Modification of gut microbiome to treat psychiatric disorders or diseases of central nervous system

Abstract

To provide modification of the gut microbiome to treat psychiatric disorders or diseases of the central nervous system.SOLUTION: The present disclosure relates to a method of treating at least one symptom of a psychiatric disorder or disease of the central nervous system in a subject by modulating the amount of GABA produced in the gut of the subject. The present disclosure also relates to a method of culturing novel bacterial strains. A method of identifying bacterial strains capable of producing GABA and engineering strains to produce GABA are also disclosed. The present disclosure also describes a method of identifying bacterial strains that can produce, for example, GABA under physiologically appropriate conditions, such as, physiologically appropriate pH.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 307,991, filed March 14, 2016, the contents of which are incorporated by reference in their entirety.

[0002] Sequence Listing Reference The contents of the text file named "HOBE001001WOSeqList.txt," created on March 13, 2017, and measuring 7.6 MB in size, are hereby incorporated by reference in their entirety.

[0003] Government funding This invention was made with government support under 3R01HG005824-02S1 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] FIELD OF THE INVENTION The present disclosure relates to compositions and methods for treating at least one symptom of a disease in a subject. In some cases, the disease is a psychiatric disorder or a disease of the central nervous system. The present disclosure teaches the treatment of the disease by modulating (altering) (e.g., increasing) the amount of endogenous GABA in the body of a subject. In some embodiments, the present disclosure teaches modulating (e.g., increasing) the amount of GABA produced in the intestine by bacteria in the intestine of a subject. For example, the present disclosure teaches the administration of bacteria that can produce GABA (e.g., in the intestine of a human) to a subject in need thereof.

[0005] The present disclosure also relates to a method for culturing a previously uncultured bacterial strain. For example, the present disclosure teaches a previously uncultured bacterial strain, Evtepia gabavorous KLE1738. As shown herein, an uncultured bacterial strain, such as Evtepia gabavorous KLE1738, can be newly cultured by providing growth factors necessary for bacterial growth and proliferation.

[0006] Also disclosed are methods for identifying bacterial strains capable of producing certain growth factors, e.g., GABA, under physiologically relevant conditions, such as physiologically relevant pH. [Background technology]

[0007] background The gut microbiome influences certain gastrointestinal and metabolic disorders, such as irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, celiac disease, obesity, heart disease, type I and type II diabetes, and colon cancer.

[0008] Microbiological research has, by necessity, been limited to cultivable microorganisms. By some estimates, 99% of bacteria in the external environment have never been cultured. Therefore, the development of new techniques for culturing previously uncultivable or uncultivable bacteria could help expand the scope of microbiological investigations. Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention The present disclosure provides compositions and methods for treating disorders such as psychiatric disorders or disorders of the central nervous system. In some embodiments, the present disclosure teaches therapeutic compositions comprising one or more bacteria (e.g., purified bacteria) capable of producing GABA. The bacteria can produce GABA under physiologically relevant conditions, including in the human intestine. The present disclosure also provides methods of treating a subject in need thereof, comprising administering to the subject a therapeutic composition comprising GABA-producing bacteria. As demonstrated herein, GABA-producing bacteria can produce GABA in the subject's intestine. GABA can diffuse to other systems within the subject's body (e.g., the circulatory system and nervous system), where endogenous GABA can act as a neurotransmitter. In some embodiments, increasing GABA levels (e.g., in the nervous system) can ameliorate symptoms of psychiatric disorders or disorders of the central nervous system.

[0010] In some embodiments, the present disclosure also provides methods for identifying bacteria that produce GABA in humans in a physiologically relevant pH range, and the use of these bacteria to modulate GABA levels in humans to treat psychiatric disorders.

[0011] The present disclosure also relates to methods for culturing previously uncultured bacterial species. For example, the present disclosure teaches the isolation and characterization of the bacterial species KLE1738, tentatively designated Evtepia gabavorous. Growth of E. gabavorous requires the presence of the growth factor GABA, which can be supplied by GABA-producing bacteria such as Bacteroides fragilis KLE1758.

[0012] In one aspect, the present disclosure provides a therapeutic composition comprising at least one purified bacterial population of bacteria capable of producing GABA in a subject in need thereof.

[0013] In some embodiments, at least one bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 1-31 shown in Table 1. In some embodiments, the at least one purified bacterial population is Bacteroides caccae KLE1911;Bacteroides clarus KLE1930;Bacteroides dorei KLE1912;Bacteroides finegoldii KLE1931;Bacteroides fragilis KLE1958;Bacteroides massiliensis KLE1932;Bacteroides ovatus KLE1770;Bacteroides stercoris KLE1933;Bacteroides thetaiotaomicron KLE1934;Bacteroides uniformis KLE1913;Bacteroides vulgatus KLE1910;Bacteroides xylanisolvens KLE1935;Bifidobacterium adolescentis KLE 1879;Blautia obeum KLE1914;Blautia wexlerae KLE1916;Butyricimonas virosa KLE1938;Clostridium perfringens KLE1937;Clostridium sordellii KLE1939;Clostridium sp. KLE1862;Clostridium sp. KLE1918;Coprobacillus sp. KLE1779;Coprococcus sp. KLE1880;Dorea longicatena KLE1917; Eggerthella lenta KLE1926; Eubacterium rectale KLE1922; Gordonibacter pamelaeae KLE1915; Oscillibacter sp. KLE1928; Parabacteroides distasonis KLE2020; Parabacteroides merdae KLE1863; Ruminococcus gnavus KLE1940; Turicibacter sanguinis KLE1941, and combinations thereof.

[0014] In some embodiments, at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 32-274 set forth in Table 2. In some embodiments, at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence that has at least 95% similarity to a 16S rDNA sequence selected from one of SEQ ID NOs: 305-2217 set forth in Table 10. In some embodiments, at least one purified bacterial population consists of bacteria comprising a DNA sequence encoding an enzyme selected from glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof. In some embodiments, the glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof, is encoded by a DNA sequence that is at least 70% similar in DNA sequence to any one of SEQ ID NOs:275-304 shown in Table 3.

[0015] In some embodiments, the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 4. In some embodiments, the putrescine aminotransferase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 5. In some embodiments, the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 6. In some embodiments, the arginine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 7. In some embodiments, the agmatinase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 8. In some embodiments, the ornithine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 9.

[0016] In some embodiments, the at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence having at least 95% similarity to a reference bacterium selected from the group consisting of Escherichia coli MG1655, Escherichia coli Nissle 1917, or a combination thereof.

[0017] In some embodiments, the bacterial population comprises bacteria capable of producing GABA at physiologically relevant pH. In some embodiments, the bacterial population comprises bacteria capable of producing GABA at a pH range of about 4.5 to about 7.5. In some embodiments, the bacterial population comprises bacteria capable of producing GABA inside the human intestine.

