is a capsule made from a thermogelling material.
- the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC).
- the capsule comprises a shell that does not contain any film forming polymer.
- the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [57 ]).
- the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel). Culturing methods
- the bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [58-60].
- 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), NaHCO 3 (0.4 g), cysteine (0.1 g), K 2 HPO 4 (0.045 g), KH 2 PO 4 (0.045 g), NaCl (0.09 g), (NH 4 ) 2 SO 4 (0.09 g), MgSO 4 ⁇ 7H 2 O (0.009 g), CaCl 2 (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 ⁇ g), cobalamin (1 ⁇ g), p-aminobenzoic acid (3 ⁇ g), folic acid (5 ⁇ g), and pyridoxamine (15 ⁇ g).
- Bacterial strains for use in vaccine compositions The inventors have identified that the bacterial strains of the invention are useful for treating or preventing inflammatory neurodegenerative disorders. This is likely to be a result of the effect that the bacterial strains of the invention have on the host immune system. Therefore, the compositions of the invention may also be useful for preventing inflammatory neurodegenerative disorders, when administered as vaccine compositions.
- the bacterial strains of the invention may be killed, inactivated or attenuated.
- the compositions may comprise a vaccine adjuvant.
- the compositions are for administration via injection, such as via subcutaneous injection.
- compositions which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
- References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides 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. [69].
- a preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 5 and a gap extension penalty of 2, and optionally a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [70].
- 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).
- a “disease” results from a pathophysiological response to external or internal factors.
- a “disorder” is a disruption of the disease to the normal or regular functions in the body or a part of the body.
- a “condition” is an abnormal state of health that interferes with the usual activities or feeling of wellbeing. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Any reference to a method for treatment comprising administering an agent to a patient, also covers that agent for use in said method for treatment, as well as the use of the agent in said method for treatment, and the use of the agent in the manufacture of a medicament. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. MODES FOR CARRYING OUT THE INVENTION Example 1 – Efficacy of strains of the genus Bacteroides to decrease IL-6 secretion.
- Cell line U373 is a human glioblastoma astrocytoma cell line.
- Cells (used between passage 20 and passage 37) were maintained in 25ml MEME supplemented with 10% heat inactivated FBS, 4mM L-Glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 5 ⁇ g/ml plasmocin, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate (referred to throughout as full growth media).
- Method In preparation for the different treatments cells were plated in 24 well plates at a density of 100,000 cells/well in 1ml of full growth media. They were left to rest at 37°C/5% CO 2 for 72 h.
- IL-6 was measured using the Human IL-6 ABTS ELISA Development Kits (Peprotech (London, UK). Samples were analysed in accordance with the manufacturer’s protocol, absorbance at 405 nm with wavelength correction set at 655 nm was recorded using the iMark microplate reader (Bio-Rad). Raw data were plotted and analysed using GraphPad Prism 7 software. Results Figure 1 shows that the majority of the Bacteroides strains tested decreased the secretion of IL-6 in U373 cells after stimulation with LPS. Only one strain, B.
- xylanisolvens NCIMB 43596 (Ref 1) was found to increase the secretion of IL-6 in U373 cells. 13 strains significantly down-regulated the production of IL-6 in U373 cells, including B. fragilis (Ref 12 and Ref 22) B. vulgatus (Ref 23 and Ref 24), B. eggherthii (Ref 11), B. stercoris NCIMB 43597 (Ref 10) and B. caccae (Ref 28). Different strains of B. fragilis, B. ovatus and B. dorei isolated from different donors (Table 1) showed the same significant down-regulation of IL-6 (Fig.1).
- IL-6 LPS is a general inflammatory mediator
- Chronic inflammation induced by IL-6 can ultimately lead to cell death. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prevention of central nervous system disorders or conditions.
- IL-6 can act as an anti-inflammation regulator which stimulates potent anti-inflammatory cytokines such as IL-10, and it is known that IL-6 and IL-10 share STAT-3 signalling pathway [15, 16].
- PBMCs peripheral blood mononuclear cells
- xylanisolvens - NCIMB 43596 (Ref 1) Method Frozen healthy human PBMCs were purchased from Stem cells. Briefly cells were thawed and left to rest overnight in full growth media (RPMI 1640 with 10% FBS, 2mM L. Glutamine and 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 55 ⁇ M mercaptoethanol) in a CO 2 incubator at 37°C. For the experiment cells were plated at a density of 750,000 cells/well in 48 well plates and treated in full growth media with 10% bacterial supernatants in the presence or absence of 1 ng/ml LPS. Cell culture media was added to untreated wells.
- full growth media RPMI 1640 with 10% FBS, 2mM L. Glutamine and 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 55 ⁇ M mercaptoethanol
- Results Figure 2 shows the level of IL-6 and IL-10 secretion of PBMCs after treatment with bacterial strains of the genus Bacteroides. All of the tested strains induced the per se production of IL-6 and none reduced secretion of the cytokine after treatment with LPS. Interestingly, all of the strains tested increased the secretion of IL-10 after treatment with LPS, with NCIMB 43595 (Ref 25) being the best strain.
- HEK-Blue cells stably expressing TLR4 and the inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the NF-kB-AP1 promoter, were used to investigate the ability of different strains of Bacteroides to activate NF-kB via TLR4 activation.
- HEK-TLR4 cells were maintained in DMEM 4.5g/L D-glucose supplemented with 10% (v/v) heat inactivated FBS, 4mM L-Glutamine, 100U/ml penicillin, 100 ⁇ g/ml streptomycin, 100 ⁇ g/ml normocin, 1x HEK-Blue selection media.
- FBS v/v
- 4mM L-Glutamine 100U/ml penicillin
- 100 ⁇ g/ml streptomycin 100 ⁇ g/ml normocin
- 1x HEK-Blue selection media 1x HEK-Blue selection media.
- cells were washed with PBS, dissociated in PBS and collected in growth media. Cells were plated in 96 well plates at a density of 25,000 cells/well.
- Cells were treated with 10 ng/ml LPS as a positive control, ⁇ -synuclein, or treated with 10% bacterial supernatant and incubated in a CO 2 incubator for 22h. Thereafter detection of Secreted Embryonic Alkaline Phosphatase (SEAP) activity from cell culture supernatant was performed using QUANTI-blue solution according to manufacturer’s instructions. Briefly, 20 ⁇ l of cell culture media was collected and analysed for the presence of secreted alkaline phosphatase by mixing with 200 ⁇ l of sterile-filtered QUANTI-Blue detection media. After 2h incubation at 37°C optical density was measured at 655nm (iMark microplate, Bio-Rad).
- SEAP Secreted Embryonic Alkaline Phosphatase
- LPS is a known activator of the NF- ⁇ B pathway which promote the proinflammatory cascade and regulates multiple aspects of the innate and adaptive immune system.
- the NF- ⁇ B pathway is known to be upregulated in inflammatory diseases.
- Figure 3 shows that all the strains tested induced NF-kB activation in a TLR4 dependent manner, with NCIMB 43597 (Ref 10) and NCIMB 43593 (Ref 27) doing so to a slightly lesser extent than the positive control.
- an inflammatory stimulus e.g. LPS or ⁇ -synuclein
- Bacteroides stercoris to reduce NF- ⁇ B activation after stimulation a- synuclein mutant makes it particularly useful in the treatment or prevention of inflammatory diseases.
- Example 4 Anti-oxidant potential of Bacteroides strains Bacteria can produce and release antioxidants into the growth media that can interfere for example, with aging or neurodegenerative processes. The intrinsic capacity of Bacteroides strains to produce anti-oxidant molecules was investigated. Material and Methods Bacterial strain tested B. ovatus - NCIMB 43600 (Ref 7) B.
- koreensis – NCIMB 43594 Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp - NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1) Bacteria culture conditions Bacterial indole production was quantified using an assay described previously [18]. Bacteria were cultured to stationary phase of growth.
- a sterile 5 ⁇ L inoculation loop was used to take a loopful of culture to streak a purity plate.
- Culture and YCFA+ media were streaked onto pre-equilibrated YCFA+ agar plates and incubated for 24h inside the anaerobic hood at 37°C.
- QC checks were performed using the MALDI ToF.
- MALDI ToF Biomaterial of grown colonies of each sample- was put on MALDI Biotarget 96 (6 colonies in duplicate) with autoclaved wooden toothpicks.
- HCCA ⁇ -cyano-4-hydroxybenzoic acid
- TFA trifluoroacetic acid
- Samples were measured on Bruker Microflex® MALDI-ToF-Mass Spectrometer using the method “MBT_AutoX”. Laser was set at a shot rate of 60 Hz with the measuring raster (spiral_small). Spectra accumulated in MS/Parent Mode (240 shots).
- Peaks were evaluated with the processing method ‘MBT_Process’ flexControl version: 3.4, Bruker Daltonics (Bremen, Germany) peak detection algorithm ‘centroid’ (signal to noise threshold of 2, a minimum intensity threshold of 600 a.u. and a maximum number of peaks of 300, a peak width of 4 m/z and height of 90%, baseline subtraction with the method ‘TopHat’ for a peak resolution >400). All spectra were compared with reference spectra of the BDAL database (Version 7.0.0.0).
- Example 4A Indole biosynthesis in Bacteroides strains Method - Indole assay
- Indole standards were prepared in YCFA+ media and included 0.2mM, 0.4mM, 0.8mM, and 1.6mM. A positive chemical control was used in this assay, 0.5mM Indole in YCFA+ media.
- the Indole assay was performed using 24-well (non-treated) assay plates. 100mM tryptophan solution in HCL was dispensed into each well to give a final concentration of 6mM in each well. 1mL stationary phase bacterial culture was added to each well and incubated for a further 48 hours. Assay plates were centrifuged at 3,500xg at room temperature for 10 minutes.
- DPPH assay is based on the reduction of DPPH, a stable free-radical, by an antioxidant molecule according to the following reaction DPPH* + AH ⁇ DPPHH + A*
- DPPH* is the stable form of the free radical
- AH is the donor molecule
- DPPHH is the reduced form of DPPH
- A* is the free radical obtained in the reaction.
- the methanolic solution of DPPH turns from a deep violet to light yellow in colour.
- sample/standard/control was added, in triplicate, to corresponding wells of a 96-well plate.
- 200 ⁇ L 200 ⁇ mol/L DPPH was added to three empty wells as a control.
- 190 ⁇ L 200 ⁇ mol/L DPPH was added to sample/standard/control wells and plates incubated in the dark for 30 minutes at room temperature.
- Absorbance was read at 515nm using a BioRad iMark microplate absorbance reader.
- the three Bacteroides dorei strains demonstrated varying degrees of radical scavenging activity, with NCIMB 43599 (Ref 20) and NCIMB 43601 (Ref 8) showing a comparable scavenging activity, second only to NCIMB 43597 (Ref 10) and significantly higher than NCIMB 43595 (Ref 25) (p ⁇ 0.0001).
- the four Bacteroides ovatus strains demonstrated comparable radical scavenging activity.
- Example 4C Antioxidant capacity of Bacteroides strains Method - 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay
- the total antioxidant capacity assay was performed using the Antioxidant Assay Kit (Sigma Aldrich, CS0790).
- the principle of the antioxidant assay is the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, which oxidizes the ABTS to produce a radical cation, ABTS.+, a soluble chromogen that is green in colour and can be spectrophotometrically at 405nm. Solutions and standards were prepared according to the instructions supplied with the assay kit. The method was performed according to the manufacturer’s instructions.
- Trolox standards were prepared from a 1.5mM Trolox working solution.
- 0mM, 0.015mM, 0.045mM, 0.105mM, 0.21mM and 0.42mM solutions were prepared in 1x assay buffer.
- a bacterial strain which demonstrated consistent efficacy (Ref A) was included in the assay as a reference/positive control. All samples were diluted 1 in 4 in 1 x assay buffer. In a 96-well plate, 10 ⁇ L standard/control/sample was added in triplicate. 20 ⁇ L myoglobin working solution was added to all standard/control/sample wells.
- ABTS substrate solution 150 ⁇ L ABTS substrate solution was added to each well and the absorbance measured at 405nm using a BioRad iMark microplate absorbance reader.
- the standard curve was prepared by plotting absorbance as a function of final Trolox concentration (mM).
- Antioxidant concentration of the test sample was calculated using the equation extrapolated from linear regression of the standard curve.
- X(mM) antioxidant concentration (mM) relative to the concentration of trolox standard
- y(A405) is the average absorbance of the test sample at 405nm
- intercept is the Y axis by the standard curve
- slope is the slope of the standard curve ( a negative value)
- dilution factor is the fold dilution of the original sample.
- Example 5 Protection of differentiated neuroblastoma cells from ROS by Bacteroides strains Methods Bacterial strains B. ovatus - NCIMB 43600 (Ref 7) B. koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp - NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B.
- SHSY-5Y cell differentiation and ROS measurement SHSY-5Y is a neuroblastoma cell line. Differentiated SHSY-5Y cells recapitulate in vitro most of the features of neurons. The cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2 mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin. Cells in growth medium were plated in black flat bottom 96 well plate at density of 5,000 cells/well and placed in the CO 2 incubator.
- differentiation medium growth medium containing 1% FBS
- 10 ⁇ M retinoic acid RA 10 ⁇ M retinoic acid RA
- the differentiation medium was removed, cells were washed with pre-warmed PBS and stained with 10 ⁇ M DCFDA molecular probe for 20 min in growth medium containing 1% FBS. Then cells were washed with pre-warmed PBS again and treated with 100 ⁇ M TBHP in the presence or absence of 10% bacteria supernatant for 2h. Fluorescence intensity was measured using TECAN plate reader at Ex/Em 485/530 nm. Results The anti-oxidant potential of nine strains of Bacteroides was investigated in vitro on neuroblastoma cells.
- Oxidative stress was induced with tert-butyl hydrogen peroxide (TBHP) in the SHSY-5Y neuroblastoma cell line.
- NCIMB 43597 (Ref 10) was able to significantly reduce ROS production (p ⁇ 0.0001, Figure 5).
- NCIMB 43600 Ref 7
- NCIMB 43599 Ref 20
- NCIMB 43598 Ref 2
- compositions comprising bacterial strains of the genus Bacteroides are particularly effective at treating neurodegenerative diseases.
- Trunk blood was collected in potassium EDTA (Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at ⁇ 80 °C for further analysis. Intestinal tissue (2 cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm of tissue from the caecum were used) were mounted into the Ussing chambers for intestinal permeability assay. A further 1cm of ileum and colon tissue was taken for tight junction gene expression analysis. The caecum was removed, weighed and stored at ⁇ 80 °C for SCFAs analysis.
- potassium EDTA Ethylene Diamine Tetra Acetic Acid
- probes (6 carboxy fluorescein – FAM
- Amplification reactions contained 1 ⁇ l cDNA, 5 ⁇ l of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to a total of 10 ⁇ l by the addition of Rnase-free water. All reactions were performed in triplicate using 96-well plates on the LightCycler®480 System. Thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for amplicon contamination, each run contained no template controls in triplicate for each probe used. Cycle threshold (Ct) values were recorded. Data was normalized using ⁇ -actin and transformed using the 2 ⁇ CT method and presented as a fold change vs. control group.
- FIGS 8 and 9 show changes in gene expression of tight junction proteins in the ileum and the colon after treatment with bacterial strains from the genus Bacteriodes.
- Tight junction proteins including TJP1 and Occludin help to regulate the permeability of the gut epithelium.
- An increase in expression of these genes indicates a decrease in gut permeability, which is desirable.
- An increase in TJP1 gene expression was observed in the ileum after treatment with Bacteroides sp NCIMB 43598 (Ref 2) ( Figure 8A) and in the colon after treatment with Bacteroides sp NCIMB 43598 (Ref 2) and Ref 31, Bacteroides dorei NCIMB 43599 (Ref 20) and Bacteroides intestinalis Ref 33 ( Figure 8B).
- Example 7 Efficacy of strains of the genus Bacteroides on the tryptophan/kynurenine system Summary The effect of bacterial strains from the genus Bacteroides on the tryptophan/kynurenine system was investigated.
- the metabolism of the amino acid tryptophan is a highly regulated physiological process leading to the generation of several neuroactive compounds within the central nervous system, including 5-hydroxytryptamine, 5-HT. Changes in gene expression of two enzymes involved in tryptophan catabolism in the ileum and the colon after treatment with bacterial strains from the genus Bacteroides was investigated.
- the first is Indoleamine 2,3 dioxygenase-1 (IDO1) which initiates tryptophan catabolism along a pathway that generates several bioactive kynurenine-based metabolites.
- the second is tryptophan hydroxylase-1 (TPH-1) which catalyses the formation of 5-hydroxy-L- tryptophan (5-HTP) from L-tryptophan, the first and rate-limiting step in the biosynthesis of 5-HT.
- TPH-1 tryptophan hydroxylase-1
- 5-HTP 5-hydroxy-L- tryptophan
- TPH-1 gene expression was observed in the ileum after treatment with Bacteroides fragilis Ref 22, Bacteroides vulgatus Ref 24, Bacteroides sp NCIMB 43598 (Ref 2) and Bacteroides xylanisolvens Ref 1.
- a significant decrease in TPH-1 gene expression was observed in ileum after treatment with Bacteroides cellulosilyticus Ref 30.
- Treatment with Bacteroides intestinalis Ref 33 and Bacteroides uniformis Ref 32 also resulted in a decrease in TPH-1 gene expression in the ileum ( Figure 11A).
- FIG 11B shows that treatment with Bacteroides dorei NCIMB 43599 (Ref 20,) Bacteroides sp NCIMB 43598(Ref 2) and Ref 31, Bacteroides uniformis Ref 32 and Bacteroides intestinalis Ref 33 resulted in an increase in TPH-1 gene expression in the colon. A significant decrease in TPH-1 gene expression was observed in the colon after treatment with Bacteroides stercoris NCIMB 43597 (Ref 10). These data suggest that bacterial strains from the genus Bacteriodes play a role in regulating the tryptophan/kynurenine system.
- koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp- NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1) Study design Analysis of GABA The amount of GABA produced was analysed by LC-MS. Stock GABA calibration standards prepared in HPLC grade water: 0, 10, 25, 50, 100, 200, 300 ng/ml.
- the internal standard (d6-GABA) prepared as a 800ng/ml stock in HPLC grade water.
- the standards and samples were prepared by adding 200 ⁇ l calibration standard/sample to 20 ⁇ l 3M perchloric acid, mixed and then centrifuged.
- Derivatisation solution 50 ⁇ l supernatant, 20 ⁇ l internal standard (4ng), 50 ⁇ l 2M KHCO3/KOH buffer, pH9.8, 50 ⁇ l dansyl chloride (20mg/ml in acetonitrile) was mixed then incubated at 80°C for 30 minutes. After the samples were cooled 20 ⁇ l acetic acid was added and then centrifuged.
- the cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2 mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin. Cells were plated in 6 well plates at density of 0.5*10 ⁇ 6 cells/well. after 24h, media were replaced with growth medium containing 1% FBS and treated with 10% (v/v) bacterial supernatants for 3 days. Thereafter total RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. cDNA was prepared from 2000 ng of total RNA using the High-Capacity cDNA reverse transcription kit (Thermo Fisher) according to the manufacturer's instructions.
- FIG. 12A demonstrates that bacterial strains from various different species of Bacteroides were able to induce GABA production.
- bacterial strains from the species Bacteroides xylanisolvens NCIMB 43596 (Ref 1) and Bacteroides ovatus NCIMB 43600 (Ref 7) were able induce over 200 ⁇ g of GABA.
- Figure 12B demonstrates that bacterial strains from the genus Bacteroides were capable of upregulating GABAergic biomarkers in neuronal cells, even for those strains which did not show an induction of GABA production. This suggests that GABA production may be dependent on the amount of bacteria used or the sensitivity of cells to GABA induction. These data indicate that bacteria from the genus Bacteroides are capable of inducing the production of GABA and upregulating the expression of GABAergic biomarkers in neuronal cells, therefore they may be useful in the treatment or prevention of central nervous system diseases or disorders, such as epilepsy and multiple sclerosis.
- a composition comprising a bacterial strain of the genus Bacteriodes, for use in a method of treating or preventing a central nervous system disease or disorder.