[0018] In some embodiments, the composition is in the form of a probiotic, prebiotic, capsule, tablet, caplet, pill, troche, lozenge, powder, granule, medical food, or a combination thereof, hi some embodiments, the composition is administered as a fecal transplant.

[0019] In some embodiments, the bacterium is capable of producing GABA through expression of any combination of glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, and / or ornithine decarboxylase.

[0020] In some embodiments, the therapeutic composition further comprises a purified bacterial strain that is cytotoxic or cytostatic to a GABA-consuming bacterium, hi some embodiments, the GABA-consuming bacterium is Evtepia gabavorous or Firmicutes bacterium MGS:114.

[0021] In some embodiments, the therapeutic composition further comprises a prebiotic capable of stimulating the growth of GABA-producing bacteria or GABA production levels.

[0022] In one aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutic composition comprising at least one purified bacterial population of bacteria capable of producing GABA in the subject in need thereof.

[0023] In one aspect, the present disclosure provides for the use of a therapeutic composition comprising at least one purified bacterial population of bacteria capable of producing GABA in the manufacture of a medicament for the treatment of a disease.

[0024] In one aspect, the present disclosure provides the use of a therapeutic composition comprising at least one purified bacterial population of bacteria capable of producing GABA for the treatment of disease.

[0025] In some embodiments, the disease or disorder is a psychiatric disease or disorder. In some embodiments, the psychiatric disease or disorder is selected from the group consisting of depression, bipolar disorder, schizophrenia, anxiety, anxiety disorders, addiction, social phobia, treatment-resistant major depressive disorder (TR-MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), neurodegenerative amyloid disorders (Parkinson's, Alzheimer's, and Huntington's disease), orthostatic tremor, Lafora's disease, restless legs syndrome, neuropathic pain, pain disorders, dementia, epilepsy, stiff-person syndrome, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD), and combinations thereof. In some embodiments, treating the disease or disorder comprises reducing at least one symptom of the disease or disorder, such as fatigue, insomnia, motor dysfunction, stress, persistent anxiety, persistent sadness, social withdrawal, substance withdrawal, irritability, suicidal ideation, self-harm ideation, restlessness, decreased libido, lack of concentration, convulsions, memory loss, anger, fits of emotional response, confusion, pain, and muscle spasms, loss of appetite, altered bowel motility, and combinations thereof.

[0026] In some embodiments, at least one bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 1-31 shown in Table 1. In some embodiments, the at least one purified bacterial population is Bacteroides caccae KLE1911;Bacteroides clarus KLE1930;Bacteroides dorei KLE1912;Bacteroides finegoldii KLE1931;Bacteroides fragilis KLE1958;Bacteroides massiliensis KLE1932;Bacteroides ovatus KLE1770;Bacteroides stercoris KLE1933;Bacteroides thetaiotaomicron KLE1934;Bacteroides uniformis KLE1913;Bacteroides vulgatus KLE1910;Bacteroides xylanisolvens KLE1935;Bifidobacterium adolescentis KLE 1879;Blautia obeum KLE1914;Blautia wexlerae KLE1916;Butyricimonas virosa KLE1938;Clostridium perfringens KLE1937;Clostridium sordellii KLE1939;Clostridium sp. KLE1862;Clostridium sp. KLE1918;Coprobacillus sp. KLE1779;Coprococcus sp. KLE1880;Dorea longicatena KLE1917;Eggerthella lenta KLE1926;Eubacterium rectale KLE1922;Gordonibacter pamelaeae KLE1915;Oscillibacter sp. KLE1928; Parabacteroides distasonis KLE2020; Parabacteroides merdae KLE1863; Ruminococcus gnavus KLE1940; Turicibacter sanguinis KLE1941, and combinations thereof.

[0027] In some embodiments, at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 32-274 shown in Table 2.

[0028] In some embodiments, at least one bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to any one of SEQ ID NOs: 305-2217 set forth in Table 10.

[0029] In some embodiments, the at least one purified bacterial population consists of bacteria that comprise a DNA sequence encoding an enzyme selected from glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof.

[0030] In some embodiments, the glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof, is encoded by a DNA sequence that is at least 70% similar to a DNA sequence selected from one of SEQ ID NOs:275-304 shown in Table 3.

[0031] In some embodiments, the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 4. In some embodiments, the putrescine aminotransferase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 5. In some embodiments, the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 6. In some embodiments, the arginine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 7. In some embodiments, the agmatinase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 8. In some embodiments, the ornithine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 9.

[0032] In some embodiments, the bacterium is genetically engineered to produce GABA, ie, through expression of glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof.

[0033] In some embodiments, the glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof, is encoded by a DNA sequence at least 70% similar to a DNA sequence selected from one of SEQ ID NOs:275-304 set forth in Table 3.

[0034] In some embodiments, the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 4. In some embodiments, the putrescine aminotransferase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 5. In some embodiments, the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 6. In some embodiments, the arginine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 7. In some embodiments, the agmatinase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 8. In some embodiments, the ornithine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 9.

[0035] In some embodiments, the at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence having at least 95% similarity to a reference bacterium selected from the group consisting of Escherichia coli MG1655, Escherichia coli Nissle 1917, or a combination thereof.

[0036] In some embodiments, the bacterial population comprises bacteria capable of producing GABA at a physiologically relevant pH. In some embodiments, the bacterial population comprises bacteria capable of producing GABA at a pH range of about 4.5 to about 7.5. In some embodiments, the bacterial population comprises bacteria capable of producing GABA inside the human intestine. In some embodiments, the composition is administered as a fecal transplant. In some embodiments, the composition is administered as a probiotic. In some embodiments, the bacteria are capable of producing GABA through expression of any combination of glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, and combinations thereof.

[0037] In some embodiments, at least one bacterial strain is cytotoxic or cytostatic to the GABA-consuming bacterium. In some embodiments, the GABA-consuming bacterium is Evtepia gabavorous or Firmicutes bacterium MGS:114.

[0038] In some embodiments, the method for treating a subject further comprises determining whether the subject will benefit from increasing endogenous GABA by measuring the initial amount of GABA in the subject's stool, thereby identifying the subject who needs treatment.In some embodiments, the initial amount of GABA in the subject's stool is less than about 8 μg per gram of wet or dry stool.In some embodiments, the amount of GABA in the subject's stool increases relative to the initial amount after administering therapeutic composition.

[0039] In some embodiments, the method for treating a subject further comprises identifying a subject in need of treatment by determining whether the subject would benefit from increased endogenous GABA by measuring the initial amount of GABA-producing bacteria in the subject's stool. In some embodiments, the initial amount of GABA-producing bacteria in the subject's stool is less than about 10% of the total bacteria measured by 16S sequence mapping. In some embodiments, at least one GABA-producing bacterium is increased in the subject's stool relative to the initial amount of GABA-producing bacteria in the subject's stool after administering the therapeutic composition.