- composition for use according to embodiments 1-3 wherein the central nervous system disease or disorder is selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele- Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocere
- compositions for use according to embodiments 1-4 wherein the composition is for use in a method of treating or preventing a neurodegenerative disease.
- the neurodegenerative disease is selected from the group consisting of Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia and spinal muscular atrophy.
- Parkinson’s disease including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-
- composition for use according to embodiment 6, wherein the Parkinson’s disease is including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson- Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism.
- the Alzheimer’s disease is Benson's syndrome.
- the composition for use according to embodiments 1-4, wherein the composition is for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition. 10.
- composition for use according to embodiment 9, wherein the neurodevelopmental disorder or neuropsychiatric condition is selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment, and corticobasal degeneration. 11.
- ASSDs autism spectrum disorders
- OCD obsessive compulsive disorder
- major depressive disorder depression
- seasonal affective disorder anxiety disorders
- chronic fatigue syndrome myalgic encephalomyelitis
- stress disorder post-traumatic stress disorder
- schizophrenia spectrum disorders schizophrenia
- bipolar disorder psychosis
- composition for use according to embodiments 1-4 wherein the composition is for use in a method of treating or preventing a brain injury.
- the brain injury is a result of a stroke, a traumatic brain injury, an acquired brain injury, a trauma, a brain haemorrhage, a tumour, encephalitis, cerebral hypoxia and/or cerebral anoxia.
- the composition for use according to embodiments 1-4, wherein the composition is for use in a method of treating or preventing epilepsy.
- the composition of any preceding embodiment, wherein the composition modulates the microbiota-gut-brain axis. 15.
- composition of any preceding embodiment wherein the composition is for oral administration. 18.
- composition comprises one or more pharmaceutically acceptable excipients or carriers.
- the composition of any preceding embodiment, wherein the bacterial strain is lyophilised.
- the composition of any preceding embodiment, wherein the bacterial strain is viable and capable of partially or totally colonising the intestine.
- 21. The composition of any preceding embodiment, wherein the composition comprises a single strain of Bacteroides.
- the composition of any preceding embodiment which comprises the Bacteroides bacterial strain as part of a microbial consortium.
- a food product comprising the composition of any preceding embodiment, for the use of any preceding embodiment. 24.
- a vaccine composition comprising the composition of any preceding embodiment, for the use of any preceding embodiment.
- 25. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the genus Bacteroides to a patient in need thereof.
- 26. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides dorei to a patient in need thereof.
- 27. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides ovatus to a patient in need thereof.
- a method of treating or preventing a central nervous system disorder or condition comprising administering a composition comprising a bacterial strain of the species Bacteroides stercoris to a patient in need thereof. 29. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides xylanisolvens to a patient in need thereof. 30.
- a pharmaceutical composition comprising the cell of embodiment 30.
- Gennaro edit.1985) [57] US 2016/0067188 [58] Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press. [59] Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press [60] Strobel (2009) Methods Mol Biol.581:247-61. [61] Gennaro (2000) Remington: The Science and Practice of Pharmacy.20th edition, ISBN: 0683306472. [62] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press). [63] Methods In Enzymology (S. Colowick and N.
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Abstract
The invention provides compositions comprising one or more bacterial strains for treating or preventing a central nervous system disease or disorder.
Description
COMPOSITIONS COMPRISING BACTERIAL STRAINS TECHNICAL FIELD
This invention is in the field of bacterial strains and the use of such bacterial strains in the treatment of disease.
BACKGROUND TO THE INVENTION
The human intestine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1],
The human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2], The successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5],
Over 2, 170 species of bacteria have been isolated from the gut of human donors. The bacteria identified can be classified into 12 different phyla, of which 93.5% belonged to Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes [6], The phylum Bacteroidetes is composed of three classes of gram negative bacteria, including the Bacteroidia class which includes the genus Bacteroides. The most common species of Bacteroides present in the gut are Bacteroides caccae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis, Bacteroides dorei, Bacteroides koreensis, Bacteroides kribbi and Bacteroides xylanisolvens [7],
Like other constituents of the gut flora, Bacteroides spp. play a role in the intestine by digesting complex molecules or producing nutrients for the host. Bacteroides spp. can secrete inflammatory neurotoxins, metalloproteinases and pro-inflammatory molecules, including lipopolysaccharides (LPS) [8], These metalloproteinases can disrupt the gut epithelium and facilitate the propagation of inflammatory neurotoxins and LPS throughout the body. Once the LPS is translocated from the gut to the systemic circulation it is recognised by TLR receptors. The TLR receptors subsequently activate the systemic immune system, resulting in the release of pro-inflammatory cytokines [9,10],
Certain bacterial strains have been proposed for use in the treatment of various diseases (see, for example, [11-14]). For example, Lactobacillus and Bifidobacterium strains have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the gastrointestinal tract (see [15] and [16] for reviews). WO2016/102950 describes the efficacy of the Bacteroides species, BT2013, against gastrointestinal inflammatory disorders and the use of BT2013 as a therapeutic agent in preventing inflammatory, autoimmune and allergic disorders. The exact relationship between different diseases and different bacterial strains is poorly understood. In particular, the precise effects of a specific bacterial species on the gut, at the systemic level and on any particular type of disease are poorly characterised, particularly for inflammatory neurodegenerative disorders. Recently, there has been increased interest in the art regarding alterations in the gut microbiome that may play a pathophysiological role in human brain diseases [17]. Preclinical and clinical evidence suggests a link between brain development and microbiota [18]. A growing body of preclinical literature has demonstrated bidirectional signalling between the brain and the gut microbiome, involving multiple neurocrine and endocrine signalling systems. Indeed, increased levels of Clostridium species in the microbiome have been linked to brain disorders [19], and an imbalance of the Bacteroidetes and Firmicutes phyla has also been implicated in brain development disorders [20]. Bacteroides fragilis NCTC 9343 was found to correct the gut permeability and improve Autism Spectrum Disorder related effects in C57BL/6N mice [21]. However, the suggested links between the levels of bacteria from the genera Bifidobacterium, Lactobacillus, Sutterella, Prevotella and Ruminococcus and the family Alcaligenaceae and immune-mediated central nervous system (CNS) disorders, have been questioned, however, by studies suggesting a lack of alteration in the microbiota between patients and healthy subjects [21]. Studies have suggested the use of Bacteroides species, including Bacteroides fragilis, Bacteroides theta, Bacteroides thetaiotaomicron, Bacteroides vulgatus and Bacteroides faecalis for treating autism spectrum disorders, epilepsy and schizophrenia [22-27]. WO02/07741 suggests that compositions comprising Clostridia in combination with other bacterial strains can be used to treat diseases associated with the presence of abnormal microflora in the gastrointestinal tract, including neurological syndromes and psychiatric disorders. WO2009/149149 suggests the use of bacteria from the genus Bacteroides in the treatment or prevention of immune-related disorders and in particular the use of Bacteroides fragilis to protect against experimental autoimmune encephalomyelitis. However, this document does not demonstrate the ability of other species of Bacteroides to treat experimental autoimmune encephalomyelitis or the ability of any other bacteria from the genus Bacteroides to treat inflammatory central nervous system disorders or neurodegenerative disease.
US2008/254009 discloses the manipulation of the gut microbiome, either through the administration of antibiotics or the administration of probiotic mixtures comprising organisms from the Clostridium genus for treating neurological and gastrointestinal disorders. WO2017160711 discusses engineering bacteria, and the utilisation of bacteria, to produce GABA for the treatment of mental illnesses or diseases of the central nervous system. A number of bacteria are identified which may be able to inherently do so. Additionally, Table 10 of that document sets out a significantly longer list of 2219 bacterial species which are potentially capable of being engineered to produce GABA but do not appear to possess the ability to do so naturally. There is a need in the art to provide further treatments for treating central nervous system diseases or disorders. SUMMARY OF THE INVENTION The inventors have developed new therapies for treating central nervous system diseases or disorders, in particular those associated with inflammation and oxidative stress. The inventors have demonstrated that bacterial strains from the genus Bacteroides have immuno-inhibitory and anti-oxidant properties. As shown in the examples, the administration of compositions comprising bacterial strains from the genus Bacteroides can protect against reactive oxygen species and prevent inflammation and therefore have an antioxidant effect. The examples also demonstrate the ability of organisms from the genus Bacteroides to induce the production of GABA in human cell lines. The invention provides compositions comprising bacteria from the genus Bacteroides for use in treating or preventing a central nervous system disease or disorder. In preferred embodiments, the compositions are for use in treating or preventing an inflammatory central nervous system disease or disorder. In preferred embodiments, the composition comprises a bacterial strain of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides koreensis, Bacteroides kribbi, Bacteroides thetaiotaomicron Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii or Bacteroides caccae. The bacterial strain can be the strain deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601. Additionally, the invention provides methods of treating or preventing a central nervous system disease or disorder, comprising administering a composition comprising a bacterial strain of the genus Bacteroides. In preferred embodiments, the invention provides methods of treating or preventing an inflammatory central nervous system disease or disorder. Furthermore, the invention provides uses of a composition comprising a bacterial strain of the genus Bacteroides for the manufacture of a medicament for the treatment or prevention of a central nervous system disease or disorder. In preferred embodiments, the central nervous system disease or disorder is an inflammatory central nervous system disease or disorder.
The invention also provides compositions, methods and uses for treating or preventing a central nervous system disease or disorder mediated by dysfunction of the microbiota-gut-brain axis, comprising administering a composition comprising a bacterial strain of the genus Bacteroides. The central nervous system disease or disorder may be mediated by the microbiota-gut-brain axis. In certain embodiments, the composition is for use in a method of modulating the microbiota-gut-brain axis. In further embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodegenerative disease, a neurodevelopmental disorder, a neuropsychiatric condition or a brain injury. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodegenerative disease. The neurodegenerative disease may be selected from the group consisting of Parkinson’s disease (including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism); Alzheimer’s disease (including Benson's syndrome); multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia and spinal muscular atrophy. The invention also provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating a brain injury. The brain injury can be the result of a stroke, a traumatic brain injury, an acquired brain injury, a trauma, a brain haemorrhage, a tumour, encephalitis, cerebral hypoxia and/or cerebral anoxia. The invention also provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating epilepsy. The invention also provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition. The neurodevelopmental disorder or neuropsychiatric condition may be selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post- traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment, and corticobasal degeneration. The composition of the invention can be for oral administration. Oral administration of the strains of the invention can be effective for treating central nervous system diseases and disorders, in particular
those mediated by the microbiota-gut-brain axis and/or those associated with inflammation. Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or total colonisation of the intestine. The composition of the invention may comprise one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the composition of the invention has been lyophilised. The composition of the invention can also comprise a lyophilised bacteria strain of the genus Bacteroides. The bacterial strain may have been lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria. In certain embodiments, the bacterial strain is viable and capable of partially or totally colonising the intestine. The invention further provides a food product comprising a composition comprising a bacterial strain of the genus Bacteroides as described above. The invention also provides a vaccine composition comprising a bacterial strain of the genus Bacteroides as described above. In developing the above invention, the inventors have identified and characterised Bacteroides bacterial strains that are particularly useful for therapy. The Bacteroides dorei species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders. In particular, bacterial strains from the species Bacteroides dorei are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, NCIMB 43599 or NCIMB 43601, or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, NCIMB 43599 or NCIMB 43601, or a derivative thereof, for use in therapy, in particular for the diseases described herein. The Bacteroides ovatus species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders. In particular, bacterial strains from the species Bacteroides ovatus are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600 , or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof, for use in therapy, in particular for the diseases described herein. The Bacteroides stercoris species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders. In particular, bacterial strains from the species Bacteroides stercoris are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a
cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof, for use in therapy, in particular for the diseases described herein. The Bacteroides xylanisolvens species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders. In particular, bacterial strains from the species Bacteroides xylanisolvens are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof, for use in therapy, in particular for the diseases described herein. The Bacteroides koreensis species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders. In particular, bacterial strains from the species Bacteroides koreensis are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof, for use in therapy, in particular for the diseases described herein. The Bacteroides sp species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders. In particular, bacterial strains from the species Bacteroides sp are shown to be effective at treating or preventing inflammatory central nervous system diseases or disorders. Therefore, in another aspect, the invention provides a cell of the Bacteroides sp strain deposited under accession number NCIMB 43593 or NCIMB 43598, or a derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides sp.strain deposited under accession number NCIMB 43593 or NCIMB 43598, or a derivative thereof, for use in therapy, in particular for the diseases described herein. BRIEF DESCRIPTION OF DRAWINGS Figure 1: Inhibition of IL-6 secretion in U373 cells by strains of Bacteroides. Figure 1 represents an average of 4 replicates (mean±SEM) and IL-6 secretion is expressed as a percentage related to LPS in YCFA+ media. *= p≤0.05; **= p≤0.01; ***= p≤0.001 and ****= p≤0.0001.
Figure 2: Secretion of IL-6 and IL-10 in peripheral blood mononuclear cells (PBMCs) after treatment with different strains of Bacteroides from an average of ten healthy human donors. Figure 3: Comparison of NF-kB activation after treatment with LPS, a-synuclein mutant A53T and different strains of Bacteroides . Figure 3 represents an average of 3 replicated (mean±SEM). N=2 for Ref 7 and REF 8. Figure 4: A) The anti-oxidant potential of different strains of Bacteroides; B) the DPPH radical scavenging activity of different strains of Bacteroides; C) the antioxidant concentration of different strains of Bacteroides. Figure 4 represents three biological replicates (mean±SEM). Indole production is expressed as concentration calculated from the standard curve. Figure 5: Antioxidant capacity of different strains of Bacteroides in SHSY-5Y cells. Figure 6 - Effect of bacteria from the genus Bacteroides on intestinal ileum permeability Figure 7 - Effect of bacteria from the genus Bacteroides on intestinal colon permeability Figure 8 - Effect of bacteria from the genus Bacteroides on the gene expression of the tight junction protein TJP1 in (A) the ileum and (B) the colon. Figure 9 - Effect of bacteria from the genus Bacteroides on the gene expression of the tight junction protein Occulin in (A) the ileum and (B) the colon. Figure 10 - Effect of bacteria from the genus Bacteroides on the gene expression of Indoleamine 2,3 dioxygenase-1 (IDO1) (A) the ileum and (B) the colon. Figure 11 - Effect of bacteria from the genus Bacteroides on the gene expression Tryptophan hydroxylase-1 (TPH-1) in (A) the ileum and (B) the colon. Figure 12 – Effect of bacteria from the genus Bacteroides on GABA production (A) and the production of GABAergic neuron markers (B) DISCLOSURE OF THE INVENTION Bacterial strains The compositions of the invention comprise a bacterial strain of the genus Bacteroides. The examples demonstrate that bacteria of this genus are useful for treating or preventing central nervous diseases and disorders, such as neurodegenerative diseases. In particular, bacterial strains from this genus are useful for treating or preventing inflammatory central nervous system diseases or disorders. Bacteroides species are gram-negative, obligate anaerobic, non-spore-forming, rod shaped, bile resistant and can be motile or non-motile [28]. Bacteroides species are one of the major species present in the human microbiome. Examples of Bacteroides species for use in the invention include Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,
Bacteroides salyersae, Bacteroides cellulosilyticus, Bacteroides intestinalis, Bacteroides koreensis, Bacteroides kribbi and Bacteroides xylanisolvens. The examples show that bacterial strains of the genus Bacteroides have immuno-inhibitory, GABAergic and anti-oxidant properties. The bacterial strains of the invention can be selected from the species: Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae. In further preferred embodiments, the composition can comprise a bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis or Bacteroides kribbi. Therefore, in preferred embodiments of the invention the bacterial strain in the composition is Bacteroides dorei. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides dorei. 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: 5 or 12. Preferably, the sequence identity is to SEQ ID NO:5 or 12. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 5 or 12. In other preferred embodiments of the invention, the bacterial strain in the composition is Bacteroides ovatus. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides ovatus. 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: 11. Preferably, the sequence identity is to SEQ ID NO: 11. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 11. In further preferred embodiments of the invention, the bacterial strain in the composition is Bacteroides stercoris. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides 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:7. Preferably, the sequence identity is to SEQ ID NO:7. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:7. In further preferred embodiments of the invention, the bacterial strain in the composition is Bacteroides xylanisolvens. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides xylanisolvens. 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:8.
Preferably, the sequence identity is to SEQ ID NO:8. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:8. In further preferred embodiments of the invention, the bacterial strain in the composition is Bacteroides koreensis. Closely related strains may also be used, such as bacterial strains that have 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 Bacteroides koreensis. 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: 9. Preferably, the sequence identity is to SEQ ID NO: 9 . Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:9. In further preferred embodiments of the invention, the bacterial strain in the composition is from the genus Bacteroides sp and has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:6 or 10. Preferably, the sequence identity is to SEQ ID NO:6 or 10. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:6 or 10. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides dorei. In preferred embodiments, the bacterial strains are NCIMB 43595, NCIMB 43599 or NCIMB 43601. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides ovatus. In preferred embodiments, the bacterial strains are NCIMB 43600. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides koreensis. In preferred embodiments, the bacterial strains are NCIMB 43594. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides sp. In preferred embodiments, the bacterial strains are. NCIMB 43593 or NCIMB 43598.In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides stercoris. In preferred embodiments, the bacterial strain is NCIMB 43597. In some embodiments, the compositions of the invention comprise bacterial strains of the species Bacteroides xylanisolvens. In preferred embodiments, the bacterial strain is NCIMB 43596. In other embodiments, the bacterial strain is not of the species Bacteroides fragilis. In some embodiments, the compositions of the invention may not contain bacterial strains from the species Bacteroides fragilis. In addition, compositions of the invention may not contain bacterial strains from the genus Clostridia. Examples of Bacteroides vulgatus strains are DSM 1447, DSM 28735, DSM 23289. The GenBank accession number for the 16S rRNA gene sequence of Bacteroides vulgatus strain DSM 1447 is HQ012024 (disclosed herein as SEQ ID NO:1). An exemplary strain of Bacteroides dorei is DSM
17855. The GenBank accession number for the 16S rRNA gene sequence of Bacteroides dorei is DSM 17855 is NZ_DS995567.1 (disclosed herein as SEQ ID NO:2). An exemplary strain of Bacteroides ovatus DSM1896. Exemplary Bacteroides xylanisolvens strains include DSM 18836. DSM 19555 is an example of a Bacteroides stercoris and DSM2151 is an example of a Bacteroides fragilis strain. Bacteroides thetaiotaomicron strain BT2013 has been deposited under accession number NCIMB 42341 on 3rd December 2014 at the National Collections of Industrial, Food and Marine Bacteria (NCIMB) at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, UK, AB21 9YA. The deposit was made under the terms of the Budapest Treaty. The deposit was made by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). GT Biologics Ltd. Has subsequently changed its name to 4D Pharma Research Limited. The genome sequence of BT2013 was provided as SEQ ID NO:1 in WO2016/102950, the contents of which is incorporated herein by reference (and referred to herein as SEQ ID NO:3). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 42408, which is also referred to herein as strain 675. A 16S rRNA sequence for the 675 strain that was tested is provided in SEQ ID NO:4. The deposit was made under the terms of the Budapest Treaty. Strain 675 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroidales 675” on 13th May 2015 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43593. Also referred to herein as Ref 27. A 16S rRNA sequence for this strain is provided in SEQ ID NO:10. The deposit was made under the terms of the Budapest Treaty. NCIMB 43593 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides sp.” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43594. Also referred to herein as Ref 17. A 16S rRNA sequence for this strain is provided in SEQ ID NO:9. The deposit was made under the terms of the Budapest Treaty. NCIMB 43594 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland) as “Bacteroides koreensis” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43595. The deposit was made under the terms of the Budapest Treaty. NCIMB 43595 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides dorei” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland).
A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43596. Also referred to herein as Ref 1. A 16S rRNA sequence for this strain is provided in SEQ ID NO:8. The deposit was made under the terms of the Budapest Treaty. NCIMB 43596 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides xylanisolvens” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43597. Also referred to herein as Ref 10. A 16S rRNA sequence for this strain is provided in SEQ ID NO:7. The deposit was made under the terms of the Budapest Treaty. NCIMB 43597 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland) as “Bacteroides stercoris” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43598. Also referred to herein as Ref 2. A 16S rRNA sequence for this strain is provided in SEQ ID NO:6. The deposit was made under the terms of the Budapest Treaty. NCIMB 43598was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides sp” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43599. Also referred to herein as Ref 20. A 16S rRNA sequence for this strain is provided in SEQ ID NO:5. The deposit was made under the terms of the Budapest Treaty. NCIMB 43599 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides dorei” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43600. Also referred to herein as Ref 7. A 16S rRNA sequence for this strain is provided in SEQ ID NO:11. The deposit was made under the terms of the Budapest Treaty. NCIMB 43600 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides ovatus” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB252ZS, Scotland). A preferred Bacteroides strain is the strain deposited under accession number NCIMB 43601. Also referred to herein as Ref 8. A 16S rRNA sequence for this strain is provided in SEQ ID NO:12. The deposit was made under the terms of the Budapest Treaty. NCIMB 43601 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA, Scotland) as “Bacteroides dorei” on 14th April 2020 by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland). Preferably, the bacterial
strain has a 16s rRNA sequence that is at least 98.65% sequence similarity to SEQ ID NO:5-12. Pairwise similarities between 16S rRNA gene sequences can be calculated based on robust global sequence alignment algorithms such as the EzTaxon server described in [29]. Bacterial strains closely related to the strain tested in the examples are also expected to be effective for treating or preventing central nervous system diseases and disorders, and in particular inflammatory neurodegenerative disorders and can thus be used in the invention. 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 Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae. 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: 5-12. Preferably, the sequence identity is to SEQ ID NO: 5-12. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 5-12. Bacterial strains that are biotypes of the bacteria disclosed herein (for example, the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 and NCIMB 43601 ) are also expected to be effective for treating or preventing central nervous system diseases and disorders, and in particular inflammatory neurodegenerative disorders. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics. Strains that are biotypes of the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may have 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). Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG)5, or REP or [30]. Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601. In certain embodiments, the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to
the genome of Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of the genome. For example, in certain embodiments, the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:3 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:3. For example, the bacterial strain for use in the invention may have a genome with at least 90% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:33, or at least 90% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 30% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 30% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 30% of SEQ ID NO:3. Alternatively, strains that are biotypes of the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 , NCIMB 43601, NCIMB 42408 or NCIMB 42341 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive 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 Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides kribbi, Bacteroides koreensis or Bacteroides caccae strains. In certain embodiments, strains that are biotypes of the bacteria deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 and that are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599,
NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example [31]). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341. The examples demonstrate that bacteria from the genus Bacteroides are capable of inducing the production of GABA, which is useful in the treatment or prevention of central nervous system diseases or disorders, such as epilepsy. In certain embodiments, the bacterial strain for use in the invention can induce GABA production to a greater extent than media alone using the assay described in Example 11. For example, bacterial strains from the genus Bacteroides or the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides kribbi, Bacteroides koreensis or Bacteroides caccae that can induce GABA production to a greater extent than media alone. In certain embodiments, strains that are biotypes of the bacteria deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 and that are suitable for use in the invention are strains which can induce the production of GABA to a greater extent than media. Other Bacteroides strains that are useful in the compositions and methods of the invention, such as biotypes of the bacteria deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341, may be identified using any appropriate method, including the assays described in the examples. For instance, strains for use in the invention may be identified by culturing a candidate strain with inflammatory neuroblastoma cells and then assessing cytokine levels and levels of inflammatory neuroprotection or inflammatory neuro- proliferation. In particular, bacterial strains that have similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 may be useful in the invention. A useful strain will have comparable immune modulatory activity to the NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600, NCIMB 43601, NCIMB 42408 or NCIMB 42341 strain. In particular, a biotype strain will elicit comparable effects on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples. For example,
a biotype strain will be able to reduce IL-6 in neuroblastoma cells, increase IL-6 and IL-10 secretion in peripheral blood mononuclear cell (PBMCs) or have a high antioxidant capacity compared to compared to YCFA+ media. The Bacteroides dorei species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders. A particularly preferred strain of the invention is the Bacteroides dorei strain deposited under accession number NCIMB 43595. This is the exemplary strain Ref 25 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides dorei strain deposited under accession number NCIMB 43595, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides dorei strain deposited under accession number NCIMB 43595The invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43595, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. A particularly preferred strain of the invention is the Bacteroides dorei strain deposited under accession number NCIMB 43599. This is the exemplary strain Ref 20 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides dorei strain deposited under accession number NCIMB 43599, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43599, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides dorei strain deposited under accession number NCIMB 43599. The invention also provides a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43599, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. A particularly preferred strain of the invention is the Bacteroides dorei strain deposited under accession number NCIMB 43601. This is the exemplary strain Ref 8 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides dorei strain deposited under accession number NCIMB 43601, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides dorei strain deposited under accession number NCIMB 43601, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides dorei strain deposited under accession number NCIMB 43601. The invention also provides a cell of the Bacteroides dorei strain deposited
under accession number NCIMB 43601, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. A particularly preferred strain of the invention is the Bacteroides ovatus strain deposited under accession number NCIMB 43600. This is the exemplary strain Ref 7 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides ovatus strain deposited under accession number NCIMB 43600. The invention also provides a cell of the Bacteroides ovatus strain deposited under accession number NCIMB 43600, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. A particularly preferred strain of the invention is the Bacteroides koreensis strain deposited under accession number NCIMB 43594. This is the exemplary strain Ref 17 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 43594, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides koreensis strain deposited under accession number NCIMB 43594. The invention also provides a cell of the Bacteroides koreensis strain deposited under accession number NCIMB 435934 or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. Furthermore, the invention provides a composition comprising the species Bacteroides koreensis for use in therapy. The Bacteroides species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders. A particularly preferred strain of the invention is the Bacteroides strain deposited under accession number NCIMB 43593. This is the exemplary strain Ref 27 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides strain deposited under accession number NCIMB 43593, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides strain deposited under accession number NCIMB 43593, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides strain deposited under accession number NCIMB 43593. The invention also provides a cell of the Bacteroides strain
deposited under accession number NCIMB 43593, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. A particularly preferred strain of the invention is the Bacteroides strain deposited under accession number NCIMB 43598. This is the exemplary strain Ref 2 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides strain deposited under accession number NCIMB 43598, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides strain deposited under accession number NCIMB 43598, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides strain deposited under accession number NCIMB 43598. The invention also provides a cell of the Bacteroides strain deposited under accession number NCIMB 43598, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. The Bacteroides xylanisolvens species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders. A particularly preferred strain of the invention is the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596. This is the exemplary strain Ref 1 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof. The invention also provides a biologically pure culture of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596. The invention also provides a cell of the Bacteroides xylanisolvens strain deposited under accession number NCIMB 43596, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. The Bacteroides stercoris species of the invention are shown to be effective for treating the diseases described herein, such as central nervous system diseases and disorders, and in particular inflammatory central nervous system diseases or disorders. A particularly preferred strain of the invention is the Bacteroides stercoris strain deposited under accession number NCIMB 43597. This is the exemplary strain Ref 10 tested in the examples and has been shown to be effective at treating central nervous system diseases or disorders, and in particular inflammatory central nervous system diseases or disorders. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof. The invention also provides a composition comprising a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof. The invention also provides a biologically pure culture
of the Bacteroides stercoris strain deposited under accession number NCIMB 43597. The invention also provides a cell of the Bacteroides stercoris strain deposited under accession number NCIMB 43597, or a derivative thereof, for use in therapy, in particular for central nervous system diseases and disorders. A derivative of the strain deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A derivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a derivative strain of the invention is therapeutically active. A derivative strain will have comparable immune modulatory activity to the original NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601strain. In particular, a derivative strain will elicit comparable effects on the central nervous system disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples. A derivative of the NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601 strain will generally be a biotype of the strain NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601. The bacterial strain may also be a strain that has the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601, and such cells are encompassed by the invention. In preferred embodiments, the bacterial strains in the compositions of the invention are viable and/or live. They may be capable of partially or totally colonising the intestine. In preferred embodiments, the bacterial strain for use in the invention is naturally-occurring. For example, in certain embodiments, the bacterial strain may have been isolated from the mammalian digestive tract. In preferred embodiments, the bacterial strain for use in the invention has not been not genetically engineered. For example, in certain embodiments, the bacterial strain may not have been transformed with recombinant DNA.
Therapeutic uses The examples demonstrate that bacterial strains from the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae can decrease the secretion of the pro-inflammatory cytokine IL-6 in human glioblastoma astrocytoma cells. In addition, the inventors have shown that bacteria from the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides koreensis, Bacteroides kribbi and Bacteroides xylanisolvens have high antioxidant capacities and can protect neuroblastoma cells from oxidative damage. Therefore, the inventors have shown that bacteria from the genus Bacteroides are particularly effective in preventing or treating central nervous system diseases or disorders, in particular those associated with inflammation. Central nervous system diseases or disorders include neurodegenerative diseases, neurodevelopmental disorders, neuropsychiatric conditions and brain injuries. In preferred embodiments, the invention provides compositions comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a neurodegenerative disease. The neurodegenerative disease may be selected from the group consisting of Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia and spinal muscular atrophy. In other embodiments the compositions of the invention may be useful for treating central nervous system diseases or disorders in adult patients and in particular inflammatory central nervous system diseases or disorders. The patients may be between 18 and 65 years old, for example between 40 to 65 years old; or they may be older than 40 years. The compositions of the invention may be for use in treating a disease associated with old age, for example, a disease diagnosed in a patient who is older than 50 years. The compositions of the invention may be effective at treating for treating central nervous system diseases or disorders, such as neurodegenerative diseases that occur in elderly patients, for example, the patients are 65 years or older. The inventors have found that bacterial strains of the genus Bacteroides can reduce the secretion of pro-inflammatory cytokines, such as IL-6. As shown in the examples, Bacteroides strains according to the invention can decrease the secretion of IL-6 in human glioblastoma astrocytoma cells. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prevention of central nervous system diseases or disorders, in particular those associated with inflammation. In some embodiments, the bacterial strains are useful in the treatment of central nervous system diseases or disorders characterised by the enhanced activation of IL-6, such as inflammatory central nervous
system diseases or disorders. Therefore, the compositions of the invention may be useful for decreasing the levels of IL-6, particular in patients with a neurodegenerative disease. IL-10 is an anti-inflammatory cytokine that is synthesised in the CNS and can act to limit clinical symptoms of stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioural changes that occur during bacterial infections [32]. The examples show that the bacterial strains of the invention can increase the per se production of IL-10. In preferred embodiments, the compositions of the invention may be useful for treating or preventing central nervous system diseases or disorders, including stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioural changes that occur during bacterial infections. In some embodiments, the bacterial strains may be useful for treating or preventing central nervous system diseases or disorders that are characterised by a reduction in the production of IL-10, such as inflammatory central nervous system diseases or disorders. The inventors have demonstrated that bacterial strains of the genus Bacteroides can decrease the gut permeability. Bacteria can modulate signalling of the microbiota-gut-brain axis by modulating the levels of gastrointestinal permeability. Abrogation of the gut epithelial barrier allows harmful substances to leave the intestine and is linked to the induction of inflammatory and autoimmune diseases. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prevention of central nervous system diseases or disorders characterised by increased gut permeability. The bacterial strains of the invention are also useful in the treatment or prevention of inflammatory central nervous system diseases or disorders characterised by increased gut permeability. Therefore, the compositions of the invention may be useful for decreasing gut permeability, particularly in patients with a neurodegenerative disease. The examples show that bacterial strains of the genus Bacteroides have potent antioxidant activity. For example, bacterial strains from the genus Bacteroides produce high levels of the known antioxidant indole, have high radical scavenging activity (e.g. against DPPH radicals) and can reduce the production of reactive oxygen species (ROS). In some embodiments, the bacterial strains are useful in the treatment of central nervous system diseases or disorders characterised by oxidative stress, and in particular those associated with inflammation. The compositions of the invention may be useful for reducing the oxidative stress by increasing the production of antioxidants, particularly in patients with a neurodegenerative disease. For example, in some embodiments the bacterial strains of the invention useful in the treatment of central nervous system diseases or disorders characterised by increased ROS levels, and in particular those associated with inflammation. The compositions of the invention may be useful for reducing the production of ROS, particularly in patients with a neurodegenerative disease. The compositions of the invention may be for use in reducing or preventing the loss of dopaminergic cells in the substantia nigra. The compositions of the invention may be used for reducing or preventing the degeneration of dopaminergic inflammatory neurons in the substantia nigra pars compacta. The
compositions of the invention may be used for reducing or preventing the degeneration of dopaminergic inflammatory neurons in the substantia nigra pars compacta and the consequent loss of their projecting nerve fibres in the striatum. The compositions of the invention may be used for reducing or preventing loss of nigrostriatal dopaminergic inflammatory neurons. The compositions of the invention may be for use in treating a central nervous system disease or disorder mediated or characterised by the accumulation of protein, in particular mis-folded protein, such as an inflammatory central nervous system disease or disorder. The compositions of the invention are for use in treating a central nervous system disease or disorder associated with grey matter inflammatory neuronal loss, such as an inflammatory central nervous system disease or disorder. In certain embodiments, the compositions of the invention are for treating a central nervous system disease or disorder that is not associated with white matter lesions, such as an inflammatory central nervous system disease or disorder. The compositions of the invention may be for use in treating a central nervous system disease or disorder associated with permanent symptoms, such as an inflammatory central nervous system disease or disorder. Permanent symptoms are present on clinical examination and expected to last throughout the person’s life. Permanent neurological symptoms include numbness, hyperaesthesia (increased sensitivity), paralysis, localised weakness, dysarthria (difficulty with speech), aphasia (inability to speak), dysphagia (difficulty in swallowing), visual impairment, difficulty in walking, lack of co- ordination, tremor, seizures, lethargy, dementia, delirium and coma. The neuroprotective properties of the compositions of the invention, as shown in the examples, mean that the compositions may be particularly effective for preventing or delaying onset or progression of a central nervous system disease or disorder, such as an inflammatory central nervous system disease or disorder. In certain embodiments, the compositions of the invention are for use in delaying onset or progression of a central nervous system disease or disorder, and in particular an inflammatory central nervous system disease or disorder. In addition, the inventors have shown that bacteria from the species Bacteroides stercoris have anti- inflammatory and anti-oxidant properties. These properties make bacteria strains from the species Bacteroides stercoris particularly effective at treating or preventing an inflammatory disease, an autoimmune disease and cancer. Additionally, the invention provides a method of treating or preventing an inflammatory disease, an autoimmune disease and cancer, comprising administering a composition comprising a bacterial strain of the species Bacteroides stercoris to a subject. Furthermore, the invention provides a use of a composition comprising a bacterial strain of the species Bacteroides stercoris for the manufacture of a medicament for the treatment or prevention of an inflammatory disease, an autoimmune disease and cancer.
The invention provides a composition comprising a bacterial strain of the species Bacteroides stercoris for use in a method of treating or preventing an inflammatory disease. The inflammatory disease can be selected from the group consisting of: adult-onset Still's disease, amyloid A amyloidosis, polymyalgia rheumatica, remitting seronegative symmetrical synovitis with pitting edema, Behcet's disease, uveitis, graft-versus-host diseases, and tumor necrosis factor receptor-associated periodic syndrome, Systemic sclerosis, ANCA-associated vasculitis, Takayasu arteritis, osteoarthritis, osteoporosis, multiple sclerosis, asthma, atopic dermatitis, sciatica, Polymyalgia rheumatica, chronic glomerulonephritis, inflammatory bowel disease and cardiovascular disease (such as pulmonary arterial hypertension, atherosclerosis and Non-ST elevation myocardial infarction). The invention provides compositions comprising a bacterial strain of the species Bacteroides stercoris for use in a method of treating or preventing an autoimmune disease, wherein the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, systemic sclerosis, polymyositis, vasculitis syndrome including giant cell arteritis, Takayasu arteritis, cryoglobulinemia, myeloperoxidase-antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis and rheumatoid vasculitis; organ-specific, rheumatoid arthritis, juvenile idiopathic arthritis; systemic onset juvenile idiopathic arthritis, Graves ophthalmopathy, Relapsing polychondritiis, ankylosing spondylitis, Type II diabetes, obesity, Crohn's disease, relapsing polychondritis, Castleman’s disease, Neuromyelitis optica (Devic's disease), acquired hemophilia A and autoimmune hemolytic anemia. The invention provides compositions comprising a bacterial strain of the species Bacteroides stercoris for use in a method of treating or preventing an cancer, wherein the cancer is selected from the group consisting of: multiple myeloma, prostate cancer, metastatic renal cell carcinoma, metastatic kidney cancer, non-small cell lung cancer, colorectal cancer,;andpancreatic cancer, T-cell acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukaemia, multiple myeloma, colorectal cancer and myeloma (such as multiple myeloma). The compositions according to the invention may also be for use in a method of treating or preventing an inflammatory disease, an autoimmune disease or cancer associated with oxidative stress. In preferred embodiments, the inflammatory disease, autoimmune disease or cancer associated with oxidative stress is selected from gastrointestinal diseases; (such as peptic ulcers) gastrointestinal cancers; inflammatory bowel disease; cardiovascular diseases; (such as atherosclerosis), ischemia; hypertension; cardiomyopathy; cardiac hypertrophy and congestive heart failure; renal diseases, such as glomerulonephritis and tubulointerstitial nephritis, chronic renal failure, proteinuria, uremia; inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD); ocular diseases (such as cataracts and retinal diseases) and joint diseases (such as rheumatoid arthritis and rheumatism).
Neurodegenerative diseases The examples demonstrate the neuroprotective properties of compositions comprising bacterial strains of the genus Bacteroides. The inventors have also shown that treatment with bacterial strains from the genus Bacteroides can reduce the activation of proinflammatory molecules, such as NFκB and IL-6, by LPS and mutant ^-synuclein A53T. This decrease is particularly effective in human glioblastoma astrocytoma cells. The compositions of the invention, therefore, may be particularly effective at preventing or delaying the onset or progression of neurodegenerative diseases. In certain embodiments, the compositions of the invention are for use in delaying the onset or progression of neurodegenerative diseases. The prevention of neurodegenerative diseases after administration of compositions of the invention can be measured relative to the disease state or disorder level observed in the patient before treatment, a healthy individual or an individual with a more severe form of the neurodegenerative disease or disorder. Chronic inflammation induced by IL-6 can ultimately lead to cell death. Bacterial strains of the invention can be used to reduce or prevent neuronal cell death. Therefore, in preferred embodiments, the compositions of the invention are for use in reducing or preventing neuron death, in particular, in the treatment of neurodegenerative diseases. The reduction in neuron cell death observed after administration of compositions of the invention can be measured relative to a healthy individual, an individual with a more severe form of the neurodegenerative disease or to the rate of neuron cell death observed in the patient before treatment with the compositions of the invention. It is known that administration of LPS can replicate some characteristics of Parkinson’s disease, including extensive activation of microglia and selective loss of dopaminergic neurons in the nigrostriatal system [33]. The inventors have demonstrated that bacterial strains of the genus Bacteroides can decrease the secretion of the pro-inflammatory cytokine IL-6 after induction with LPS. Therefore, in preferred embodiments, compositions comprising bacterial strains from the genus Bacteroides may be particularly effective at treating environmental Parkinson’s. IL-6 can act as an anti-inflammatory regulator by stimulating potent anti-inflammatory cytokines such as IL-10 [34, 35]. The examples show that bacterial strains of the genus Bacteroides have anti- inflammatory activity and can induce the per se production of IL-6 and IL-10 in peripheral blood mononuclear cells. Therefore, the compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are the result of inflammation. In preferred embodiments, bacterial strains of the genus Bacteroides are useful in the treatment of neurodegenerative diseases characterised by the reduced production of IL-6. IL-10 limits inflammation in the brain via three major pathways: (1) reducing the synthesis of proinflammatory cytokines, (2) suppressing cytokine receptor expression, and (3) inhibiting receptor activation [Error! Bookmark not defined.]. Therefore, in certain embodiments the composition of the invention may be useful for treating or preventing inflammation in the brain.