[0040] In some embodiments, the method for treating a subject further comprises identifying a subject in need of treatment by measuring the initial amount of GABA in the subject's blood or serum to determine whether the subject will benefit from increasing endogenous GABA. In some embodiments, the amount of GABA in the subject's blood or serum is less than about 10 μg per liter of blood. In some embodiments, the amount of GABA in the subject's blood or serum is increased relative to the initial amount after administering the therapeutic composition.

[0041] In some embodiments, the method for treating a subject further comprises identifying a subject in need of treatment by measuring the amount of GABA in the subject's brain to determine whether the subject would benefit from increasing endogenous GABA. In some embodiments, the amount of GABA in the subject's brain is less than about 1.0 mM / kg. In some embodiments, the amount of GABA in the subject's brain increases relative to the initial amount after administering the therapeutic composition.

[0042] In some embodiments, the method for treating a subject further comprises identifying a subject in need of treatment by determining whether the subject will benefit from increased endogenous GABA by measuring the initial level of GABA-producing enzyme expression in the subject's stool. In some embodiments, the GABA-producing enzyme is selected from glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, and combinations thereof. In some embodiments, the initial level of enzyme expression is measured by qPCR. In some embodiments, the enzyme expression is increased relative to the initial level of enzyme expression after administering the therapeutic composition.

[0043] In some embodiments, the method for treating a subject further comprises identifying a subject in need of treatment by measuring the initial amount of GABAergic response in the subject's brain to determine whether the subject will benefit from increasing endogenous GABA.In some embodiments, the amount of GABAergic response in the subject's brain increases relative to the initial amount after administering the therapeutic composition.In some embodiments, the therapeutic composition comprises a prebiotic that can stimulate the growth of GABA-producing bacteria or GABA production.

[0044] In one aspect, the present disclosure provides a method of culturing GABA-dependent bacteria, the method comprising disposing at least one viable GABA-dependent bacterial cell on a suitable substrate and providing a source of GABA.

[0045] In some embodiments, the suitable substrate is agar. In some embodiments, the step of providing a source of GABA comprises co-cultivating with another bacterial strain, said strain being capable of producing GABA. In some embodiments, GABA is added to the substrate. In some embodiments, the GABA-dependent bacterium is E. gabavorous.

[0046] In one aspect, the disclosure provides a method for identifying a bacterial strain or strains capable of producing GABA at the physiologically relevant pH of the human intestinal tract, comprising: (a) dispersing a sample suspected of containing GABA-producing bacteria in a matrix, the matrix being at least partially permeable to GABA; (b) contacting the substrate containing the potentially GABA-producing bacteria with the GABA-dependent bacteria; (c) identifying GABA-producing bacteria by observing the formation of colonies of GABA-dependent bacteria around potential GABA-producing bacteria in the substrate; The present invention provides a method comprising:

[0047] In some embodiments, the substrate is buffered to maintain a pH in the physiological range found in the human gastrointestinal tract. In some embodiments, the GABA-dependent bacterium is E. gabavorous. In some embodiments, the pH range is between about 4.5 and about 7.5.