IL-10 promotes the survival of neurons and all glial cells in the brain by blocking the effects of proapoptotic cytokines and by promoting expression of cell survival signals [Error! Bookmark not defined.]. Therefore, in certain embodiments the compositions of the invention can increase the survival of neurons in the brain. In other embodiments, the compositions of the invention can increase the survival of glial cells in the brain. The compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are the result of neuronal or glial cell death in the brain. The cytokines IL-6 and IL-10 are known to share the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signalling pathway [34,35]. Activation of the JAK/STAT3 signalling pathway by IL-10 promotes cell survival by inhibiting both ligand- and mitochondrial-induced apoptotic pathways [Error! Bookmark not defined.]. The JAK/STAT3 pathway is a common mediator of astrocyte reactivity which is a hallmark of neurodegenerative diseases, such as Alzheimer's and Huntington's diseases [36]. Therefore, in certain embodiments, the bacterial strains of the invention can activate the JAK/STAT3 pathway. The compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are mediated by the STAT-3 signalling pathway. In other embodiments, the bacterial strains of the invention can promote astrocyte reactivity. Therefore, in certain embodiments, the composition of the invention may be useful for treating or preventing neurodegenerative diseases, such as Alzheimer's and Huntington's disease that are the result of astrocyte reactivity. The examples demonstrate that compositions of the invention can decrease the activation of the NF-κB promoter which activates cytokine production, for example the cytokines IL-1β, IL-1 ^, IL-18, TNF ^ and IL-6. Activation of the NF-κB promoter is mediated through the TLR4 ligand. TLR4 has been found to be upregulated in brains of Parkinson’s disease patients and is known to mediate cell death in the mouse model MPTP, which simulates Parkinson’s disease [37]. Therefore, compositions of the invention may be effective at treating Parkinson’s disease by inhibiting the ability of TLR4 signalling to activate the NF-κB promoter. Oxidative damage is implicated in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease and stroke (brain ischemia/reperfusion injury) [38]. The examples show that the bacterial strains of the invention have anti-oxidant activity, including the ability to protect inflammatory neuroblastoma cells from reactive oxygen species (ROS). Therefore, in certain embodiments the compositions of the invention may be particularly effective at treating neurodegenerative diseases by reducing the oxidative damage by free radicals. The compositions of the invention can therefore be effective for treating neurodegenerative diseases with a pathogenesis of oxidative damage, including Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease and stroke (brain ischemia/reperfusion injury).
Tau associated neurodegenerative diseases Tauopathies are neurodegenerative diseases associated with the pathological aggregation of tau protein in neurofibrillary or gliofibrillary tangles in the human brain. Alzheimer’s disease is an example of a tauopathology. Synucleinopathies (also called α-Synucleinopathies) are neurodegenerative diseases characterised by the abnormal accumulation of aggregates of α-synuclein in neurons, nerve fibres or glial cells. Parkinson’s disease is an example of a synucleinopathology. There is clinical and pathological overlap between these two pathologies. Parkinson's disease patients frequently have dementia and Alzheimer's disease patients often manifest parkinsonism [39]. For example, progressive supranuclear palsy (also known as Steele-Richardson-Olszewski syndrome) has a tauopathology, but also leads to prominent parkinsonism [40]. Mutations in LRRK2 known to cause parkinsonism are associated with the accumulation of synuclein, tau, neither, or both proteins [41]. Lewy body disease (LBD) is a neurodegenerative disease that is one of the most common causes of dementia in the elderly. LBD exemplifies the existence of a continuum between tau- and synuclein- pathologies. LBD shares clinical and pathological features with Parkinson disease, Parkinson disease dementia and Alzheimer disease [39]. The compositions of the invention may be useful for treating or preventing tauopathies and/or synucleinopathies. In particular, the compositions of the invention may be useful for treating or preventing tauopathies. The compositions of the invention may be useful for treating or preventing Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson- Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism. In preferred embodiments, the compositions of the invention may be useful for treating or preventing Alzheimer’s disease, including Benson's syndrome. In further preferred embodiments, the compositions of the invention may be useful for treating or preventing dementia; including Lewy body; vascular and frontotemporal dementia. Parkinson’s disease Parkinson’s disease is a common neurodegenerative disease neuropathologically characterised by degeneration of heterogeneous populations of neural cells (dopamine-producing cells). The clinical diagnosis of Parkinson’s disease requires bradykinesia and at least one of the following core symptoms: resting tremor; muscle rigidity and postural reflex impairment. Other signs and symptoms that may be present or develop during the progression of the disease are autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature. Parkinson’s disease is a neurodegenerative disease that may develop or persist due to dysfunction of
the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing Parkinson’s disease in a subject. The invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing Parkinson’s disease. Compositions comprising a bacterial strain of the genus Bacteroides may improve motor and cognitive functions in models of Parkinson’s disease. Treatment with Bacteroides strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of Parkinson’s disease. The composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens. Compositions using Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating Parkinson’s disease and are thus preferred. The compositions of the invention may prevent, reduce or alleviate one or more of the symptoms of Parkinson’s disease in a subject. For example, the compositions of the invention may prevent, reduce or alleviate one or more core symptoms, such as bradykinesia, resting tremor; muscle rigidity and/or postural reflex impairment in a subject. In addition or alternatively, the compositions of the invention may prevent, reduce or alleviate one or more symptoms associated with Parkinson’s disease progression selected from autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature. The compositions of the invention may prevent, reduce or alleviate depressive symptoms comorbid with Parkinson’s disease. For example, the compositions of the invention may improve verbal memory, executive functions, attention, working memory, verbal fluency and/or anxiety. In addition, compositions of the invention may prevent, reduce or alleviate cognitive dysfunctions comorbid with Parkinson’s disease. The compositions of the invention may prevent, reduce or alleviate Parkinson’s disease progression. For example, the compositions of the invention may prevent, reduce or alleviate late motor complications, late motor fluctuations and/or neuronal loss. The compositions of the invention may improve symptoms of Parkinson’s disease dementia (PDD). Symptoms of PDD include changes in memory, concentration and judgment, trouble interpreting visual information, muffled speech, visual
hallucinations, delusions, especially paranoid ideas, depression, irritability, anxiety and sleep disturbances, such as excessive daytime drowsiness and rapid eye movement (REM) sleep disorder. In some embodiments, the compositions of the invention improve the symptoms associated the PDD according to a symptomatic or diagnostic test and/or scale. In certain embodiments, the test or scale is selected from the Hopkins Verbal Learning Test – Revised (HVLT-R); the Delis-Kaplan Executive Function System (D-KEFS) Color-Word Interference Test; the Hamilton Depression Rating Scale (HAM-D 17; depression); the Hamilton Anxiety Rating Scale (HAM-A; anxiety) and the Unified Parkinson’s Disease Rating Scale (UPDRS; PD symptom severity). The compositions of the invention may prevent, reduce or alleviate impairment of executive function, attention and/or working memory. The compositions of the invention may improve dopaminergic neurotransmission, or prevent, reduce or alleviate impaired dopaminergic neurotransmission. The compositions of the invention may improve the symptoms of Parkinson’s disease. This can be assessed according to a symptomatic or diagnostic scale. For example, the test for assessing symptomatic improvement of motor function in Parkinson’s disease may be the Unified Parkinson’s Disease Rating Scale. In particular, UPDRS II considers the activity of daily life and UPDRS III considers motor-examination. The compositions of the invention may improve the Clinical Global Impression – Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. The compositions of the invention may display a positive effect on global social and occupational impairment of the subject with Parkinson’s disease. Alzheimer’s disease and dementia In DSM-5, the term dementia was replaced with the terms major neurocognitive disorder and mild neurocognitive disorder. Neurocognitive disorder is a heterogeneous class of psychiatric diseases. The most common neurocognitive disorder is Alzheimer’s disease, followed by vascular dementias or mixed forms of the two. Other forms of neurodegenerative disease (e.g. Lewy body disease, frontotemporal dementia, Parkinson’s dementia, Creutzfeldt-Jakob disease, Huntington’s disease, and Wernicke-Korsakoff syndrome) are accompanied by dementia. Alzheimer’s disease and dementia are also characterised by neuronal loss, so the neuroprotective effects shown in the examples for the compositions of the invention indicate that they may be useful for treating or preventing these conditions. In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing Alzheimer’s disease. The composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron,
Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens. Compositions using Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating Alzheimer’s disease, and thus bacterial strains from these species are preferred. In some embodiments, the composition for use in preventing or treating Alzheimer’s disease does not comprise Bacteroides fragilis. In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing dementia. The composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens. Compositions using Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating dementia, and thus bacterial strains from these species are preferred. The symptomatic criteria for dementia under DSM-5 are evidence of significant cognitive decline from a previous level of performance in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. The cognitive deficits must interfere with independence in everyday activities. In addition, the cognitive deficits do not occur exclusively in the context of a delirium and are not better explained by another mental disorder (for example MDD or schizophrenia). In addition to the primary symptom, subjects with neurodegenerative disease display behavioural and psychiatric symptoms including agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances. Neurodegenerative diseases may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing neurodegenerative diseases in a subject. In preferred embodiments, the neurodegenerative disease is Alzheimer’s disease. In other embodiments, the neurodegenerative disease is selected from vascular dementias; mixed form Alzheimer’s disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson’s dementia; Creutzfeldt-Jakob disease; Huntington’s disease; and Wernicke-Korsakoff syndrome.
The compositions of the invention may prevent, reduce or alleviate one or more of the symptoms of neurodegenerative diseases in a subject. For example, the compositions of the invention may prevent, reduce or alleviate the occurrence of cognitive decline in a subject and/or improve the level of performance of a subject with neurodegenerative disease or disorder in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual- motor and social cognition. The compositions of the invention may prevent, reduce or alleviate the occurrence of one or more behavioural and psychiatric symptoms associated with neurodegenerative disorders selected from agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances. The compositions of the invention may prevent, reduce or alleviate symptomatic disease by intervention in the suspected pathogenic mechanisms at a preclinical stage. The compositions of the invention may improve disease modification, by slowing or arrest the symptom progression. For example, the slowing or arresting of symptom progression correlates with evidence in delaying the underlying neuropathological process. The compositions of the invention may improve symptoms of neurodegenerative disorders comprising enhanced cognitive and functional improvement, for example, the compositions of the invention may improve the behavioural and psychiatric symptoms of dementia (BPSD). The compositions of the invention may improve the ability of a subject with neurodegenerative disease to undertake everyday activities. The compositions of the invention may improve both cognition and functioning in a subject with Alzheimer’s disease. For example, the composition of the invention improve the cognitive endpoint in a subject with Alzheimer’s disease and/or the functional endpoint in a subject with Alzheimer’s disease. The compositions of the invention may improve the overall clinical response (the global endpoint) in a subject with Alzheimer’s disease. The compositions of the invention may improve the symptoms of neurodegenerative disorders according to a symptomatic or diagnostic test. For example, the tests for assessing symptomatic improvement of Alzheimer’s disease (and other neurodegenerative disorders) are selected from objective cognitive, activities of daily living, global assessment of change, health related quality of life tests and tests assessing behavioural and psychiatric symptoms of neurodegenerative disorders. The objective cognitive tests for assessment of symptomatic improvement use the Alzheimer’s disease may be the Assessment Scale cognitive subscale (ADAS-cog) and the classic ADAS scale. Symptomatic improvement of cognition is assessed using the neurophysiological Test Battery for Use in Alzheimer’s Disease (NTB). The global assessment of change test uses the Clinical Global Impression – Global Improvement (CGI- I) scale for assessing psychiatric and neurological disorders. The global scale can be the Clinician's Interview Based Impression of Change plus (CIBIC-plus) or the Alzheimer’s Disease Cooperative Study Unit Clinician’s Global Impression of Change (ADCS-CGIC). For example, the health related
quality of life measures are the Alzheimer’s Disease-Related QOL (ADRQL) and the QOL- Alzheimer’s Disease (QOL-AD). The tests assessing behavioural and psychiatric symptoms of neurodegenerative disorders can be selected from the Behavioural pathology in Alzheimer’s Disease Rating Scale (BEHAVE-AD); the Behavioural Rating Scale for Dementia (BRSD); the neuropsychiatric Inventory (NPI); and the Cohen- Mansfield Agitation Inventory (CMAI). The compositions of the invention may be particularly effective at preventing, reducing or alleviating neurodegenerative diseases and disorders when used in combination with another therapy for treating neurodegenerative disorders. Such therapies include acetylcholinesterase inhibitors including donepezil (Aricept™), galantamine (Razadyne™) and rivastigmine (Exelon™), and memantine. Multiple Sclerosis Multiple sclerosis (MS) is a demyelinating disease in which the myelin sheath surrounding neurons in the brain and spinal cord are damaged. The exact underlying causes of MS are unknown, but are thought to vary between individuals. Certain forms of MS are hereditary. Environmental factors are also thought to contribute to MS. In some individuals, a combination of both genetic and environmental factors may trigger the onset of MS. There are a wide variety of symptoms associated with MS. Subjects may exhibit almost any neurological symptom associated with the impairment of autonomic, visual, motor or sensory control. The exact symptoms will vary depending on the site of neuronal damage/demyelination. The compositions of the invention may prevent, reduce or alleviate one or more symptoms of MS in a subject. For example, the compositions of the invention may prevent, reduce or alleviate fatigue, resting tremor, muscle weakness, muscle spasms, muscle stiffness, paraesthesia and/or ataxia in a subject. In addition, the compositions of the invention may prevent, reduce or alleviate one or more symptoms associated with MS progression selected from the list consisting of autonomic disturbances, constipation, micturition disturbances, sexual functioning, dysphagia, dysarthria, syncope, vertigo and/or dizziness, sleep disturbances, and disturbances in the sense of smell or sense of temperature. The compositions of the invention may prevent, reduce or alleviate one or more ocular symptoms associated with MS. The ocular symptom can be selected from the list consisting of loss of vision, eye pain, colour blindness, double vision and/or involuntary eye movements in a subject. The invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing MS. The composition may comprise a bacterial strain of the species Bacteroides caccae, Bacteroides coprocola, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides koreensis, Bacteroides kribbi or Bacteroides xylanisolvens. Compositions using
Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae may be particularly effective for treating MS and thus bacterial strains from these species are preferred. In some embodiments, the composition for use in a method of preventing or treating MS does not comprise bacteria strains of the genus Bacteroides which have a 16s rRNA sequence that is at least 98%, 99%, 99.5%, 99.9% or 100% identical to SEQ ID NO:4. The compositions of the invention may prevent, reduce or alleviate dizziness, vertigo, neuropathic pain, musculoskeletal pain, cognitive dysfunction, bowel incontinence, dysphagia, dysarthria, or any combination thereof. The compositions of the invention may prevent, reduce or alleviate depressive symptoms or anxiety in a patient diagnosed with MS. The improvement of symptoms can be determined using the 2017 McDonald criteria for diagnosing MS. Treatment with the compositions of the invention may result in a reduction in MS incidence or MS severity. The compositions of the invention may be for use in reducing relapse incidence or relapse severity. Treatment with the compositions of the invention may prevent a decline in motor function or results in improved motor function associated with MS. The compositions of the invention are therefore for use in preventing a decline in motor function or for use in improving motor function in the treatment of MS. Treatment with the compositions of the invention may prevent the development of paralysis in MS. Therefore, the compositions of the invention are for use in preventing paralysis in the treatment of MS. In certain embodiments the compositions of the invention are for use in preventing multiple sclerosis in a patient that has been identified as at risk of multiple sclerosis, or that has been diagnosed with early-stage multiple sclerosis or “relapsing-remitting” multiple sclerosis. The compositions of the invention may be useful for preventing the development of MS. The compositions of the invention may be useful for preventing the progression of MS. In certain embodiments, the compositions of the invention are for use in a patient identified as having a genetic predisposition to MS, such as major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2 or HLA- DR4. The compositions of the invention may be useful for managing or alleviating MS. The compositions of the invention may be particularly useful for reducing symptoms associated with MS. Treatment or prevention of MS may refer to, for example, an alleviation of the severity of symptoms or a reduction 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 slow or stop progression of the disease. In certain embodiments, the compositions of the invention are for use in treating relapsing-remitting MS. In alternative embodiments, the compositions of the invention are for use in treating progressive MS, such as secondary progressive MS (SPMS), which develops over time following diagnosis of
RRMS, primary progressive MS (PPMS) which exhibits gradual continuous neurologic deterioration and progressive relapsing MS (PRMS), which is similar to PPMS but with overlapping relapses. In certain embodiments, the compositions of the invention are for use in treating one or more symptoms of MS selected from the group consisting of: fatigue, vision problems, numbness, tingling, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain, problems with thinking, learning and planning, depression and anxiety, sexual problems, bladder problems, bowel problems, speech and swallowing difficulties. Modulation of the microbiota-gut-brain axis Communication between the gut and the brain (the gut-brain axis) occurs via a bidirectional neurohumoral communication system. Recent evidence shows that the microbiota that resides in the gut can modulate brain development and produce behavioural phenotypes via the microbiota-gut-brain axis. Indeed, a number of reviews suggest a role of the microbiota-gut-brain axis in maintaining central nervous system functionality and implicate dysfunction of the microbiota-gut-brain axis in the development of central nervous system disorders and conditions [42]. The bidirectional communication between the brain and the gut (i.e. the-gut-brain axis) includes the central nervous system, neuroendocrine and neuroimmune systems, including the hypothalamus- pituitary-adrenal (HPA) axis, sympathetic and parasympathetic arms of the autonomic nervous system (ANS), including the enteric nervous system (ENS) and the vagus nerve, and the gut microbiota. The compositions of the invention are useful for modulating the microbiota-gut-brain axis to reduce the cell death associated with central nervous system diseases or disorders, such as inflammatory central nervous system diseases or disorders. Accordingly, the compositions of the invention may be used for treating or preventing neurodegenerative disease sand disorders, in particular those associated with dysfunction of the microbiota-gut-brain axis. For example, it has been suggested that IL-6 signalling and alterations in the compositions of the microbiota play key roles in the pathogenesis of depression. The intravenous administration of an IL-6 receptor antibody (MR16-1) resulted in the induction of depression in a rat model, but intracerebroventricular injection of MR16-1 did not induce any anti-depressant effects. In addition, the intravenous administration of MR16-1 lead to a decrease in the number of Firmicutes and the Firmicutes/Bacteroidetes ratio. It was suggested that MR16-1 may show antidepressant-like effects by normalizing abnormalities in Firmicutes (or Firmicutes/Bacteroidetes) [43]. The examples demonstrate the ability of compositions comprising bacteria from the genus Bacteroides to decrease the secretion of IL-6 in human glioblastoma astrocytomas, but to increase IL-6 secretion in PMBCs. Therefore, the compositions of the invention may modulate the microbiota-gut-brain axis by increasing the levels of IL-6 in the periphery but not in the brain. Therefore, the compositions of
the invention may be useful for treating or preventing depression by modulating the microbiota-gut- brain-axis. In other embodiments, the compositions of the invention may be useful for treating or preventing a disease or condition selected from the group consisting of: Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia; including Lewy body; vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration. In other embodiments, the compositions of the invention may be useful for treating or preventing epilepsy. The compositions of the invention may be useful for treating or preventing chronic disease, treating or preventing disease in patients that have not responded to other therapies (such as treatment with Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, and/or anticholinergics), and/or treating or preventing the tissue damage and symptoms associated with dysfunction of the microbiota-gut-brain axis. The compositions of the invention may be useful for modulating the CNS. For example, the compositions of the invention may modulate the autonomic nervous system (ANS), enteric nervous system (ENS), the hypothalamic, pituitary, adrenal (HPA) axis, neuroendocrine, dopaminergic and/or neuroimmune pathway. The signalling of the microbiota-gut-brain-axis is modulated by neural systems. Accordingly, the compositions of the invention may modulate signalling in neural systems, in the central nervous system in sensory neurons, and/or in motor neurons. The compositions of the invention may modulate the signalling in the ANS, for example in the parasympathetic or sympathetic ANS nervous system. In addition, the compositions of the invention may modulate the signalling in the vagus nerve or ENS. The compositions of the invention may also module the levels of commensal metabolites and/or the gastrointestinal permeability of a subject. The signalling of ANS and ENS neurons may respond directly to luminal contents of the gastrointestinal tract or indirectly to neurochemicals produced by luminal bacteria. In addition, the signalling of ANS and ENS neurons may respond to neurochemicals produced by luminal bacteria or enteroendocrine cells. The neurons of the ENS may activate vagal afferents that influence the functions of the CNS. Also the compositions of the invention may regulate the activity of enterochromaffin cells.
Neurochemical factors, neuropeptides and neurotransmitters and the microbiota-gut-brain axis As outlined above, the microbiota-gut-brain axis is modulated by a number of different physiological systems. For example, the microbiota-gut-brain axis is modulated by a number of signalling molecules. Alterations in the levels of these signalling molecules results in central nervous system diseases and disorders, such as inflammatory central nervous system diseases and disorders. The signalling of the microbiota-gut-brain axis is modulated by levels of neurochemical factors, neuropeptides and neurotransmitters. Dysregulation of the productions of these metabolites can lead to central nervous system diseases and disorders, such as Parkinson’s disease. Accordingly, in certain embodiments, the compositions of the invention modulates levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain preferred embodiments, the compositions of the invention directly alter CNS biochemistry. - Indole The inventors have further demonstrated that bacterial strains from the genus Bacteroides can produce indole, which can attenuate inflammation and oxidative stress. Therefore, bacteria from the genus Bacteroides, and in particular bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae are useful in treating or preventing central nervous system diseases or disorders that are associated with inflammation and oxidative stress. In preferred embodiments, the bacterial strain is from the species Bacteroides dorei, Bacteroides koreensis, Bacteroides sp or Bacteroides ovatus, for example NCIMB 43598 (Ref 2), NCIMB 43601 (Ref 8), NCIMB 43599 (Ref 20) or NCIMB 43593 (Ref 27). Oxidative stress has been implicated in the development of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, epileptic seizures and stroke [44]. Both the production of reactive oxygen species and autophagy have been linked to the onset and development of neurological disorders. Due to their high antioxidant activity indole compounds have been suggested to act as neuroprotectants [45]. The experiments performed by the inventors demonstrate that bacteria from the genus Bacteroides can increase the level of indole. In certain embodiments, the compositions of the invention can modulate the level of indole. In preferred embodiments the compositions of the invention can increase the level of indole. - GABA The signalling of the microbiota-gut-brain axis is modulated by levels of γ-aminobutyric acid (GABA). Accordingly, the compositions of the invention may be able to modulate the levels of GABA. GABA is an inhibitory neurotransmitter that reduces neuronal excitability. The compositions of the invention
are effective at increasing the levels of GABA. The compositions of the invention may alter GABAergic neurotransmission. The compositions of the invention may modulate the level of GABA transcription in different regions of the central nervous system. The commensal derived GABA crosses the blood-brain barrier and affects neurotransmission directly. The compositions of the invention may lead to a reduction of GABA in the hippocampus, amygdala and/or locus coeruleus. The compositions of the invention may lead to an increase of GABA in cortical regions. The levels of neuroactive molecules, such as GABA, are linked to the pathophysiology of central nervous system diseases such as dementia, Alzheimer’s disease, Huntington’s disease and epilepsy. Thus, the compositions of the invention may be used for treating or preventing a disease mediated by GABA. In preferred embodiments, the compositions of the invention are for use in a method of treating epilepsy. Compositions which modulate the levels of GABA have been found to be useful in the treatment of epilepsy. As demonstrated in the examples, the claimed compositions can increase the production of GABA. Consequently, in some embodiments, the compositions of the invention are for use in the treatment of epilepsy. In some embodiments, the treatment reduces the frequency and/or the intensity of epileptic seizures in a subject. The frequency and/or intensity of epileptic seizures can vary greatly in different subjects. In some embodiments, the treatment comprises reducing the frequency and/or intensity of seizures by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%. Modes for measuring the reduction in frequency and/or intensity of epileptic seizures are known in the art. For example, the intensity of a seizure may be assessed using the Chalfont Seizure Severity Scale. In some subjects it may be appropriate to measure the reduction in frequency and/or intensity over the course of one day. In some subjects it may be appropriate to measure the reduction in frequency and/or intensity over either a day or a longer period, e.g. two days, a week, a month, six months, a year or longer. Thus, in some embodiments, the percentage reduction in the frequency of the seizures is measured over a period of one day, two days, three days, a week, two weeks, a month, three months, six months, a year, two years or longer than two years. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is awake. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is asleep. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is awake but not whilst the subject is asleep, or whilst the subject is asleep but not whilst the subject is awake. In some embodiments, the treatment comprises eliminating epileptic seizures. In some embodiments, the frequency of epileptic seizures is reduced to once a day or less, once every two days or less, once every week or less, once every two weeks or less, once a month or less, once every three months or less, once every six months or less, once a year or less, once every two years or less, or once every three years or less. In some
embodiments, the treatment results in the subject experiencing an epileptic seizure free period of at least a day, at least two days, at least a week, at least a month, at least three months, at least six months, at least a year, at least two years, or at least three years. In some embodiments, the compositions of the invention are particularly effective at treating epilepsy when used in combination with another therapy for treating epilepsy. In some embodiments, the other therapy for use in combination with the composition of the invention is selected from one or more of: Acetazolamide, Brivaracetam, Carbamazepine, Clobazam, Clonazepam, Eslicarbazepine acetate, Ethosuximide, Everolimus, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Oxcarbazepine, Perampanel, Phenobarbital, Phenytoin, Piracetam, Pregabalin, Primidone, Rufinamide, Sodium valproate, Stiripentol, Tiagabine, Topiramate, Valproic acid, Vigabatrin, and Zonisamide. In some embodiments, the treatment results in the subject being able to reduce the dose or stop taking one or more other anti-epileptic drugs without seeing a deterioration in symptoms. For example, in some embodiments, the dose of a GABA-mediator anti-epileptic drug may be reduced or the GABA- mediator anti-epileptic drug may no longer be taken. In some embodiments, the GABA-mediator anti- epileptic drug is selected from valproic acid, sodium valproate and pregabalin. - Immune response The signalling of the microbiota-gut-brain axis is modulated by alterations in the immune response and inflammatory factors and markers. The compositions of the invention may modulate the immune response. For example, the compositions of the invention may modulate the systemic levels of circulating neuroimmune signalling molecules, pro-inflammatory cytokine production and/or inflammation. The compositions of the invention may modulate the inflammatory state. The compositions of the invention may decrease IL-6 production and secretion and/or decrease the activation of the NFκB promoter. As shown in the examples the compositions of the invention are able to modulate the activation of IL-6 production by the potent pro-inflammatory endotoxin LPS. Therefore, the compositions of the invention are able to modulate the activation of the NFκB promoter by LPS and ^-synuclein mutant proteins such as A53T. Increased levels of circulating cytokines are closely associated with various central nervous system diseases and disorders, including Parkinson’s, dementia and Alzheimer’s. Thus, the compositions of the invention are for use in reducing IL-6 levels and/or NFκB levels in the treatment of a central nervous system disease and disorder, such as inflammatory central nervous system diseases and disorders. The signalling of the microbiota-gut-brain axis is modulated by levels of gastrointestinal permeability. The compositions of the invention may alter the integrity of the gastrointestinal tract epithelium, modulate the permeability of the gastrointestinal tract and/or modulate gastrointestinal tract motility. The compositions of the invention may modulate the barrier function and integrity of the gastrointestinal tract. The compositions of the invention may modulate the translocation of commensal
metabolites and inflammatory signalling molecules into the bloodstream from the gastrointestinal tract lumen. The signalling of the microbiota-gut-brain axis is modulated by the composition of the microbiome in the gastrointestinal tract. The compositions of the invention may modulate the microbiome composition of the gastrointestinal tract. The compositions of the invention may prevent microbiome dysbiosis and associated increases in toxic metabolites (e.g. LPS). The compositions of the invention may modulate the levels of Clostridium in the gastrointestinal tract. For example, the compositions of the invention may reduce the level of Clostridium in the gastrointestinal tract. The compositions of the invention may reduce the levels of Campylobacter jejuni. The compositions of the invention may modulate the proliferation of harmful anaerobic bacteria and the production of inflammatory neurotoxins produced by these bacteria. The compositions of the invention may modulate the microbiome levels of Lactobacillus and/or Bifidobacterium. In certain embodiments, the compositions of the invention modulate the microbiome levels of Sutterella, Prevotella, Ruminococcus genera and/or the Alcaligenaceae family. The compositions of the invention may increase the level of Lactobacillus plantarum and/or Saccharomyces boulardii. Brain injury The examples demonstrate that the compositions of the invention have antioxidant activity and have neuroprotective properties. Oxidative damage is implicated in the pathogenesis of central nervous system diseases and disorders such as stroke (brain ischemia/reperfusion injury) [38]. The compositions of the invention are for use in treating a brain injury. The brain injury can be for example a traumatic brain injury, an acquired brain injury, a brain injury resulting from trauma, a brain injury resulting from a tumour, a brain injury resulting from a stroke, a brain injury resulting from a brain haemorrhage, a brain injury resulting from encephalitis, a brain injury resulting from cerebral hypoxia or a brain injury resulting from cerebral anoxia. The compositions of the invention may be for use in treating stroke. The effects shown in the examples are particularly relevant to the treatment of stroke. Stroke occurs when blood flow to at least a part of the brain is interrupted. Without an adequate supply of blood to provide oxygen and nutrients to the brain tissue and to remove waste products from the brain tissue, brain cells rapidly begin to die. The symptoms of stroke are dependent on the region of the brain which is affected by the inadequate blood flow. Symptoms include paralysis, numbness or weakness of the muscles, loss of balance, dizziness, sudden severe headaches, speech impairment, loss of memory, loss of reasoning ability, sudden confusion, vision impairment, coma or even death. A stroke is also referred to as a brain attack or a cerebrovascular accident (CVA). The symptoms of stroke may be brief if adequate blood flow is restored within a short period of time. However, if inadequate blood flow continues for a significant period of time, the symptoms can be permanent.
In some embodiments, the stroke is cerebral ischemia. Cerebral ischemia results when there is insufficient blood flow to the tissues of the brain to meet metabolic demand. The cerebral ischemia may be focal cerebral ischemia, i.e. confined to a specific region of the brain or the cerebral ischemia may be global cerebral ischemia, i.e. encompassing a wide area of the brain tissue. Focal cerebral ischemia commonly occurs when a cerebral vessel has become blocked, either partially or completely, reducing the flow of blood to a specific region of the brain. In some embodiments the focal cerebral ischemia is ischemic stroke. In some embodiments, the ischemic stroke is thrombotic, i.e. caused by a thrombus or blood clot, which develops in a cerebral vessel and restricts or blocks blood flow. In some embodiments the ischemic stroke is a thrombotic stroke. In some embodiments, the ischemic stroke is embolic, i.e. caused by an embolus, or an unattached mass that travels through the bloodstream and restricts or blocks blood flow at a site distant from its point of origin. In some embodiments the ischemic stroke is an embolic stroke. Global cerebral ischemia commonly occurs when blood flow to the brain as a whole is blocked or reduced. The global cerebral ischemia can be caused by hypoperfusion, i.e. due to shock or is a result of a cardiac arrest. The subject diagnosed with brain injury may have suffered from cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered focal cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered an ischemic stroke. In some embodiments, the subject diagnosed with brain injury has suffered a thrombotic stroke. In some embodiments, the subject diagnosed with brain injury has suffered an embolic stroke. In some embodiments, the subject diagnosed with brain injury has suffered global cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered hypoperfusion. In some embodiments, the subject diagnosed with brain injury has suffered a cardiac arrest. In some embodiments, the compositions of the invention are for use in treating a brain injury caused by a cerebral ischemia. The compositions of the invention may be for use in treating a brain injury caused by a focal cerebral ischemia. The compositions of the invention may be for use in treating a brain injury caused by a ischemic stroke. The compositions of the invention may be for use in treating a brain injury caused by a thrombotic stroke. The compositions of the invention may be for use in treating a brain injury caused by a embolic stroke. The compositions of the invention may be for use in treating a brain injury caused by a global cerebral ischemia. The compositions of the invention may be for use in treating a brain injury caused by a hypoperfusion. The stroke can be a hemorrhagic stroke. Hemorrhagic stroke is caused by bleeding into or around the brain resulting in swelling, pressure and damage to the cells and tissues of the brain. Hemorrhagic stroke is commonly a result of a weakened blood vessel that ruptures and bleeds into the surrounding brain. In some embodiments, the hemorrhagic stroke is an intracerebral hemorrhage, i.e. caused by bleeding within the brain tissue itself. The intracerebral hemorrhage can be caused by an intraparenchymal haemorrhage or an intraventricular hemorrhage. The hemorrhagic stroke can be a subarachnoid hemorrhage i.e. bleeding that occurs outside of the brain tissue but still within the skull.
The hemorrhagic stroke may be a result of cerebral amyloid angiopathy. In some embodiments, the hemorrhagic stroke is a result of a brain aneurysm. In some embodiments, the hemorrhagic stroke is a result of cerebral arteriovenous malformation (AVM). In some embodiments the subject diagnosed with brain injury has suffered hemorrhagic stroke. For example, the subject diagnosed with brain injury may have suffered from an intracerebral haemorrhage or an intraparenchymal hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered an intraventricular hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered a subarachnoid hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with brain injury has suffered a brain aneurysm. In some embodiments, the subject diagnosed with brain injury has suffered cerebral AVM. In some embodiments, the compositions of the invention are for use in treating hemorrhagic stroke. In some embodiments, the compositions of the invention are for use in treating an intracerebral hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraparenchymal hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraventricular hemorrhage. In some embodiments, the compositions of the invention are for use in treating a subarachnoid hemorrhage. In some embodiments, the compositions of the invention are for use in treating a cerebral amyloid angiopathy. In some embodiments, the compositions of the invention are for use in treating a brain aneurysm. In some embodiments, the compositions of the invention are for use in treating cerebral AVM. Restoration of adequate blood flow to the brain after a period of interruption, though effective in alleviating the symptoms associated with stroke, can paradoxically result in further damage to the brain tissue. During the period of interruption, the affected tissue suffers from a lack of oxygen and nutrients, and the sudden restoration of blood flow can result in inflammation and oxidative damage through the induction of oxidative stress. This is known as reperfusion injury, and is well documented not only following stroke, but also following a heart attack or other tissue damage when blood supply returns to the tissue after a period of ischemia or lack of oxygen. In some embodiments the subject diagnosed with brain injury has suffered from reperfusion injury as a result of stroke. In some embodiments, the compositions of the invention are for use in treating reperfusion injury as a result of stroke. A transient ischemic attack (TIA), often referred to as a mini-stroke, is a recognised warning sign for a more serious stroke. Subjects who have suffered one or more TIAs are therefore at greater risk of stroke. In some embodiments the subject diagnosed with brain injury has suffered a TIA. In some embodiments, the compositions of the invention are for use in treating a TIA. In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has suffered a TIA.
High blood pressure, high blood cholesterol, a familial history of stroke, heart disease, diabetes, brain aneurysms, arteriovenous malformations, sickle cell disease, vasculitis, bleeding disorders, use of nonsteroidal anti-inflammatory drugs (NSAIDs), smoking tobacco, drinking large amounts of alcohol, illegal drug use, obesity, lack of physical activity and an unhealthy diet are all considered to be risk factors for stroke. In particular, lowering blood pressure has been conclusively shown to prevent both ischemic and hemorrhagic strokes [46, 47]. In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has at least one risk factor for stroke. In some embodiments the subject has two risk factors for stroke. In some embodiments the subject has three risk factors for stroke. In some embodiments the subject has four risk factors for stroke. In some embodiments the subject has more than four risk factors for stroke. In some embodiments the subject has high blood pressure. In some embodiments the subject has high blood cholesterol. In some embodiments the subject has a familial history of stroke. In some embodiments the subject has heart disease. In some embodiments the subject has diabetes. In some embodiments the subject has a brain aneurysm. In some embodiments the subject has arteriovenous malformations. In some embodiments the subject has vasculitis. In some embodiments the subject has sickle cell disease. In some embodiments the subject has a bleeding disorder. In some embodiments the subject has a history of use of nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments the subject smokes tobacco. In some embodiments the subject drinks large amounts of alcohol. In some embodiments the subject uses illegal drugs. In some embodiments the subject is obese. In some embodiments the subject is overweight. In some embodiments the subject has a lack of physical activity. In some embodiments the subject has an unhealthy diet. The examples indicate that the compositions of the invention may be useful for treating brain injury and aiding recovery when administered before the injury event occurs. Therefore, the compositions of the invention may be particularly useful for treating brain injury when administered to subjects at risk of brain injury, such as stroke. The compositions of the invention may be for use in reducing the potential damage caused by a potential brain injury, preferably a stroke. The compositions may reduce the damage caused when they are administered before the potential brain injury occurs, in particular when administered to a patient identified as at risk of a brain injury. In some embodiments, the compositions of the invention treat brain injury by reducing motoric damage. In some embodiments, the compositions of the invention treat brain injury by improving motor function. In some embodiments, the compositions of the invention treat brain injury by improving muscle strength. In some embodiments, the compositions of the invention treat brain injury by improving memory. In some embodiments, the compositions of the invention treat brain injury by improving social recognition. In some embodiments, the compositions of the invention treat brain injury by improving inflammatory neurological function.
Treatment of brain injury may refer to, for example, an alleviation of the severity of symptoms. Treatment of brain injury may also refer to reducing the inflammatory neurological impairments following stroke. Compositions of the invention for use in treating stroke may be provided to the subject in advance of the onset of stroke, for example in a patient identified as being at risk of stroke. Compositions of the invention for use in treating stroke may be provided after a stroke has occurred, for example, during recovery. Compositions of the invention for use in treating stroke may be provided during the acute phase of recovery (i.e. up to one week after stroke). Compositions of the invention for use in treating stroke may be provided during the subacute phase of recovery (i.e. from one week up to three months after stroke). Compositions of the invention for use in treating stroke may be provided during the chronic phase of recovery (from three months after stroke). In certain embodiments, the compositions of the invention are for use in combination with a secondary active agent. In certain embodiments, the compositions of the invention are for use in combination with aspirin or tissue plasminogen activator (tPA). Other secondary agents include other antiplatelets (such as clopidogrel), anticoagulants (such as heparins, warfarin, apixaban, dabigatran, edoxaban or rivaroxaban), antihypertensives (such as diuretics, ACE inhibitors, calcium channel blockers, beta- blockers or alpha-blockers) or statins. The compositions of the invention may improve the patient’s response to the secondary active agent. In certain embodiments, the compositions of the invention reduce the effect of ischemia on tissues. In certain embodiments, the compositions of the invention reduce the amount of damage to tissues caused by ischemia. In certain embodiments, the tissues damaged by ischemia are the cerebral tissues. In certain embodiments, the compositions of the invention reduce necrosis or the number of necrotic cells. In certain embodiments, the compositions of the invention reduce apoptosis or the number of apoptotic cells. In certain embodiments, the compositions of the invention reduce the number of necrotic and apoptotic cells. In certain embodiments, the compositions of the invention prevent cell death by necrosis and/or apoptosis. In certain embodiments, the compositions of the invention prevent cell death by necrosis and/or apoptosis caused by ischemia. In certain embodiments, the compositions of the invention improve the recovery of the tissue damaged by ischemia. In certain embodiments, the compositions of the invention improve the speed of clearance of necrotic cells and/or apoptotic cells. In certain embodiments, the compositions of the invention improve the efficacy of the clearance of necrotic cells and/or apoptotic cells. In certain embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues. In certain embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues damaged by ischemia. In certain embodiments, the compositions of the invention improve the overall histology of the tissue (for example upon a biopsy). 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 other words, the bacteria may have colonised some or all of the gastrointestinal tract and / or such colonisation may be transient or permanent. More specifically, in some embodiments, the “total colonisation of the intestine” means that bacteria have colonised all parts of the intestine (i.e. the small intestine, large intestine and rectum). Additionally or alternatively, the term “total colonisation” means that the bacteria engraft permanently in the some or all parts of the intestine. In some embodiments, “partial colonisation of the intestine” means that bacteria have colonised some but not all parts of the intestine. Additionally or alternatively, the term “partial colonisation” means that the bacteria engraft transiently in some or all parts of the intestine. The transience of engraftment can be determined by assessing (e.g. in a fecal sample) the abundance of the bacterial strain of the invention periodically (e.g. daily) following the end of a dosing interval to determine the washout period, i.e. the period between conclusion of the dosing interval and there being no detectable levels of the bacterial strain of the invention present. In embodiments of the invention, the washout period is 14 days or less, 12 days or less, 10 days or less, 7 days or less, 4 days or less, 3 days or less, 2 days or less or 1 day or less. In embodiments of the invention, the bacteria of the present invention engraft transiently in the large intestine. The compositions of the invention may be administered as a tablet, a foam, as a spray or a gel. 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. The compositions of the invention may be administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum 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. The compositions of the invention may be administered daily, weekly or monthly (either once or several times). 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.