[0048] The present disclosure provides compositions and methods for treating a subject's psychiatric or central nervous system disorders, as well as therapeutic compositions for the same purpose. The methods may include administering to the subject one or more bacteria capable of producing endogenous GABA in the subject's intestines at a physiologically appropriate pH. The technology of the present invention may be beneficial in alleviating symptoms of psychiatric or central nervous system disorders without the aid of synthetic medications (e.g., antidepressants), which may have unwanted side effects, or in combination with existing medications. Furthermore, the technology of the present invention may have the advantage of further improving the subject's digestive health, such as improving intestinal motility and reducing gastrointestinal pain. Further features and advantages of the technology of the present invention will be apparent to those skilled in the art upon reading the following detailed description of the invention. The present invention provides, for example, the following items. (Item 1) A therapeutic composition comprising at least one purified bacterial population of bacteria capable of producing GABA in a subject in need thereof. (Item 2) 2. The therapeutic composition of item 1, wherein the at least one bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 1 to 31 set forth in Table 1. (Item 3) The at least one purified bacterial population is Bacteroides caccae KLE1911;Bacteroides clarus KLE1930;Bacteroides dorei KLE1912;Bacteroides finegoldii KLE1931;Bacteroides fragilis KLE1958;Bacteroides massiliensis KLE1932;Bacteroides ovatus KLE1770;Bacteroides stercoris KLE1933;Bacteroides thetaiotaomicron KLE1934;Bacteroides uniformis KLE1913;Bacteroides vulgatus KLE1910;Bacteroides xylanisolvens KLE1935;Bifidobacterium adolescentis KLE 1879;Blautia obeum KLE1914;Blautia wexlerae KLE1916;Butyricimonas virosa KLE1938;Clostridium perfringens KLE1937;Clostridium sordellii KLE1939;Clostridium sp. KLE1862;Clostridium sp. KLE1918;Coprobacillus sp. KLE1779;Coprococcus sp. KLE1880;Dorea longicatena KLE1917;Eggerthella lenta KLE1926;Eubacterium rectale KLE1922;Gordonibacter pamelaeae KLE1915;Oscillibacter sp. KLE1928;Parabacteroides distasonis KLE2020;Parabacteroides merdae KLE1863;Ruminococcus gnavus KLE1940;Turicibacter sanguinis 2. The therapeutic composition of claim 1, comprising a bacterium selected from the group consisting of: KLE1941, KLE1941, and combinations thereof. (Item 4) Item 1, wherein the at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 32 to 274 set forth in Table 2. (Item 5) Item 1, wherein the at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence having at least 95% similarity to a 16S rDNA sequence selected from one of SEQ ID NOs: 305 to 2217 set forth in Table 10. The therapeutic composition of item 1. (Item 6) 2. The therapeutic composition of claim 1, wherein the at least one purified bacterial population consists of bacteria comprising a DNA sequence encoding an enzyme selected from glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof. (Item 7) 7. The therapeutic composition of item 6, wherein the glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof is encoded by a DNA sequence that is at least 70% similar in DNA sequence to any one of SEQ ID NOs: 275 to 304 shown in Table 3. (Item 8) 7. The therapeutic composition of item 6, wherein the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 4. (Item 9) 7. The therapeutic composition of item 6, wherein the putrescine aminotransferase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 5. (Item 10) 7. The therapeutic composition of item 6, wherein the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 6. (Item 11) 7. The therapeutic composition of item 6, wherein the arginine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 7. (Item 12) 7. The therapeutic composition of item 6, wherein the agmatinase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 8. (Item 13) 7. The therapeutic composition of item 6, wherein the ornithine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 9. (Item 14) the at least one purified bacterial population Escherichia coli MG1655, Escherichia coli Nissle 1917, or a combination of these 2. The therapeutic composition of claim 1, comprising a bacterium comprising a 16S rDNA sequence having at least 95% similarity to a reference bacterium selected from the group consisting of: (Item 15) 2. The therapeutic composition of claim 1, wherein the bacterial population consists of bacteria capable of producing GABA at a physiologically relevant pH. (Item 16) 2. The therapeutic composition of claim 1, wherein the bacterial population consists of bacteria capable of producing GABA in a pH range between about 4.5 and about 7.5. (Item 17) 2. The therapeutic composition of claim 1, wherein the bacterial population consists of bacteria capable of producing GABA inside the human intestine. (Item 18) 10. The therapeutic composition of item 1, in the form of a probiotic, a prebiotic, a capsule, a tablet, a caplet, a pill, a troche, a lozenge, a powder, a granule, a medical food, or a combination thereof. (Item 19) 2. The therapeutic composition of item 1, administered as a fecal transplant. (Item 20) 2. The therapeutic composition of claim 1, wherein the bacterium is capable of producing GABA through expression of any combination of glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, and / or ornithine decarboxylase. (Item 21) 2. The therapeutic composition of item 1, further comprising a purified bacterial strain that is cytotoxic or cytostatic to GABA-consuming bacteria. (Item 22) 22. The therapeutic composition of claim 21, wherein the GABA-consuming bacterium is Evtepia gabavorous or Firmicutes bacterium MGS:114. (Item 23) 10. The therapeutic composition of claim 1, further comprising a prebiotic capable of stimulating the growth of GABA-producing bacteria or GABA production levels. (Item 24) A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutic composition comprising at least one purified bacterial population of bacteria capable of producing GABA in the subject in need thereof. (Item 25) 25. The method of item 24, wherein the disease or disorder is a psychiatric disease or disorder. (Item 26) 25. The method of item 24, wherein the psychiatric disease or disorder is selected from the group consisting of depression, bipolar disorder, schizophrenia, anxiety, anxiety disorders, addiction, social phobia, treatment-resistant major depressive disorder (TR-MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), neurodegenerative amyloid disorders (Parkinson's, Alzheimer's, and Huntington's disease), orthostatic tremor, Lafora's disease, restless legs syndrome, neuropathic pain, pain disorders, dementia, epilepsy, stiff-person syndrome, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD), and combinations thereof. (Item 27) 25. The method of item 24, wherein treating a disease or disorder comprises reducing at least one symptom of said disease or disorder such as fatigue, insomnia, motor dysfunction, stress, persistent anxiety, persistent sadness, social withdrawal, substance withdrawal, irritability, suicidal ideation, self-harm ideation, restlessness, decreased libido, lack of concentration, convulsions, memory loss, anger, attacks of emotional response, confusion, pain, and muscle spasms, loss of appetite, altered bowel motility, and combinations thereof. (Item 28) 25. The method according to item 24, wherein the at least one bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 1 to 31 set forth in Table 1. (Item 29) The at least one purified bacterial population consists of bacteria selected from the group consisting of Bacteroides caccae KLE1911; Bacteroides clarus KLE1930; Bacteroides dorei KLE1912; Bacteroides finegoldii KLE1931; Bacteroides fragilis KLE1958; Bacteroides massiliensis KLE1932; Bacteroides ovatus KLE1770; Bacteroides stercoris KLE1933; Bacteroides thetaiotaomicron KLE1934; Bacteroides uniformis KLE1913; Bacteroides vulgatus KLE1910; Bacteroides xylanisolvens KLE1935; Bifidobacterium adolescentis KLE 1879; Blautia obeum KLE1914; Blautia wexlerae KLE1916; Butyricimonas virosa KLE1938; Clostridium perfringens KLE1937; Clostridium sordellii KLE1939; Clostridium sp. KLE1862; Clostridium sp. KLE1918; Coprobacillus sp. KLE1779; Coprococcus sp. KLE1880; Dorea longicatena KLE1917; Eggerthella lenta KLE1926; Eubacterium rectale KLE1922; Gordonibacter pamelaeae KLE1915; Oscillibacter sp. KLE1928; Parabacteroides distasonis KLE2020; Parabacteroides merdae KLE1863; Ruminococcus gnavus KLE1940; Turicibacter sanguinis KLE1941, and combinations thereof, according to the method of item 24. (Item 30) 25. The method according to item 24, wherein the at least one purified bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to a 16S rDNA sequence selected from one of SEQ ID NOs: 32 to 274 set forth in Table 2. (Item 31) 25. The method of item 24, wherein the at least one bacterial population consists of bacteria comprising a 16S rDNA sequence that is at least about 95% identical to any one of SEQ ID NOs: 305 to 2217 set forth in Table 10. (Item 32) 25. The method of claim 24, wherein the at least one purified bacterial population consists of bacteria comprising a DNA sequence encoding an enzyme selected from glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof. (Item 33) 33. The method of item 32, wherein the glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof is encoded by a DNA sequence at least 70% similar to a DNA sequence selected from one of SEQ ID NOs: 275 to 304 set forth in Table 3. (Item 34) 33. The method of item 32, wherein the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 4. (Item 35) 33. The method of item 32, wherein the putrescine aminotransferase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 5. (Item 36) 33. The method of item 32, wherein the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 6. (Item 37) 33. The method of claim 32, wherein the arginine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 7. (Item 38) 33. The method of item 32, wherein the agmatinase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 8. (Item 39) 33. The method of claim 32, wherein the ornithine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 9. (Item 40) 25. The method of claim 24, wherein the bacterium is genetically engineered to produce GABA. (Item 41) 41. The method of claim 40, wherein the bacterium is engineered to produce GABA through expression of glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof. (Item 42) 42. The method of item 41, wherein the glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof is encoded by a DNA sequence at least 70% similar to a DNA sequence selected from one of SEQ ID NOs: 275 to 304 set forth in Table 3. (Item 43) 42. The method of claim 41, wherein the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 4. (Item 44) 42. The method of claim 41, wherein the putrescine aminotransferase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 5. (Item 45) 42. The method of claim 41, wherein the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 6. (Item 46) 42. The method of claim 41, wherein the arginine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 7. (Item 47) 42. The method of claim 41, wherein the agmatinase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 8. (Item 48) 42. The method of claim 41, wherein the ornithine decarboxylase is encoded by a DNA sequence that is at least 95% similar in DNA sequence to a gene having an EMBL / GENBANK / DDBJ ID found in Table 9. (Item 49) the at least one purified bacterial population Escherichia coli MG1655, Escherichia coli Nissle 1917, or a combination of these 25. The method according to item 24, wherein the bacterium comprises a bacterium having a 16S rDNA sequence having at least 95% similarity to a reference bacterium selected from the group consisting of: (Item 50) 25. The method of claim 24, wherein the bacterial population consists of bacteria capable of producing GABA at a physiologically relevant pH. (Item 51) 25. The method of claim 24, wherein the bacterial population consists of bacteria capable of producing GABA in a pH range between about 4.5 and about 7.5. (Item 52) 25. The method of claim 24, wherein the bacterial population consists of bacteria capable of producing GABA inside the human intestine. (Item 53) 25. The method of claim 24, wherein the composition is administered as a fecal transplant. (Item 54) 25. The method of claim 24, wherein the composition is administered as a probiotic. (Item 55) 25. The method of claim 24, wherein the bacterium is capable of producing GABA through expression of any combination of glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, and combinations thereof. (Item 56) 25. The method of claim 24, wherein the at least one bacterial strain is cytotoxic or cytostatic to GABA-consuming bacteria. (Item 57) 57. The method of claim 56, wherein the GABA-consuming bacterium is Evtepia gabavorous or Firmicutes bacterium MGS:114. (Item 58) 25. The method of claim 24, further comprising identifying a subject in need of treatment by determining whether the subject would benefit from increasing endogenous GABA by measuring the initial amount of GABA in the subject's stool. (Item 59) 59. The method of claim 58, wherein the initial amount of GABA in the subject's stool is less than about 8 μg per gram of wet or dry stool. (Item 60) 59. The method of claim 58, wherein the amount of GABA in the subject's stool is increased relative to the initial amount after administering the therapeutic composition. (Item 61) 25. The method of claim 24, further comprising identifying a subject in need of treatment by determining whether the subject would benefit from increasing endogenous GABA by measuring the initial amount of GABA-producing bacteria in the subject's stool. (Item 62) 62. The method of claim 61, wherein the initial amount of GABA-producing bacteria in the subject's stool is less than about 10% of the total bacteria as measured by 16S sequence mapping. (Item 63) 62. The method of claim 61, wherein at least one GABA-producing bacterium is increased in the subject's stool relative to the initial amount of GABA-producing bacteria in the subject's stool after administering the therapeutic composition. (Item 64) 25. The method of claim 24, further comprising identifying a subject in need of treatment by determining whether the subject would benefit from increasing endogenous GABA by measuring the initial amount of GABA in the subject's blood or serum. (Item 65) 65. The method of claim 64, wherein the amount of GABA in the subject's blood or serum is less than about 10 μg per liter of blood. (Item 66) 65. The method of claim 64, wherein the amount of GABA in the subject's blood or serum is increased relative to the initial amount after administration of the therapeutic composition. (Item 67) 25. The method of claim 24, further comprising identifying a subject in need of treatment by determining whether the subject would benefit from increasing endogenous GABA by measuring the amount of GABA in the subject's brain. (Item 68) Item 68. The method of item 67, wherein the amount of GABA in the subject's brain is less than about 1.0 mM / kg. (Item 69) 68. The method of claim 67, wherein the amount of GABA in the subject's brain is increased relative to the initial amount after administration of the therapeutic composition. (Item 70) 25. The method of claim 24, further comprising identifying a subject in need of treatment by determining whether the subject would benefit from an increase in endogenous GABA by measuring an initial amount of GABA-producing enzyme expression in the subject's stool. (Item 71) 71. The method of claim 70, wherein the GABA-producing enzyme is selected from glutamic acid decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, and combinations thereof. (Item 72) 71. The method of claim 70, wherein the initial amount of enzyme expression is measured by qPCR. (Item 73) 71. The method of claim 70, wherein the enzyme expression is increased relative to the initial amount of enzyme expression after administering the therapeutic composition. (Item 74) 25. The method of claim 24, further comprising identifying a subject in need of treatment by determining whether the subject would benefit from an increase in endogenous GABA by measuring an initial amount of GABAergic response in the subject's brain. (Item 75) 75. The method of claim 74, wherein the amount of the GABAergic response in the subject's brain is increased relative to the initial amount after administering the therapeutic composition. (Item 76) 25. The method of claim 24, wherein the therapeutic composition comprises a prebiotic capable of stimulating the growth of GABA-producing bacteria or GABA production. (Item 77) A method for culturing GABA-dependent bacteria, comprising the steps of placing at least one viable GABA-dependent bacterial cell on a suitable substrate and providing a source of GABA. (Item 78) 78. The method of claim 77, wherein the suitable substrate is agar. (Item 79) 78. The method of claim 77, wherein the step of providing a source of GABA comprises co-cultivating with another bacterial strain, said bacterial strain being capable of producing GABA. (Item 80) 78. The method of item 77, wherein GABA is added to the substrate. (Item 81) Item 78. The method of item 77, wherein the GABA-dependent bacterium is E. gabavorous. (Item 82) 1. A method for identifying a bacterial strain or strains capable of producing GABA at the physiologically relevant pH of the human intestinal tract, comprising: (a) dispersing a sample suspected of containing GABA-producing bacteria in a matrix, the matrix being at least partially permeable to GABA; (b) contacting the substrate containing the added potential GABA-producing bacteria with GABA-dependent bacteria; (c) identifying GABA-producing bacteria by observing the formation of colonies of the GABA-dependent bacteria around potential GABA-producing bacteria in the substrate; A method comprising: (Item 83) 83. The method of claim 82, wherein the substrate is buffered to maintain a pH in the physiological range found in the human gastrointestinal tract. (Item 84) Item 83. The method of item 82, wherein the GABA-dependent bacterium is E. gabavorous. (Item 85) 83. The method of claim 82, wherein the pH range is between about 4.5 and about 7.5. [Brief explanation of the drawings]

[0049] [Figure 1] Figure 1A shows a co-culture assay to grow and identify previously uncultivable bacteria.

[0050] FIG. 1B shows the growth of Evtepia gabavorous KLE1738 in the presence of the helper bacterium Bacteroides fragilis KLE1758.

[0051] [Figure 2A] FIG. 2A shows the growth of Evtepia gabavorous KLE1738 in the presence of supernatant from Bacteroides fragilis.

[0052] [Figure 2B] FIG. 2B shows the lack of growth of Evtepia gabavorous KLE1738 in the presence of empty vehicle.

[0053] [Figure 2C] FIG. 2C shows the growth of Evtepia gabavorous in the presence of the most polar fraction of the supernatant from Bacteroides fragilis KLE1758.

[0054] [Figure 2D] FIG. 2D shows a close up view of the lack of growth of Evtepia gabavorous KLE1738 in the presence of empty vehicle.

[0055] [Figure 2E] FIG. 2E shows a close-up view of the growth of Evtepia gabavorous KLE1738 in the presence of supernatant from Bacteroides fragilis KLE1758.

[0056] [Figure 2F] FIG. 2F shows a close up view of the lack of growth of Evtepia gabavorous KLE1738 in the presence of empty vehicle (bacterial medium).

[0057] [Figure 2G] Figure 2G shows a table of potential growth factors for E. gabavorous KLE1738, with green indicating growth induction.

[0058] [Figure 3] Figure 3 shows the proposed GABA metabolism of E. gabavorous KLE1738.