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 some embodiments the compositions of the invention are administered for 16 days. Treatment according to the invention may be accompanied by 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. The composition of the invention may be administered to a pregnant animal, for example a mammal such as a human in order to prevent a central nervous system disease or disorder, such as a neurodegenerative disease, a neurodevelopmental disorder, a neuropsychiatric condition or a brain injury developing in her child in utero and / or after it is born. The compositions of the invention may be administered to a patient that has been diagnosed with a central nervous system disease or disorder, or that has been identified as being at risk of a central nervous system disease or disorder. The compositions may also be administered as a prophylactic measure to prevent the development of a central nervous system disease or disorder in a healthy patient. The compositions of the invention may be administered to a patient that has been diagnosed with a neurodegenerative disease, or that has been identified as being at risk of a neurodegenerative disease. The compositions may also be administered as a prophylactic measure to prevent the development of neurodegenerative disease in a healthy patient. The compositions disclosed herein may be administered to a patient that has been diagnosed with a central nervous system disorder or condition, in particular a central nervous system disorder or condition mediated by the microbiota-gut-brain axis, or that has been identified as being at risk of a central nervous system disorder or condition, in particular central nervous system disorder or condition mediated by the microbiota-gut-brain axis. The compositions may also be administered as a prophylactic measure to prevent the development of central nervous system disorders or conditions, in particular central nervous system disorders or conditions mediated by the microbiota-gut-brain axis 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 have reduced or absent colonisation by Bacteroides, and in particular Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae. The compositions of the invention may be administered as a food product, such as a nutritional supplement.
Generally, the compositions of the invention are for the prevention or treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used. 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 The compositions of the invention comprise bacteria. The inventors have identified the surprising ability of bacteria from the genus Bacteroides to treat central nervous system diseases or disorders, such as inflammatory central nervous system diseases or disorders. However, in order for bacteria from the genus Bacteroides to exert their beneficial effect they need to be effectively delivered alive and/or viable to the small intestine. In general, a composition of the invention therefore does not comprise inactivated bacteria of the species Bacteroides, in particular heat-inactivated bacteria of the species Bacteroides. The invention provides compositions which are formulated to prevent the bacteria from being degraded or absorbed in the upper digestive tract and being unable to exert their effect. For example, the compositions may comprises oxygen scavengers and/or prebiotic substrates, such as vitamin C and non-digestible carbohydrates. In addition, the composition can be enterically formulated to ensure that the bacteria are not degraded on route to the small intestine. Therefore, 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 degradation 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 [48] and [49]. 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 of the 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 [50-52]. The examples demonstrate that lyophilised compositions are particularly effective. 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. The bacterial strain in the composition of the invention may not have been inactivated, for example, may not have been heat-inactivated. The bacterial strain in the composition of the invention may not have been killed, for example, not been heat-killed. The bacterial strain in the composition of the invention may not have been attenuated, for example, not been heat-attenuated. For example, the bacterial strain in the composition of the invention may not have been killed, inactivated and/or attenuated. For example, the bacterial strain in the composition of the invention is live. For example, the bacterial strain in the composition of the invention is viable. For example, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine. The bacterial strain in the composition of the invention may be viable and capable of partially or totally colonising the intestine. The bacterial strain in the composition of the invention may be live and capable of partially or totally colonising the intestine. The bacterial strain in the composition of the invention may be live and viable. The bacterial strain in the composition of the invention may be live, viable and capable of partially or totally colonising the intestine. The composition can comprises a mixture of live bacterial strains and bacterial strains that have been killed. The invention provides compositions which are formulated to prevent the bacteria from being degraded or absorbed in the upper digestive tract and being unable to exert their effect. For example, the compositions may comprises oxygen scavengers and/or prebiotic substrates, such as vitamin C and non-digestible carbohydrates. In addition, the composition can be enterically formulated. This ensures that the bacteria are not degraded on the way to the small intestine. Encapsulation protects the composition from degradation 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 [53-54]. The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Bacteroides are anaerobes.
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 beneficial effect upon a patient. A therapeutically effective amount of a bacterial strain may be sufficient to result 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 1010 CFU; in another example from about 1 x 106 to about 1 x 1010 CFU; 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 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. A dose of the composition may comprise the bacterial strain from about 1 x 106to about 1 x 1011 colony forming units (CFU) /g, respect to the weight of the composition. The dose may be suitable for an adult human. For example, the composition may comprise the bacterial strain from about 1 x 103 to about 1 x 1011CFU/g; for example, from about 1 x 107 to about 1 x 1010 CFU/g; in another example from about 1 x 106 to about 1 x 1010 CFU/g; in another example from about 1 x 107 to about 1 x 1011 CFU/g; in another example from about 1 x 108 to about 1 x 1010 CFU/g; in another example from about 1 x 108 to about 1 x 1011 CFU/g, from about 1 x 108 to about 1 x 1010 CFU/g. The dose may be, for example, 1g, 3g, 5g, and 10g. The composition may be formulated as a probiotic. A probiotic is defined by the FAO/WHO as a live microorganism that, when administered in adequate amounts, confers a health benefit on the host. Typically, a probiotic, such as a composition of the invention, is optionally combined with at least one suitable prebiotic compound. In certain embodiments, the probiotic composition of the present invention may include 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. Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides. 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. Potential prebiotics can have a number of beneficial physiological properties including: resistance to digestion in the upper sections of the alimentary tract, can be fermented by the intestinal microbiota, have a beneficial effect on the host’s health, can selectively stimulate the growth of the probiotic and is stable in various food or feed processing conditions. The 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 resistant
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 molecule to which three glucose molecules are bonded. The prebiotic can stimulate the growth and activity of bacteria from the genus Bacteroides in the gastrointestinal tract. This allows the bacteria to exert their beneficial effect on the host of treating central nervous system diseases or disorders, such as inflammatory central nervous system diseases or disorders. Other prebiotic compounds (such as vitamin C, for example), may be included as oxygen scavengers and to improve the delivery and/or partial or total colonisation and survival in vivo. Alternatively, 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 compositions of the invention may be used in combination with another therapeutic compound for treating or preventing central nervous system diseases and disorders, such as inflammatory central nervous system diseases or disorders. The compositions of the invention may be administered with nutritional supplements that modulate neuroprotection or neuroproliferation. The nutritional supplements comprise or consist of nutritional vitamins. For example, the vitamins can be vitamin B6, magnesium, dimethylglycine (vitamin B16) and vitamin C. The compositions of the invention may be administered in combination with another probiotic. In certain embodiments, the compositions of the invention are for use in enhancing the effect of a second agent on a central nervous system disease and disorder, such as inflammatory central nervous system diseases or disorders. The immune modulatory effects of the compositions of the invention may make the brain more susceptible to conventional therapies such as Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, or anticholinergics, which are exemplary secondary agents to be administered in combination (sequentially or contemporaneously) with the compositions of the invention. The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [55]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [56]. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, 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 stearate, magnesium stearate, 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 and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. 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 supplement. 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. The compositions of the invention may comprise one or more bacterial strains of the genus Bacteroides and do not contain bacteria from any other genera, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another genera. Thus, the invention provides a composition comprising one or more bacterial strains of the genus Bacteroides, which does not contain bacteria from any other genera or which comprises only de minimis or biologically irrelevant amounts of bacteria from another genera, for use in therapy. The compositions of the invention may comprise one or more bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola and do not contain bacteria from any other species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another species. Thus, the invention provides a composition comprising one or more bacterial strains of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola, which does not contain bacteria from any other species or which comprises only de minimis or biologically irrelevant amounts of bacteria from another species, for use in therapy. In some embodiments, the composition does not comprise bacteria of the species Clostridium and/or does not comprise bacteria of the species Bacteroides fragilis. In some embodiments the composition does not comprise Bacteroides thetaiotaomicron. In some embodiments, the composition does not
comprise bacteria of the phylum Actinobacteria and/or does not comprise bacteria of the phylum Tenericutes, or comprises only de minimis or biologically irrelevant amounts of bacteria from the phylum Actinobacteria and/or the phylum Tenericutes for use in therapy. The compositions of the invention may contain a single bacterial species and do not contain any other bacterial species. In certain embodiments, the compositions of the invention contain a single bacterial strain and do not contain any other bacterial strains. For example, the compositions of the invention may comprise a bacterial strain only of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola. Such compositions may comprise only de 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 organism. In some embodiments, such compositions may be a lyophilisate that is substantially free from other species of organism. The invention also provides a composition comprising a single bacterial strain of the genus Bacteroides, which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy. The invention also provides a composition comprising a single bacterial strain of the species Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy. The compositions of the invention may comprise more than one bacterial strain. 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, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains from within the same species and, optionally, do not contain bacteria from any other species. The invention comprises any combination of the foregoing. The composition may comprise a microbial consortium. For example, the composition may comprises a Bacteroides strain as part of a microbial consortium. For example, the Bacteroides 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 the genus Bacteroides and/or other genera with which it can live symbiotically in vivo in the intestine. For example, the composition comprises a bacterial strain of the genus Bacteroides in combination with a bacterial strain from a different genus. In another example, the composition comprises a bacterial strain
of Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola in combination with a bacterial strain from the genus Bacteroides or the composition comprises a bacterial strain of Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron. Bacteroides koreensis, Bacteroides kribbi or Bacteroides coprocola in combination with a bacterial strain from the genus Bacteroides and 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. The microbial consortium may not found together in nature. For example, the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms. The microbial consortium can comprises bacteria from two different organisms which are from the same species, e.g. two different humans. For example, the two different organisms are an infant human and an adult human. The microbial consortium can comprises bacteria from two different organisms, which are not from the same species, such as a human and a non-human mammal. In some embodiments, the composition comprises the Bacteroides bacterial strain as part of a microbial consortium which contains fewer than 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 bacterial species. The bacterial species may be from the genus Bacteroides and/or from other genera with which it can live symbiotically in vivo in the intestine. Those skilled in the art will recognise that a bacterial population comprising 200 or fewer different species is significantly less complex and more controlled than material derived from a faecal sample which will contain many thousands of different species of bacteria. In preferred embodiments, the composition is not a faecal sample, such as a human faecal sample. In preferred embodiments, the composition may be formulated as part of a microbial consortium which contains fewer than 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, 20, 30, 40 or 50 bacterial strains. The bacterial strains may be from the genus Bacteroides and/or from other genera with which it can live symbiotically in vivo in the intestine. In some embodiments, the microbial consortium contains fewer than 30 bacterial strains. In some embodiments, the microbial consortium contains fewer than 20 bacterial strains. In some embodiments, the microbial consortium contains fewer than 10 bacterial strains. The composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43600, but which is not NCIMB 43600 or which is not a Bacteroides ovatus. The composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as NCIMB 43595, NCIMB 43599 or NCIMB 43601, but which is not NCIMB 43595, NCIMB 43599 or NCIMB 43601, or which is not a Bacteroides dorei.
The composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43597, but which is not NCIMB 43597, or which is not a Bacteroides stercoris. The composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43596, but which is not NCIMB 43596, or which is not a Bacteroides xylanisolvens. The composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43594, but which is not NCIMB 43594 which is not a Bacteroides koreensis. The composition of the invention may additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43593 or NCIMB 43598, but which is not NCIMB 43593 or NCIMB 43598, or which is not a Bacteroides sp. 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 genera may be for separate, simultaneous or sequential administration. For example, the composition may comprise all of the more than one bacterial strain, species or genera, or the bacterial strains, species or genera may be stored separately and be administered separately, simultaneously or sequentially. In some embodiments, the more than one bacterial strains, species or genera 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, all 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 have been cultured subsequent to being obtained from the human adult faeces and being used in a composition of the invention. In some embodiments, the bacterial strain for use in the invention is obtained from human infant faeces. In some embodiments in which the composition of the invention comprises more than one bacterial strain, all of the bacterial strains are obtained from human infant faeces or if other bacterial strains are present they are present only in de minimis amounts. The bacteria may have been cultured subsequent to being obtained from the human infant faeces and being used in a composition of the invention As mentioned above, in some embodiments, the one or more Bacteroides 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 a lyophilised pharmaceutical composition comprising the bacterial strains as discussed earlier. 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 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 of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat an inflammatory neurodegenerative disorder when administered to a subject in need thereof. In certain embodiments, the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent an inflammatory neurodegenerative disorder. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 × 103 to about 1 × 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, 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, magnesium stearate, mannitol and sorbitol. In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water. In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride. In certain embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer. In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and 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 atmosphere 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 present 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 gelatine capsule (“gel-cap”). The capsule can be a hard or a soft capsule. 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, have a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gelatine 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 have 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. In some embodiments, the compositions of the invention are administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract. 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), emulsions 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-resistant formulation (for example, resistant 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 degradation 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, gastro-resistant 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 [57 ]). In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel). Culturing methods The bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [58-60]. 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), 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 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg). Bacterial strains for use in vaccine compositions The inventors have identified that the bacterial strains of the invention are useful for treating or preventing inflammatory neurodegenerative disorders. This is likely to be a result of the effect that the bacterial strains of the invention have on the host immune system. Therefore, the compositions of the invention may also be useful for preventing inflammatory neurodegenerative disorders, when administered as vaccine compositions. In 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 present 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 [61] and [62-68], 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 definition of the invention. References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides 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. [69]. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 5 and a gap extension penalty of 2, and optionally a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [70]. 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). A “disease” results from a pathophysiological response to external or internal factors. A “disorder” is a disruption of the disease to the normal or regular functions in the body or a part of the body. A “condition” is an abnormal state of health that interferes with the usual activities or feeling of wellbeing. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Any reference to a method for treatment comprising administering an agent to a patient, also covers that agent for use in said method for treatment, as well as the use of the agent in said method for treatment, and the use of the agent in the manufacture of a medicament. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. MODES FOR CARRYING OUT THE INVENTION Example 1 – Efficacy of strains of the genus Bacteroides to decrease IL-6 secretion. Summary This study used human glioblastoma astrocytoma cells as a model to test the effect of bacterial strains from the genus Bacteroides on the levels of pro-inflammatory cytokines. Activation of proinflammatory cytokines has been associated with neuron damage in inflammatory neurodegenerative disease. Lipopolysaccharide (LPS) is a known stimulator of the proinflammatory cytokine IL-6. Human glioblastoma astrocytoma cells were pre-treated with LPS for one hour before bacterial supernatant was added to observe their ability to modulate the levels of IL-6. Material and Methods Bacterial strain
Cell line U373 is a human glioblastoma astrocytoma cell line. Cells (used between passage 20 and passage 37) were maintained in 25ml MEME supplemented with 10% heat inactivated FBS, 4mM L-Glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 5 µg/ml plasmocin, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate (referred to throughout as full growth media). Method In preparation for the different treatments, cells were plated in 24 well plates at a density of 100,000 cells/well in 1ml of full growth media. They were left to rest at 37°C/5% CO2 for 72 h. On the day of treatment media was removed from each well, cells were rinsed with 0.5 ml wash media (serum free MEME) and media discarded. 0.9ml stimulation media (MEME media containing 2% FBS), supplemented with 1 µg/ml for LPS treated wells, was added and incubated at 37°C and 5% CO2. After 1 h pre-incubation, the cells were removed from the CO2 incubator and treated with 100 µl bacterialsupernatant. YCFA+ media was used as control. The cells were then incubated for 24 h at 37°C and 5% CO2, after which cell-free supernatants were collected and spun down at 10,000g at 4°C for 3 min. Samples were aliquoted in 1.5 ml microtubes and stored at -80°C for hIL-6. IL-6 was measured using the Human IL-6 ABTS ELISA Development Kits (Peprotech (London, UK). Samples were analysed in accordance with the manufacturer’s protocol, absorbance at 405 nm with
wavelength correction set at 655 nm was recorded using the iMark microplate reader (Bio-Rad). Raw data were plotted and analysed using GraphPad Prism 7 software. Results Figure 1 shows that the majority of the Bacteroides strains tested decreased the secretion of IL-6 in U373 cells after stimulation with LPS. Only one strain, B. xylanisolvens NCIMB 43596 (Ref 1) was found to increase the secretion of IL-6 in U373 cells. 13 strains significantly down-regulated the production of IL-6 in U373 cells, including B. fragilis (Ref 12 and Ref 22) B. vulgatus (Ref 23 and Ref 24), B. eggherthii (Ref 11), B. stercoris NCIMB 43597 (Ref 10) and B. caccae (Ref 28). Different strains of B. fragilis, B. ovatus and B. dorei isolated from different donors (Table 1) showed the same significant down-regulation of IL-6 (Fig.1). These results show that Bacteroides strains according to the invention can decrease the LPS-induced secretion of IL-6 (LPS is a general inflammatory mediator). Chronic inflammation induced by IL-6 can ultimately lead to cell death. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prevention of central nervous system disorders or conditions. Example 2 – Secretion of IL-6 and IL-10 in PBMCs after treatment with different strains of Bacteroides Summary Importantly IL-6 can act as an anti-inflammation regulator which stimulates potent anti-inflammatory cytokines such as IL-10, and it is known that IL-6 and IL-10 share STAT-3 signalling pathway [15, 16]. The anti-inflammatory potential of B. ovatus, B. dorei, B. stercoris and B. xylanisolvens were investigated in peripheral blood mononuclear cells (PBMCs) from healthy human donors. Material and Methods Bacterial strain B. ovatus - NCIMB 43600 (Ref 7) B. koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp - NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1)
Method Frozen healthy human PBMCs were purchased from Stem cells. Briefly cells were thawed and left to rest overnight in full growth media (RPMI 1640 with 10% FBS, 2mM L. Glutamine and 100 U/ml penicillin, 100µg/ml streptomycin, 55µM mercaptoethanol) in a CO2 incubator at 37°C. For the experiment cells were plated at a density of 750,000 cells/well in 48 well plates and treated in full growth media with 10% bacterial supernatants in the presence or absence of 1 ng/ml LPS. Cell culture media was added to untreated wells. Cells were left to rest for 72 h, thereafter cell free supernatants were collected and spun down for 3 minutes at 10,000g at 4°C. Samples were stored at -80°C for IL-6 and IL-10 cytokines analysis using the enzyme-linked immunosorbent assay (ELISA). Results Figure 2 shows the level of IL-6 and IL-10 secretion of PBMCs after treatment with bacterial strains of the genus Bacteroides. All of the tested strains induced the per se production of IL-6 and none reduced secretion of the cytokine after treatment with LPS. Interestingly, all of the strains tested increased the secretion of IL-10 after treatment with LPS, with NCIMB 43595 (Ref 25) being the best strain. These data suggest that the ability of bacterial strains of the genus Bacteroides to reduce IL-6 secretion might be specific to neuroblastoma cells. Alternatively, neutrophil accumulation following LPS injection may antagonise the action of IL-1β and / or TNFα and thus this apparent discrepancy may be due to the cell type used. In any event, a general anti-inflammatory signature is evident in PBMCs from healthy donors. Figures 2A and B show the data for IL-6 and IL-10 combined all together for 10 donors. Example 3 – Effect of different strains of Bacteroides on TLR4 activation Summary Recent studies have highlighted that Bacteroides produce a type of LPS with tolerogenic properties [14]. Therefore HEK-Blue cells, stably expressing TLR4 and the inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the NF-kB-AP1 promoter, were used to investigate the ability of different strains of Bacteroides to activate NF-kB via TLR4 activation. Material and Methods Bacterial strain B. ovatus - NCIMB 43600 (Ref 7) B. koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp - NCIMB 43593 (Ref 27)
B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1) Method HEK293-Blue reporter cells stably expressing human TLR4 (HEK-TLR4) cells were cultured, according to the manufacturer’s instructions. Briefly HEK-TLR4 cells were maintained in DMEM 4.5g/L D-glucose supplemented with 10% (v/v) heat inactivated FBS, 4mM L-Glutamine, 100U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml normocin, 1x HEK-Blue selection media. For the experiment, cells were washed with PBS, dissociated in PBS and collected in growth media. Cells were plated in 96 well plates at a density of 25,000 cells/well. Cells were treated with 10 ng/ml LPS as a positive control, α-synuclein, or treated with 10% bacterial supernatant and incubated in a CO2 incubator for 22h. Thereafter detection of Secreted Embryonic Alkaline Phosphatase (SEAP) activity from cell culture supernatant was performed using QUANTI-blue solution according to manufacturer’s instructions. Briefly, 20 µl of cell culture media was collected and analysed for the presence of secreted alkaline phosphatase by mixing with 200µl of sterile-filtered QUANTI-Blue detection media. After 2h incubation at 37°C optical density was measured at 655nm (iMark microplate, Bio-Rad). Results LPS is a known activator of the NF-κB pathway which promote the proinflammatory cascade and regulates multiple aspects of the innate and adaptive immune system. The NF-κB pathway is known to be upregulated in inflammatory diseases. Figure 3 shows that all the strains tested induced NF-kB activation in a TLR4 dependent manner, with NCIMB 43597 (Ref 10) and NCIMB 43593 (Ref 27) doing so to a slightly lesser extent than the positive control. Interestingly, in the presence of an inflammatory stimulus (e.g. LPS or α-synuclein), organisms of the Bacteroides genus down-regulated TLR4 activation and therefore induced a reduction in NF-kB activation. Therefore, the ability of Bacteroides stercoris to reduce NF-κB activation after stimulation a- synuclein mutant makes it particularly useful in the treatment or prevention of inflammatory diseases. Example 4 – Anti-oxidant potential of Bacteroides strains Bacteria can produce and release antioxidants into the growth media that can interfere for example, with aging or neurodegenerative processes. The intrinsic capacity of Bacteroides strains to produce anti-oxidant molecules was investigated.