[0059] [Figure 4A] Figure 4a shows that GABA production in B. fragilis KLE1758 is only observed at a pH of 5.5 and lower, highlighting the importance of an acidic pH for GABA production for certain strains or species of bacteria.

[0060] [Figure 4B] Figure 4B shows an assay for identifying GABA-producing bacteria capable of producing GABA at pH values ​​between 4.5 and 7.5, using the GABA requirement of E. gabavorus KLE1738. The medium is highly buffered to maintain the desired pH range and identify bacterial strains capable of producing GABA at physiological pH values ​​equivalent to those in the human intestine.

[0061] [Figure 4C] FIG. 4C shows a phylogenetic tree of GABA-producing strains identified by the methods herein that are capable of producing GABA at a pH of about 4.5 to about 7.5.

[0062] [Figure 5] Figure 5 shows the GABA production capacity of several strains identified using the screen described in Example 7. The ability of the organisms to produce GABA under a range of pH conditions is highlighted.

[0063] [Figure 6A] FIG. 6A shows the growth of E. gabavorous KLE1738 in the presence of E. coli engineered to express glutamic acid decarboxylase gadA.

[0064] [Figure 6B]FIG. 6B shows the growth of E. gabavorous KLE1738 in the presence of E. coli engineered to express glutamic acid decarboxylase gadB.

[0065] [Figure 6C] Figure 6C shows the growth of E. gabavorous KLE1738 in the presence of B. fragilis KLE1758, which is known to produce GABA.

[0066] [Figure 6D] FIG. 6D shows the absence of growth of E. gabavorous KLE1738 in the presence of E. coli engineered to express glutamic acid decarboxylase gadC.

[0067] [Figure 6E] FIG. 6E shows the absence of growth of E. gabavorous KLE1738 in the presence of the empty vector. DETAILED DESCRIPTION OF THE INVENTION

[0068] Detailed Description of the Invention The present disclosure relates to compositions and methods for treating or reducing the symptoms of a disease in a subject. The disease can be a psychiatric disease or a disease of the central nervous system. After identifying a subject with a psychiatric disease or a disease of the central nervous system, the method can include determining whether the subject will benefit from increasing endogenous GABA, for example, by measuring the amount of GABA in the subject's stool, blood, serum, or other body fluids, measuring the GABA level in various brain regions, measuring the GABAergic response in various brain regions, measuring the activity of GABA-producing enzymes in stool, or measuring the amount of GABA-producing bacteria in the subject's stool. The method can further include administering to the subject GABA-producing bacteria (single or multiple bacteria) that may be able to produce GABA in the subject's intestine (for example, at the physiologically appropriate pH of the intestine).

[0069] Some bacteria produce GABA from gamma-aminobutyrate to maintain intracellular pH homeostasis and overcome acid stress.As shown herein, the production of GABA by microorganisms (e.g., bacteria) in the human intestine can affect the health of the subject.For example, the GABA produced by bacteria in the human intestine can act as a neurotransmitter to treat psychiatric disorders, central nervous system disorders, or improve the gastrointestinal health of the subject. definition

[0070] As used herein, "administering" and "administration" encompass embodiments in which one person directs another person to consume bacteria or a bacterial composition in a particular manner and / or for a particular purpose, as well as situations in which a user uses bacteria or a bacterial composition in a particular manner and / or for a particular purpose, regardless of or contrary to any instructions received from the second person. Non-limiting examples of embodiments in which one person directs another person to consume bacteria or a bacterial composition in a particular manner and / or for a particular purpose include when a physician prescribes a course of action and / or treatment to a patient, when a parent instructs a minor user (such as a child) to consume bacteria or a bacterial composition, when a trainer encourages a user (such as an athlete) to follow a particular action and / or treatment, and when a manufacturer, distributor, or seller recommends conditions of use to the end consumer, for example, by advertising or labeling on packaging or other materials provided in connection with the sale or purchase of a product.

[0071] The term "isolated" encompasses bacteria or other entities or substances that (1) have been separated from at least some of the components with which they are associated when originally produced (in nature, such as human stool, or in a laboratory setting, such as a Petri plate of artificial growth medium) and / or (2) have been produced, prepared, purified, and / or manufactured by the hand of man. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they are originally associated. In some embodiments, isolated bacteria are greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components (such as other bacterial species). The terms "purify," "purifying," and "purified," as recognized by those skilled in the art of bacterial culture, refer to bacteria or other materials that have been separated from at least some of the associated components, either when originally produced or generated (e.g., whether naturally occurring or generated in an experimental setting), or any time after initial production. A bacterium or bacterial population may be considered purified, for example, if it is isolated during or after production from the material or environment containing the bacterium or bacterial population; a purified bacterium or bacterial population may contain up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90% other materials and still be considered "isolated." In some embodiments, purified bacteria and bacterial populations are greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. In the example bacterial compositions provided herein, one or more bacterial types present in the composition can be purified independently from one or more other bacteria present in the material or environment in which it was produced and / or contains that cell type. Bacterial compositions and their bacterial components are generally purified from residual habitat products.

[0072] As used herein, "probiotics" is understood to mean "living microorganisms that, when administered in adequate amounts, confer a health benefit on the host," as currently defined by the World Health Organization.

[0073] As used herein, "prebiotics" is understood to mean ingredients that specifically alter the composition and / or activity of the gastrointestinal flora, which may (or may not) confer a benefit to the host.

[0074] As used herein, "medical diet," as defined by Section 5(b) of the Orphan Drug Act (21 U.S.C. 360ee(b)(3)), is understood to mean "a food formulated to be consumed or administered enterally under physician supervision and intended for the specific dietary management of a disease or condition whose distinctive nutritional requirements have been established by medical evaluation based on recognized scientific principles."

[0075] As used herein, "initial amount" is understood to mean the amount of a substance, e.g., GABA, in an aliquot of a sample before administration of GABA-producing bacteria to a subject. The initial amount can be measured in terms of concentration. For example, the initial amount can be measured in terms of micrograms of substance per milliliter of sample, e.g., micrograms of GABA per milliliter of blood or serum (μg GABA / mL blood or serum). The initial amount can also be measured, for example, as the amount of GABA in a brain region, such as the prefrontal cortex, before administration of GABA-producing bacteria. The amount of GABA can be expressed in terms of millimoles of GABA per kg tissue (mmol GABA per kg brain tissue). The initial amount can also be measured, for example, as the amount of GABA in a stool sample from a subject before administration of GABA-producing bacteria to the subject. The amount of GABA can be expressed in terms of micrograms of GABA per gram of stool (μg GABA / g stool). The initial amount can also be the expression level (logarithmic change in reading) of a GABA-producing enzyme in stool, measured by qPCR or other suitable method. Unless otherwise defined herein, stool is weighed within 1 hour of production when wet or dry, i.e., without active drying. For example, stool can be weighed within 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes of production.

[0076] As used herein, "GABAergic response" refers to the response of a given organ (e.g., the brain or vagus nerve) to differences in the concentration of GABA, GABA-producing bacteria, or prebiotics to which the given organ is exposed. GABAergic responses include changes in the concentration of GABA as well as the activity of various GABAs. A , GABA B , and / or GABA C The expression level and / or activity of the receptor may be included.

[0077] "GABA-producing bacteria" is understood to mean bacteria capable of producing measurable amounts of GABA as detected by LC / MS, ELISA, or other suitable analytical assays. In some embodiments, the GABA-producing bacteria are capable of producing GABA under human physiological conditions, e.g., the pH and temperature of the human intestine.

[0078] The "physiologically relevant pH" of the human intestinal tract is understood to mean the pH range present in the body. For example, the pH range that is physiologically relevant for the human intestine may be within the range of about 4.5 to about 7.5.

[0079] The term "intestine" is understood to refer to the human gastrointestinal tract, also known as the digestive tract, which includes the oral cavity, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum and colon), and rectum.

[0080] As used herein, "bacteria" or "bacterial strain" is understood to mean a species of bacteria. "bacterium" is understood as a single bacterial cell of a given species.

[0081] With respect to a subject, the term "treating" refers to improving at least one symptom of the subject's disorder. Treating includes curing, improving, or at least partially alleviating the disorder.

[0082] The term "EMBL / GenBank / DDBJ ID" refers to an accession number that, when entered and used in relation to databases such as the European Molecular Biology Laboratory (EMBL), GenBank, or the DNA Data Bank of Japan (DDBJ) via their respective internet websites, allows access to information such as the nucleotide sequence of a gene and the bacterium that encodes that sequence in its genome. EMBL / GenBank / DDBJ ID is used in this application as a convenient means of accessing sequence information. Gamma-aminobutyric acid (GABA)

[0083] The term "GABA" is understood to mean gamma-aminobutyric acid (γ-aminobutyric acid). GABA has the chemical structure: [ka] It has.

[0084] GABA is the major inhibitory neurotransmitter in the mammalian central nervous system. GABA plays a major role in reducing neuronal excitability throughout the nervous system. GABA can be a challenging compound to deliver therapeutically due to efflux and half-life limitations. For example, in rodents, the brain efflux of GABA was found to be 17-fold higher than the influx. Furthermore, GABA has a half-life of only 17 minutes in mice. Therefore, oral GABA supplementation may be ineffective because frequent administration may be required due to the short half-life of GABA in vivo, even with sustained-release capsules.

[0085] The present disclosure provides for the delivery of one or more therapeutic compositions of bacteria capable of producing GABA in the intestinal tract, in order to consistently deliver GABA to the systemic circulation (e.g., to the nervous system).Endogenously produced GABA can also directly activate vagus nerve receptors.This can reduce the natural half-life of GABA.

[0086] In some embodiments, microbiome can affect GABA levels and GABAergic responses in the brain.For example, germ-free animals may have substantially reduced luminal and serum GABA levels.Without wishing to be bound by theory, this suggests that microbiome is important in regulating the level of this important neurotransmitter.As shown herein, GABAergic modulation by microbiome intervention (for example, using the methods and compositions described herein) may have therapeutic efficacy.

[0087] In some embodiments, GABA can play a role in psychiatric illness or central nervous system disorders.For example, in some embodiments, low GABA levels may be associated with depression, bipolar disorder, schizophrenia, anxiety, anxiety disorders, addiction, social phobia, treatment-resistant major depressive disorder (TR-MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), neurodegenerative amyloid disorders (Parkinson's, Alzheimer's, and Huntington's disease), orthostatic tremor, Lafora's disease, restless legs syndrome, neuropathic pain, pain disorders, dementia, epilepsy, stiff-person syndrome, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD).As shown herein, the present disclosure provides for increasing the amount of endogenous GABA in a subject, and can reduce the level of a subject's psychiatric illness or central nervous system disorders.

[0088] In some embodiments, GABA produced by gut bacteria may play a role in psychiatric or central nervous system disorders via the vagus nerve, which connects the gut to the peripheral and central nervous systems.

[0089] In some embodiments, GABA produced by gut bacteria may play a role in psychiatric or central nervous system disorders by affecting circulating levels of GABA throughout the host's body, which may affect the peripheral and central nervous systems. Microbial means of producing GABA

[0090] Bacteria can produce GABA using a variety of different pathways. Representative pathways that bacteria and other microorganisms can use to produce GABA (e.g., in As shown below, any of the GABA production pathways described herein may occur naturally in a given bacterium. Alternatively, the necessary enzyme or enzymes can be added to the bacterium's DNA sequence to enable the bacterium to produce GABA. Glutamate pathway

[0091] In some embodiments, the microorganism can produce GABA using a glutamate decarboxylase enzyme (e.g., glutamate decarboxylase EC 4.1.1.15). In some embodiments, the glutamate decarboxylase can directly convert glutamate to GABA. A route to 4-aminobutanal from putrescine

[0092] In some embodiments, the microorganism can produce GABA using a putrescine to 4-aminobutanal pathway. The microorganism can then convert 4-aminobutanal to GABA. In some embodiments, putrescine can be converted to 4-aminobutanal using a putrescine aminotransferase (e.g., putrescine aminotransferase EC 2.6.1.82). 4-aminobutanal can then be converted to GABA in the presence of a gamma-aminobutyraldehyde dehydrogenase (e.g., gamma-aminobutyraldehyde dehydrogenase (EC 1.2.1.19)). Pathway of arginine to agmatine and then to putrescine

[0093] In some embodiments, the microorganism can produce GABA using the arginine to agmatine and then to putrescine pathway. Once putrescine is produced, putrescine can be converted to GABA as described above (e.g., using the putrescine to 4-aminobutanal pathway). In some embodiments, an arginine decarboxylase (e.g., arginine decarboxylase (EC 4.1.1.19)) can convert arginine to agmatine. Agmatine can then be converted to putrescine using an agmatinase (e.g., agmatinase (EC 3.5.3.11)). L-ornithine pathway to putrescine In some embodiments, ornithine decarboxylase (e.g., ornithine decarboxylase (EC 4.1.1.17)) can be used to convert ornithine to putrescine. Once putrescine is produced, it can be converted to GABA as described above (e.g., using the putrescine to 4-aminobutanal pathway). Bacterial strains

[0094] The present disclosure provides bacterial strains (e.g., purified strains) and therapeutic compositions comprising the bacterial strains for administration to a subject in need thereof. The bacteria can be naturally occurring or can be engineered to produce GABA (e.g., through strain engineering or selection). In some embodiments, a single strain of GABA-producing bacteria can be administered to a subject. In some embodiments, multiple strains of GABA-producing bacteria can be administered to a subject in need thereof. In some embodiments, one or more bacteria (e.g., purified bacteria) can act synergistically. For example, multiple bacteria can act synergistically to produce high levels of GABA. In some embodiments, one or more bacteria can also help reduce the number of GABA-consuming bacteria in the human gut. Thus, any one or any combination of the GABA-producing bacteria taught herein can be administered to a subject in need thereof.