Material and Methods Bacterial strain tested B. ovatus - NCIMB 43600 (Ref 7) B. koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp - NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1) Bacteria culture conditions Bacterial indole production was quantified using an assay described previously [18]. Bacteria were cultured to stationary phase of growth. An overnight culture of three separate subbed colonies, streaked from -80°C culture collection stock, was used for the inoculation of three 40mL tubes of YCFA⁺ to yield three biological replicates. YCFA⁺ media was used a baseline control. The volume to be inoculated to each tube was first removed from the respective falcon tube, giving a final volume of 40 mL after inoculation. Confirmation that the bacteria had reached stationary phase was obtained by measurement of optical density.1mL of culture, or YCFA⁺ as a reference, was dispensed into a 1.5mL plastic cuvette and the OD recorded at 600nm. As part of the quality control process a sterile 5µL inoculation loop was used to take a loopful of culture to streak a purity plate. Culture and YCFA⁺ media were streaked onto pre-equilibrated YCFA⁺ agar plates and incubated for 24h inside the anaerobic hood at 37°C. QC checks were performed using the MALDI ToF. MALDI ToF Biomaterial of grown colonies of each sample-was put on MALDI Biotarget 96 (6 colonies in duplicate) with autoclaved wooden toothpicks. Each spot was overlaid with 1 µL of HCCA (10 mg/mL α-cyano-4-hydroxybenzoic acid (HCCA) in 50% acetonitrile, 47.5% water and 2.5% trifluoroacetic acid (TFA)) matrix. Samples were measured on Bruker Microflex® MALDI-ToF-Mass Spectrometer using the method “MBT_AutoX”. Laser was set at a shot rate of 60 Hz with the measuring raster (spiral_small). Spectra accumulated in MS/Parent Mode (240 shots). Peaks were evaluated with the processing method ‘MBT_Process’ flexControl version: 3.4, Bruker Daltonics (Bremen, Germany) peak detection algorithm ‘centroid’ (signal to noise threshold of 2, a minimum intensity threshold of 600 a.u. and a maximum number of peaks of 300, a peak width of 4 m/z and height of 90%, baseline subtraction with the method ‘TopHat’ for a peak resolution >400). All spectra were compared with reference spectra of the BDAL database (Version 7.0.0.0).
Collection of bacterial supernatants Supernatants of the bacterial cultures were tested in the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free- radical assay and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay. Supernatants were collected by centrifuging at 5,000xg for 10 minutes at room temperature. An additional centrifugation step was added at 5,000xg for 25 minutes at room temperature for NCIMB 43597 (Ref 10) to facilitate filtering of the supernatant. Filtered supernatant was dispensed into sterile 1.5mL microfuge tubes in 1mL volumes and stored at -80°C immediately. Example 4A – Indole biosynthesis in Bacteroides strains Method - Indole assay Indole standards were prepared in YCFA+ media and included 0.2mM, 0.4mM, 0.8mM, and 1.6mM. A positive chemical control was used in this assay, 0.5mM Indole in YCFA+ media. The Indole assay was performed using 24-well (non-treated) assay plates. 100mM tryptophan solution in HCL was dispensed into each well to give a final concentration of 6mM in each well. 1mL stationary phase bacterial culture was added to each well and incubated for a further 48 hours. Assay plates were centrifuged at 3,500xg at room temperature for 10 minutes. The supernatant was retained and the pellet discarded. In a 96-well plate 140µL supernatant was dispensed in triplicate. 140µL Kovac’s reagent was added and the absorbance read at 540nm immediately using a BioRad iMark microplate absorbance reader. The standard curve was prepared by plotting absorbance as a function of final Indole concentration (mM). Indole concentration of the test sample was calculated using the equation extrapolated from linear regression of the standard curve. Results Incubation of the nine Bacteroides strains with 6mM tryptophan demonstrated quantifiable indole production in all the strains tested (Figure 4A). Between the 9 different bacterial strains tested, variation in the concentration of indole produced was observed. All Bacteroides dorei strains tested were moderate or high producers of indole. The four Bacteroides ovatus strains produced comparable levels of indole. The Bacteroides stercoris NCIMB 43597 (Ref 10) produced a quantity of indole comparable to other Bacteroides strains. Example 4B – Radical scavenging activity of Bacteroides strains against DPPH radicals Method- 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free-radical assay Cell-free supernatants were thawed at 4°C for approximately 2 hours prior to use. All samples were diluted 1 in 2 in 1.5mL microfuge tubes using sterile 5mM PBS pH7 yielding a final volume of 1mL. The principle of the DPPH assay is based on the reduction of DPPH, a stable free-radical, by an antioxidant molecule according to the following reaction DPPH* + AH → DPPHH + A*
Where DPPH* is the stable form of the free radical, AH is the donor molecule, DPPHH is the reduced form of DPPH and A* is the free radical obtained in the reaction. Over the course of the reaction, the methanolic solution of DPPH turns from a deep violet to light yellow in colour. (±)-6-Hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid (synonym of which is Trolox) was included as a standard. A stock solution of 500µM Trolox in 5mM PBS, pH7 was prepared to make the standard curve. The 500µm Trolox stock solution was diluted in 5mM PBS, pH7 to prepare 0µM, 50µM, 100µM, 200µM and 400µM standards. A positive chemical control was included in the assay, U83836E (a lazaroid antioxidant), at a concentration of 200µM in 100% methanol. The DPPH assay was performed in a 96- well plate as described previously [19] with minor modifications made. In brief, 10µL sample/standard/control was added, in triplicate, to corresponding wells of a 96-well plate. 200µL 200µmol/L DPPH was added to three empty wells as a control. 190µL 200µmol/L DPPH was added to sample/standard/control wells and plates incubated in the dark for 30 minutes at room temperature. Absorbance was read at 515nm using a BioRad iMark microplate absorbance reader. DPPH radical scavenging activity was calculated as follows: DPPH radical scavenging activity (%) = [1-(Asample - Ablank)/Acontrol)]*Dilution factor*100 Where Asample was the average absorbance of sample + 200µmol/L DPPH, Acontrol was the average absorbance of methanol DPPH without sample, and Ablank is the average absorbance of YCFA media blank. Results All of the Bacteroides strains tested demonstrated radical scavenging activity (Figure 4B). Bacteroides stercoris NCIMB 43597 (Ref 10) was the only strain that had radical scavenging activity comparable to the Lazaroid antioxidant U83836E, a Vitamin E derivative, (200µM). The three Bacteroides dorei strains demonstrated varying degrees of radical scavenging activity, with NCIMB 43599 (Ref 20) and NCIMB 43601 (Ref 8) showing a comparable scavenging activity, second only to NCIMB 43597 (Ref 10) and significantly higher than NCIMB 43595 (Ref 25) (p<0.0001). The four Bacteroides ovatus strains demonstrated comparable radical scavenging activity. Example 4C - Antioxidant capacity of Bacteroides strains Method - 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay The total antioxidant capacity assay was performed using the Antioxidant Assay Kit (Sigma Aldrich, CS0790). The principle of the antioxidant assay is the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, which oxidizes the ABTS to produce a radical cation, ABTS.+, a soluble chromogen that is green in colour and can be spectrophotometrically at 405nm.
Solutions and standards were prepared according to the instructions supplied with the assay kit. The method was performed according to the manufacturer’s instructions. In brief, a set of Trolox standards was prepared from a 1.5mM Trolox working solution. For the purposes of the standard curve 0mM, 0.015mM, 0.045mM, 0.105mM, 0.21mM and 0.42mM solutions were prepared in 1x assay buffer. A bacterial strain which demonstrated consistent efficacy (Ref A) was included in the assay as a reference/positive control. All samples were diluted 1 in 4 in 1 x assay buffer. In a 96-well plate, 10µL standard/control/sample was added in triplicate. 20µL myoglobin working solution was added to all standard/control/sample wells. 150µL ABTS substrate solution was added to each well and the absorbance measured at 405nm using a BioRad iMark microplate absorbance reader. The standard curve was prepared by plotting absorbance as a function of final Trolox concentration (mM). Antioxidant concentration of the test sample was calculated using the equation extrapolated from linear regression of the standard curve.
Where X(mM) is antioxidant concentration (mM) relative to the concentration of trolox standard, y(A₄₀₅) is the average absorbance of the test sample at 405nm, intercept is the Y axis by the standard curve, slope is the slope of the standard curve ( a negative value), and dilution factor is the fold dilution of the original sample. Results Determination of a general antioxidant capacity of the bacterial supernatant of nine Bacteroides strains was conducted using the ABTS method. Of the Bacteroides strains tested most of the strains had significantly higher antioxidant capacity in comparison to the YCFA+ media control (Figure 4C). These results from the ABTS assay mirror the data from the DPPH radical scavenging assay discussed in Example 4B. The Bacteroides stercoris NCIMB 43597 (Ref 10) demonstrated the highest antioxidant capacity of the strains tested (Fig.4C). NCIMB 43601 (Ref 8) was again the second-best strain with a comparable degree of antioxidant capacity with NCIMB 43597 (Ref 10) and not significantly different from the other B. dorei strains (Fig.4C).
Example 5 - Protection of differentiated neuroblastoma cells from ROS by Bacteroides strains Methods Bacterial strains B. ovatus - NCIMB 43600 (Ref 7) B. koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp - NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1) SHSY-5Y cell differentiation and ROS measurement SHSY-5Y is a neuroblastoma cell line. Differentiated SHSY-5Y cells recapitulate in vitro most of the features of neurons. The cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2 mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin. Cells in growth medium were plated in black flat bottom 96 well plate at density of 5,000 cells/well and placed in the CO2 incubator. After 24h, media were replaced with differentiation medium (growth medium containing 1% FBS) and 10 µM retinoic acid RA). On Day 10, the differentiation medium was removed, cells were washed with pre-warmed PBS and stained with 10 µM DCFDA molecular probe for 20 min in growth medium containing 1% FBS. Then cells were washed with pre-warmed PBS again and treated with 100 µM TBHP in the presence or absence of 10% bacteria supernatant for 2h. Fluorescence intensity was measured using TECAN plate reader at Ex/Em 485/530 nm. Results The anti-oxidant potential of nine strains of Bacteroides was investigated in vitro on neuroblastoma cells. Oxidative stress was induced with tert-butyl hydrogen peroxide (TBHP) in the SHSY-5Y neuroblastoma cell line. NCIMB 43597 (Ref 10) was able to significantly reduce ROS production (p<0.0001, Figure 5). With the exception of NCIMB 43600 (Ref 7), a reduction was observed for all the other strains, in particular for NCIMB 43599 (Ref 20) and NCIMB 43598 (Ref 2). These results, in combination with the results from Example 4 demonstrate the antioxidant activity of strains of Bacteroides. Compositions comprising bacterial strains of the genus Bacteroides are therefore particularly effective at reducing oxidative stress in the neuronal cells. Therefore, compositions comprising bacterial strains of the genus Bacteroides are particularly effective at treating neurodegenerative diseases.
Example 6 – Efficacy of strains of the genus Bacteroides on gut permeability Summary The effect of bacterial strains from the genus Bacteroides on gut permeability was investigated. Abrogation of the epithelial barrier function allows harmful substances to leave the gut and is linked to the induction of inflammatory and autoimmune diseases. Material and Methods Bacterial strain Bacteroides salyersae - Ref 15 Bacteroides xylanisolvens - NCIMB 43596 (Ref 1) Bacteroides fragilis - Ref 22 Bacteroides intestinalis - Ref 33 Bacteroides stercoris - NCIMB 43597 (Ref 10) Bacteroides cellulosilyticus - Ref 30 Bacteroides uniformis - Ref 32 Bacteroides vulgatus - Ref 24 Bacteroides dorei - NCIMB 43599 (Ref 20) Bacteroides ovatus – Ref 31 Bacteroides sp – NCIMB 43598 (Ref 2) Animals BALBc (Envigo, UK) adult male mice were group-housed under a 12 h light-dark cycle; standard rodent chow and water were available ad libitum. All experiments were performed in accordance with European guidelines following approval by University College Cork Animal Ethics Experimentation Committee. Animals were 8 weeks old at the start of the experiment. Study Design Animals were allowed to habituate to their holding room for one week after arrival into the animal unit. They receive oral gavage (200μL dose) of live biotherapeutics at a dose of 1 X 109 CFU for 6 consecutive days between 15:00 and 17:00. On day 7, the animals are decapitated and tissues are harvested for experimentation. Tissue Collection Tissue collection Animals were sacrificed in a random fashion regarding treatment and testing condition; sampling occurred between 9.00 a.m. and 2:30 p.m. Trunk blood was collected in potassium EDTA (Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at −80 °C for further analysis. Intestinal tissue (2 cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm of tissue from the caecum were used) were mounted into the Ussing chambers for intestinal permeability assay. A further 1cm of ileum and colon tissue was taken for tight
junction gene expression analysis. The caecum was removed, weighed and stored at −80 °C for SCFAs analysis. Intestinal Permeability Mice were euthanized by cervical dislocation, and the distal ileum and colon were removed, placed in chilled Krebs solution opened along the mesenteric line and carefully rinsed. Preparations were then placed in Ussing chambers (Harvard Apparatus, Kent, UK, exposed area of 0.12 cm2) as described previously (Hyland and Cox, 2005) with oxygenated (95% O2, 5% CO2) Krebs buffer maintained at 37°C.4 kDa FITC-dextran was added to the mucosal chamber at a final concentration of 2.5 mg/mL; 200 μL samples were collected from the serosal chamber every 30 min for the following 3 h. Gene Expression Analysis Total RNA was extracted using the mirVana™ miRNA Isolation kit (Ambion/Llife technologies, Paisley, UK) and Dnase treated (Turbo DNA-free, Ambion/life technologies) according to the manufacturers recommendations. RNA was quantified using NanoDrop™ spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer’s instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent, Stockport, UK) according to the manufacturer’s procedure and an RNA integrity number (RIN) was calculated. RNA with RIN value >7 was used for subsequent experiments. RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA kit (Applied Biosystems, Warrington, UK) according to manufacturer’s instructions. Briefly, Multiscribe Reverse Transcriptase (50 U/μL) (1)(2)(1)(10) was added as part of RT master mix, incubated for 25°C for 10 min, 37°C for 2 h, 85°C for 5 min and stored at 4°C. Quantitative PCR was carried out using probes (6 carboxy fluorescein – FAM) designed by Applied Biosystems to mouse specific targeted genes, while using β-actin as an endogenous control. Amplification reactions contained 1 μl cDNA, 5 μl of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to a total of 10 μl by the addition of Rnase-free water. All reactions were performed in triplicate using 96-well plates on the LightCycler®480 System. Thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for amplicon contamination, each run contained no template controls in triplicate for each probe used. Cycle threshold (Ct) values were recorded. Data was normalized using β-actin and transformed using the 2−ΔΔCT method and presented as a fold change vs. control group. Statistical Analysis Normally distributed data are presented as mean ± SEM; Non-parametric datasets are presented as median with inter-quartile range. Unpaired two-tailed t-test were applied to analyse parametric data and Mann-Whitney test was used for non-parametric. Spearman’s rank correlation coefficient was employed for the correlation analysis in the pooled datasets. A p value < 0.05 was deemed significant in all cases.
Results Figures 6 and 7 show the effect of bacterial strains from the genus Bacteriodes on gut permeability in the ileum and the colon, respectively. An increase in FITC concentration indicates an increase in the leakiness of the intestinal gut epithelium. All but one of the bacterial strains tested were able to either reduce gut permeability in the ileum or demonstrated similar gut permeability compared to the control (Figure 6). In the colon, the majority of the bacterial strains tested showed similar effects on gut permeability compared to the control. Bacteroides sp NCIMB 43598 (Ref 2) was able to significantly reduce the gut permeability in the colon compared to the control (Figure 7). These data suggest that bacterial strains from the genus Bacteroides are particularly useful in treating central nervous system diseases or disorders, such as neurodegenerative diseases, by reducing the permeability of the gut epithelium and thereby preventing harmful substances from leaving the intestine. Figures 8 and 9 show changes in gene expression of tight junction proteins in the ileum and the colon after treatment with bacterial strains from the genus Bacteriodes. Tight junction proteins including TJP1 and Occludin help to regulate the permeability of the gut epithelium. An increase in expression of these genes indicates a decrease in gut permeability, which is desirable. An increase in TJP1 gene expression was observed in the ileum after treatment with Bacteroides sp NCIMB 43598 (Ref 2) (Figure 8A) and in the colon after treatment with Bacteroides sp NCIMB 43598 (Ref 2) and Ref 31, Bacteroides dorei NCIMB 43599 (Ref 20) and Bacteroides intestinalis Ref 33 (Figure 8B). An increase in Occuldin gene expression was observed in the ileum after treatment with Bacteroides dorei NCIMB 43599 (Ref 20), Bacteroides sp NCIMB 43598 (Ref 2) and Bacteroides intestinalis Ref 33 (Figure 9A), while an increase in Occuldin gene expression in the colon was seen after treatment with Bacteroides uniformis Ref 32, Bacteroides intestinalis Ref 33 (Figure 9B). A decrease in Occuldin gene expression was seen in the colon after treatment with Bacteroides dorei Ref 20 and Bacteroides sp Ref 2, which is different to the effect observed in the ileum. This suggests that the effect of bacterial strains on gene expression can be specific for the ileum or the colon. Example 7 – Efficacy of strains of the genus Bacteroides on the tryptophan/kynurenine system Summary The effect of bacterial strains from the genus Bacteroides on the tryptophan/kynurenine system was investigated. The metabolism of the amino acid tryptophan is a highly regulated physiological process leading to the generation of several neuroactive compounds within the central nervous system, including 5-hydroxytryptamine, 5-HT. Changes in gene expression of two enzymes involved in tryptophan catabolism in the ileum and the colon after treatment with bacterial strains from the genus Bacteroides was investigated. The first is Indoleamine 2,3 dioxygenase-1 (IDO1) which initiates tryptophan catabolism along a pathway that generates several bioactive kynurenine-based metabolites.
The second is tryptophan hydroxylase-1 (TPH-1) which catalyses the formation of 5-hydroxy-L- tryptophan (5-HTP) from L-tryptophan, the first and rate-limiting step in the biosynthesis of 5-HT. Material and Methods The gene expression analysis, bacterial strains tested, animals and tissue collection were conducted as described in Example 6. Results A significant increase in IDO1 gene expression was observed in the colon after treatment with Bacteroides intestinalis Ref 33 (Figure 10B). Treatment with Bacteroides salyersae Ref 15 and Bacteroides cellulosilyticus Ref 30 also lead to an increase in IDO1 gene expression in the colon. A decrease in IDO1 gene expression was observed in the ileum after treatment with Bacteroides ovatus Ref 31, Bacteroides uniformis Ref 32 and Bacteroides intestinalis Ref 33 (Figure 10A); and in the colon after treatment with Bacteroides dorei Ref 20, Bacteroides sp NCIMB 43598 (Ref 2) and Ref 31 (Figure 10B). An increase in TPH-1 gene expression was observed in the ileum after treatment with Bacteroides fragilis Ref 22, Bacteroides vulgatus Ref 24, Bacteroides sp NCIMB 43598 (Ref 2) and Bacteroides xylanisolvens Ref 1. A significant decrease in TPH-1 gene expression was observed in ileum after treatment with Bacteroides cellulosilyticus Ref 30. Treatment with Bacteroides intestinalis Ref 33 and Bacteroides uniformis Ref 32 also resulted in a decrease in TPH-1 gene expression in the ileum (Figure 11A). Figure 11B shows that treatment with Bacteroides dorei NCIMB 43599 (Ref 20,) Bacteroides sp NCIMB 43598(Ref 2) and Ref 31, Bacteroides uniformis Ref 32 and Bacteroides intestinalis Ref 33 resulted in an increase in TPH-1 gene expression in the colon. A significant decrease in TPH-1 gene expression was observed in the colon after treatment with Bacteroides stercoris NCIMB 43597 (Ref 10). These data suggest that bacterial strains from the genus Bacteriodes play a role in regulating the tryptophan/kynurenine system. This system generates several neuroactive compounds within the central nervous system, therefore the ability of bacterial strains from the genus Bacteriodes to modulate the expression of enzymes within this pathway makes them particularly useful in treating central nervous system diseases or disorders, such as neurodegenerative diseases. Example 8– Induction of GABA Production and regulation of GABAergic Biomarkers in the brain Summary The ability of bacterial strains from the genus Bacteroides to induce GABA production and to alter the expression of GABAergic neuron markers in the brain was analysed.
Material and Methods Bacterial strains B. ovatus - NCIMB 43600 (Ref 7) B. koreensis – NCIMB 43594 (Ref 17) Bacteroides sp - NCIMB 43598 (Ref 2), Bacteroides sp- NCIMB 43593 (Ref 27) B. dorei – NCIMB 435601 (Ref 8), NCIMB 43599 (Ref 20), NCIMB 43595 (Ref 25) B. stercoris - NCIMB 43597 (Ref 10) B. xylanisolvens - NCIMB 43596 (Ref 1) Study design Analysis of GABA The amount of GABA produced was analysed by LC-MS. Stock GABA calibration standards prepared in HPLC grade water: 0, 10, 25, 50, 100, 200, 300 ng/ml. The internal standard (d6-GABA) prepared as a 800ng/ml stock in HPLC grade water. The standards and samples were prepared by adding 200μl calibration standard/sample to 20μl 3M perchloric acid, mixed and then centrifuged. Derivatisation solution (50μl supernatant, 20μl internal standard (4ng), 50μl 2M KHCO3/KOH buffer, pH9.8, 50μl dansyl chloride (20mg/ml in acetonitrile) was mixed then incubated at 80°C for 30 minutes. After the samples were cooled 20μl acetic acid was added and then centrifuged. LC conditions - Column: Hichrom 5 ^m C18 (150 x 2.1 mm), Mobile phase: 55% water (containing 0.1% formic acid), 45% acetonitrile, Flow rate: 0.2ml/min, Column temp: 30 ^C and Tray temp 4 ^C MS conditions: Ionisation mode: ESI, Polarity: positive, Spray voltage: 3500, Sheath gas: 60, Auxilliary gas: 0, Capillary temp: 375, Tune lens: tuned value, Skimmer offset: 12, Collision energy: 16, Collision pressure: 1.8, Divert valve: divert to waste up to 3.5 minutes, Run time: 8 minutes SRM transitions: GABA : 337 – 170, D6-GABA : 343 – 170 Retention times: GABA : 4.9 minutes, D6-GABA : 4.9 minutes Gene expression analysis of GABA transporter, GABABR2 and GABABR3 in SHSY5Y Cells SH-SY5Y cells are a neuroblastoma cell line. The cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2 mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin. Cells were plated in 6 well plates at density of 0.5*10^6 cells/well. after 24h, media were replaced with growth medium containing 1% FBS and treated with 10% (v/v) bacterial supernatants for 3 days. Thereafter total RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. cDNA was prepared from 2000 ng of total RNA using the High-Capacity cDNA reverse transcription kit (Thermo Fisher) according to the manufacturer's instructions. QPCR was performed using SYBR-Green and products were detected on Quant Studio 6 flex real-time PCR machine (Applied Biosystems) Data was normalized using β-actin
as reference gene and transformed using the 2^−ΔΔCT method and presented as a fold change vs. untreated control
( Q ) ( Q ) Results Figure 12A demonstrates that bacterial strains from various different species of Bacteroides were able to induce GABA production. For example, bacterial strains from the species Bacteroides xylanisolvens NCIMB 43596 (Ref 1) and Bacteroides ovatus NCIMB 43600 (Ref 7) were able induce over 200μg of GABA. Figure 12B demonstrates that bacterial strains from the genus Bacteroides were capable of upregulating GABAergic biomarkers in neuronal cells, even for those strains which did not show an induction of GABA production. This suggests that GABA production may be dependent on the amount of bacteria used or the sensitivity of cells to GABA induction. These data indicate that bacteria from the genus Bacteroides are capable of inducing the production of GABA and upregulating the expression of GABAergic biomarkers in neuronal cells, therefore they may be useful in the treatment or prevention of central nervous system diseases or disorders, such as epilepsy and multiple sclerosis. Sequences
GCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATG SEQ ID NO:2 - 16S rRNA gene sequence of Bacteroides dorei strain DSM 17855
SEQ ID NO:3 – genome sequence BT2013 incorporated by reference to SEQ ID NO:1 in WO2016/102950. SEQ ID NO:4 - 16S rRNA gene sequence of Bacteroides strain (NCIMB 42408)
SEQ ID NO:5 - 16S rRNA gene sequence of Bacteroides dorei (NCIMB 43599) Ref 20
SEQ ID NO:6 - 16S rRNA gene sequence of Bacteroides sp (NCIMB 43598) Ref 2
5
SEQ ID NO:7 - 16S rRNA gene sequence of Bacteroides stercoris (NCIMB 43597) Ref 10
GATGCTGCCCCTCGACTTGCA SEQ ID NO:8 - 16S rRNA gene sequence of Bacteroides xylanisolvens (NCIMB 43596) Ref 1
5
SEQ ID NO:9 - 16S rRNA gene sequence of Bacteroides koreensis (NCIMB 43594) Ref 17
5
Embodiments The invention further provides the following embodiments: 1. A composition comprising a bacterial strain of the genus Bacteriodes, for use in a method of treating or preventing a central nervous system disease or disorder. 2. The composition for use according to embodiment 1, wherein the central nervous system disease or disorder is mediated by the microbiota-gut-brain axis. 3. The composition for use according to embodiment 1 or embodiment 2, wherein the composition is for use in a method of treating or preventing a neurodegenerative disease, a neurodevelopmental disorder, a neuropsychiatric condition or a brain injury. 4. The composition for use according to embodiments 1-3, wherein the central nervous system disease or disorder is selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele- Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia;
mild cognitive impairment; HIV-related cognitive impairment, corticobasal degeneration; and epilepsy. 5. The composition for use according to embodiments 1-4, wherein the composition is for use in a method of treating or preventing a neurodegenerative disease. 6. The composition for use according to embodiment 5, wherein the neurodegenerative disease is selected from the group consisting of Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia and spinal muscular atrophy. 7. The composition for use according to embodiment 6, wherein the Parkinson’s disease is including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson- Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism. 8. The composition for use according to embodiment 6, wherein the Alzheimer’s disease is Benson's syndrome. 9. The composition for use according to embodiments 1-4, wherein the composition is for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition. 10. The composition for use according to embodiment 9, wherein the neurodevelopmental disorder or neuropsychiatric condition is selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment, and corticobasal degeneration. 11. The composition for use according to embodiments 1-4, wherein the composition is for use in a method of treating or preventing a brain injury. 12. The composition for use according to embodiment 11, wherein the brain injury is a result of a stroke, a traumatic brain injury, an acquired brain injury, a trauma, a brain haemorrhage, a tumour, encephalitis, cerebral hypoxia and/or cerebral anoxia. 13. The composition for use according to embodiments 1-4, wherein the composition is for use in a method of treating or preventing epilepsy. 14. The composition of any preceding embodiment, wherein the composition modulates the microbiota-gut-brain axis.
15. The composition for use according to any of embodiments 1-14, 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 NOs:5-12. 16. The composition for use according to any of embodiments 1-14, 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 the 16s rRNA sequence of a bacterial strain of Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides coprocola, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides eggherthii, Bacteroides koreensis, Bacteroides kribbi or Bacteroides caccae. 17. The composition of any preceding embodiment, wherein the composition is for oral administration. 18. The composition of any preceding embodiment, wherein the composition comprises one or more pharmaceutically acceptable excipients or carriers. 19. The composition of any preceding embodiment, wherein the bacterial strain is lyophilised. 20. The composition of any preceding embodiment, wherein the bacterial strain is viable and capable of partially or totally colonising the intestine. 21. The composition of any preceding embodiment, wherein the composition comprises a single strain of Bacteroides. 22. The composition of any preceding embodiment, which comprises the Bacteroides bacterial strain as part of a microbial consortium. 23. A food product comprising the composition of any preceding embodiment, for the use of any preceding embodiment. 24. A vaccine composition comprising the composition of any preceding embodiment, for the use of any preceding embodiment. 25. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the genus Bacteroides to a patient in need thereof. 26. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides dorei to a patient in need thereof. 27. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides ovatus to a patient in need thereof. 28. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides stercoris to a patient in need thereof.
29. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Bacteroides xylanisolvens to a patient in need thereof. 30. A cell of the Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris and Bacteroides xylanisolvens Bacteroides koreensis, Bacteroides strain deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601 , or a derivative thereof, for the use of any of embodiments 1-22. 31. A pharmaceutical composition comprising the cell of embodiment 30. 32. The composition of embodiment 31, comprising a pharmaceutically acceptable carrier or excipient. 33. The pharmaceutical composition of embodiment 30 or 31 for use in therapy. 34. The pharmaceutical composition of claim 33 for the use of any one of embodiments 1-22. REFERENCES [1] Spor et al. (2011) Nat Rev Microbiol.9(4):279-90. [2] Eckburg et al. (2005) Science.10;308(5728):1635-8. [3] Macpherson et al. (2001) Microbes Infect.3(12):1021-35 [4] Macpherson et al. (2002) Cell Mol Life Sci.59(12):2088-96. [5] Mazmanian et al. (2005) Cell 15;122(1):107-18. [6] Thursby and Juge (2017) Biochem J.474(11): 1823–1836. [7] Kraal et al (2014) PLoS One 9 (5):e97279. [8] Hooper et al. (2001) Science 291 (5505):881-884. [9] Rakoff-Nahoum et al. (2004) Cell 118(2):229-241. [10] Hakansson and Molin (2011) Nutrients, 3 (6):637-682. [11] WO 2013/050792 [12] WO 03/046580 [13] WO 2013/008039 [14] WO 2014/167338 [15] Goldin and Gorbach (2008) Clin Infect Dis.46 Suppl 2:S96-100. [16] Azad et al. (2013) BMJ.347:f6471. [17] Mayer et al (2014) The Journal of Inflammatory neuroscience 34(46):15490 –15496 [18] Cryan and Dinan (2015) Inflammatory neuropsychopharmacology, 40: 241-2. [19] Zhou and Foster (2015) Inflammatory neuropsychiatric Disease and Treatment 11: 715–723. [20] Wang and Kasper (2014) Brain Behav Immun.38: 1–12. [21] Hsiao et al. (2012) Proc Natl Acad Sci, 109(31):12776-12781. [22] Hsiao et al. (2013) Cell 19; 155(7): 1451–1463 [23] US2012/087895 [24] US2014/065132 [25] US2016/120916 [26] US2016/120917 [27] US2016/375065 [28] Wexler (2007) Clin. Microbiol. Rev, 20, 4593-621 [29] Chun et al (2007). Int J Syst Evol Microbiol 57, 2259–2261. [30] Masco et al. (2003) Systematic and Applied Microbiology, 26:557-563. [31] Srůtková et al. (2011) J. Microbiol. Methods, 87(1):10-6. [32] Strle et al (2001) Crit Rev Immunol.21(5):427-49. [33] Liu and Bing (2011) Parkinsons Dis.327089. [34] Jawa RS, Anillo S, Huntoon K, Baumann H, Kulaylat M: Analytic review: Interleukin-6 in surgery, trauma, and critical care: part I: basic science. J Intensive Care Med 2011, 26(1):3-12.
[35] Jawa et al (2011) J Intensive Care Med, 26(2):73-8 [36] Haim et al. (2015) J Inflammatory neurosci. ;35(6):2817-29 [37] Pal R et al. (2016) Inflammatory neurol Res 38(12):1111-1122 [38] Kamat et al. (2009) J Alzheimers Dis.15(3): 473–493. [39] Foguem & Manckoundia (2018) Current Inflammatory neurology and Inflammatory neuroscience Reports, 18:24 [40] Ludolph et al. (2009) Eur J Inflammatory neurol.16(3): 297–309. [41] Galpern & Lang (2006) Inflammatory neurological Progress 59 (3) 449-458[ [42] Wang et al. (2016) J Inflammatory neurogastroenterol Motil 22: 589-605. [43] Zhang et al. (2017) Transl Psychiatry 7, e1138; [44] Suzen (2007) Bioactive Heterocycles V pp 145-178 [45] Stolc et al. (2006) Cellular and Molecular Neurobiology, 26, 7–8, 1493–1502 [46] Psaty et al. (2003) JAMA, 289(19):2534–44 [47] Lancet. (1995) 346(8991–8992):1647–53 [48] Mitropoulou et al. (2013) J Nutr Metab. (2013) 716861. [49] Kailasapathy et al. (2002) Curr Issues Intest Microbiol.3(2):39-48. [50] Miyamoto-Shinohara et al. (2008) J. Gen. Appl. Microbiol., 54, 9–24. [51] Cryopreservation and Freeze-Drying Protocols, ed. by Day and McLellan, Humana Press. [52] Leslie et al. (1995) Appl. Environ. Microbiol.61, 3592–3597. [53] Mitropoulou et al. (2013) J Nutr Metab. (2013) 716861. [54] Kailasapathy et al. (2002) Curr Issues Intest Microbiol.3(2):39-48. [55] Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller [56] Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.1985) [57] US 2016/0067188 [58] Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press. [59] Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press [60] Strobel (2009) Methods Mol Biol.581:247-61. [61] Gennaro (2000) Remington: The Science and Practice of Pharmacy.20th edition, ISBN: 0683306472. [62] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press). [63] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [64] Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds, 1986, Blackwell Scientific Publications) [65] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press). [66] Handbook of Surface and Colloidal Chemistry (Birdi, K.S. ed., CRC Press, 1997) [67] Ausubel et al. (eds) (2002) Short protocols in molecular biology, 5th edition (Current Protocols). [68] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag) [69] Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30 [70] Smith & Waterman (1981) Adv. Appl. Math.2: 482-489.
Claims
CLAIMS 1. A composition comprising a bacterial strain of the genus Bacteriodes, for use in a method of treating or preventing a central nervous system disease or disorder. 2. The composition for use according to claim 1, wherein: (a) the central nervous system disease or disorder is mediated by the microbiota-gut-brain axis; (b) the composition is for use in a method of treating or preventing a neurodegenerative disease, a neurodevelopmental disorder, a neuropsychiatric condition or a brain injury and/or (c) the composition is for use in a method of modulating the microbiota-gut-brain axis. 3. The composition for use according to claims 1-2, wherein the composition is for use in a method of treating or preventing a neurodegenerative disease. 4. The composition for use according to claim 3, wherein the neurodegenerative disease is be selected from the group consisting of Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor inflammatory neurone disease; prion disease; spinocerebellar ataxia or spinal muscular atrophy. 5. The composition for use according to claims 1-2, wherein the composition is for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition. 6. The composition for use according to claim 5, wherein the neurodevelopmental disorder or neuropsychiatric condition is selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic pain; Guillain-Barre syndrome and meningitis, dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration. 7. The composition for use according to claims 1-2, wherein the composition is for use in a method of treating or preventing epilepsy. 8. The composition for use according to claims 1-2, wherein the composition is for use in a method of treating or preventing a brain injury, optionally wherein the brain injury is a result of a stroke, a traumatic brain injury, an acquired brain injury, a trauma, a brain haemorrhage, a tumour, encephalitis, cerebral hypoxia and/or cerebral anoxia. 9. The composition for use according any of claims 1-8, wherein:
(a) the bacterial strain is of the species Bacteroides dorei; optionally 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 Bacteroides dorei, optionally 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 12; (b) the bacterial strain is of the species Bacteroides ovatus; optionally 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 Bacteroides ovatus, optionally 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: 11; (c) the bacterial strain is of the species Bacteroides stercoris, optionally 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 Bacteroides stercoris, optionally 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:7; or (d) the bacterial strain is of the species Bacteroides xylanisolvens, optionally 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 Bacteroides xylanisolvens, optionally 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:8; or (e) the bacterial strain is of the species Bacteroides koreensis, optionally 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 Bacteroides koreensis, optionally 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: 9 ; or (f) bacterial strain is of the genus Bacteroides and 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 Bacteroides sp, optionally 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: 6 or 10. 10. The composition of any preceding claim, wherein: (a) the composition is for oral administration;
(b) the composition comprises one or more pharmaceutically acceptable excipients or carriers; (c) the bacterial strain is lyophilised; (d) the bacterial strain s viable and/or (e) the bacterial strain is capable of partially or totally colonising the intestine; 11. The composition of any preceding claim, wherein the composition comprises: (a) a single strain of Bacteroides; and/or (b) the Bacteroides bacterial strain as part of a microbial consortium. 12. A food product or vaccine composition comprising the composition of any preceding claim, for the use of any preceding claim. 13. A cell of the Bacteroides dorei, Bacteroides ovatus, Bacteroides stercoris and Bacteroides xylanisolvens Bacteroides koreensis, Bacteroides strain deposited under accession number NCIMB 42341, NCIMB 42408, NCIMB 43593, NCIMB 43594, NCIMB 43595, NCIMB 43596, NCIMB 43597, NCIMB 43598, NCIMB 43599, NCIMB 43600 or NCIMB 43601, or a derivative thereof, for the use of any of claims 1-12. 14. A pharmaceutical composition comprising a cell according to claim 13, optionally comprising a pharmaceutically acceptable carrier or excipient. 15. The pharmaceutical composition of claim 14 for the use of any one of claims 1-12.
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