[0095] In some embodiments, the bacteria taught herein can produce GABA under physiologically relevant conditions, such as those found in the human intestine. In some embodiments, the GABA-producing bacteria taught herein can produce GABA at a pH suitable for the human intestine between about 4.5 and about 7.5. For example, the pH can be about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or any value between about 4.5 and 7.5.

[0096] The ability to produce GABA at a physiologically relevant pH is important given the pH limitations of GABA production in many bacteria. For example, E. coli cannot produce GABA above pH 4.5. Alternatively, without wishing to be bound by theory, E. coli may use, for example, the conversion of glutamate to GABA coupled with GABA export as a means to neutralize the intracellular environment. Thus, the present disclosure provides bacteria capable of producing GABA at a pH appropriate for the host environment, such as the more neutral pH of the large intestine (e.g., between about pH 4.5 and about pH 7.5). In some embodiments, the bacteria taught herein can also produce GABA under other appropriate conditions present in the human intestine. That is, the GABA-producing bacteria taught herein can produce GABA in the absence of oxygen, in a competitive and variable nutrient environment, and in the absence of light. Natural strains

[0097] In some embodiments, GABA-producing bacteria can be identified by having a 16S nucleic acid sequence substantially similar to the 16S sequence of a reference bacterium listed in Table 1 and having SEQ ID NOs: 1-31. In some embodiments, GABA-producing bacteria can have at least 90% 16S sequence similarity to a 16S sequence given in Table 1 (e.g., at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, at least 99% similarity, at least 99.5% similarity, at least 99.9% similarity, or 100% similarity).

[0098] In some embodiments, GABA-producing bacteria can be identified by the NCBI Taxon ID of the organism to which they are most related by 16S sequence. These bacteria include Bacteroides caccae KLE1911;Bacteroides clarus KLE1930;Bacteroides dorei KLE1912;Bacteroides finegoldii KLE1931;Bacteroides fragilis KLE1958;Bacteroides massiliensis KLE1932;Bacteroides ovatus KLE1770;Bacteroides stercoris KLE1933;Bacteroides thetaiotaomicron KLE1934;Bacteroides uniformis KLE1913;Bacteroides vulgatus KLE1910;Bacteroides xylanisolvens KLE1935;Bifidobacterium adolescentis KLE 1879;Blautia obeum KLE1914;Blautia wexlerae KLE1916;Butyricimonas virosa KLE1938;Clostridium perfringens KLE1937;Clostridium sordellii KLE1939;Clostridium sp. KLE1862;Clostridium sp. KLE1918;Coprobacillus sp. KLE1779;Coprococcus sp. KLE1880;Dorea longicatena KLE1917;Eggerthella lenta KLE1926;Eubacterium rectale KLE1922;Gordonibacter pamelaeae KLE1915;Oscillibacter sp. KLE1928;Parabacteroides distasonis KLE2020;Parabacteroides merdae KLE1863;Ruminococcus gnavus KLE1940;Turicibacter sanguinis KLE1941, and combinations thereof that belong to the same NCBI Taxon assignment. [Table 1]

[0099] Also disclosed herein are bacteria predicted to be capable of producing GABA (e.g., under physiologically relevant conditions and / or in the human intestine). Bacteria are identified as potential GABA-producing bacteria if their genomes encode enzymes involved in GABA biosynthesis. In some embodiments, bacteria predicted to be capable of producing GABA can be identified by having a 16S nucleic acid sequence substantially similar to the 16S sequence of a reference bacterium listed in Table 2 and having SEQ ID NOs: 32-274. In some embodiments, predicted GABA-producing bacteria may have at least 90% 16S sequence similarity (e.g., at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, at least 99% similarity, at least 99.5% similarity, at least 99.9% similarity, or 100% similarity) to the 16S sequences given in Table 2 and having SEQ ID NOs: 32-274. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] Engineered strains

[0100] In some embodiments, bacteria can be engineered to produce GABA (e.g., under human intestinal conditions). Bacteria can be engineered using molecular biology techniques or can be evolved using the process of selection to produce GABA in the human intestine.

[0101] As noted above, GABA can be produced by multiple pathways within microbial cells. For example, GABA can be produced by the glutamate pathway, the putrescine to 4-aminobutanal pathway, the arginine to agmatine to putrescine pathway, the L-ornithine to putrescine pathway, or a combination of pathways. In some embodiments, bacteria can be engineered to contain one or more enzymes in any one of the above pathways that can enable the bacteria to produce GABA or the precursors required for GABA.

[0102] A variety of different host bacteria can be engineered to produce GABA. For example, in some embodiments, Escherichia coli Nissle 1917 can be genetically modified or evolutionarily selected to produce GABA. In some embodiments, bacteria (e.g., Escherichia coli Nissle 1917) can be engineered to express or overexpress glutamate decarboxylase A or glutamate decarboxylase B. Bacteria can also be engineered to produce GABA by one or more of the other pathways described herein.

[0103] Thus, in some embodiments, an engineered GABA-producing strain can be identified as having a specific enzyme encoded in its genome, for example, the enzyme can be glutamate decarboxylase (EC 4.1.1.15), putrescine aminotransferase (EC 2.6.1.82), gamma-aminobutyraldehyde dehydrogenase (EC 1.2.1.19), arginine decarboxylase (EC 4.1.1.19), agmatinase (EC 3.5.3.11), ornithine decarboxylase (EC 4.1.1.17), or a combination thereof. In some embodiments, GABA-producing strains can be engineered to contain enzymes having at least 50% similarity (e.g., at least 60% similarity, at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, at least 99% similarity, at least 99.5% similarity, at least 99.9% similarity, or 100% similarity) to the representative sequences listed in Table 3. The enzyme classes are identified by their Enzyme Commission (EC) numbers and are listed in Table 3. [Table 3]

[0104] Representative examples of glutamate decarboxylases (EC 4.1.1.15) are provided below in Table 4 and are identified by their EMBL / GENBANK / DDBJ ID numbers. Any of the bacteria provided in Table 10 can be engineered with any version of glutamate decarboxylase shown in Table 3 or Table 4. For example, a bacterium can be engineered with a version of a glutamic acid decarboxylase enzyme that has at least 50% nucleotide similarity (e.g., at least 60% nucleotide similarity, at least 70% nucleotide similarity, at least 80% nucleotide similarity, at least 90% nucleotide similarity, at least 91% nucleotide similarity, at least 92% nucleotide similarity, at least 93% nucleotide similarity, at least 94% nucleotide similarity, at least 95% nucleotide similarity, at least 96% nucleotide similarity, at least 97% nucleotide similarity, at least 98% nucleotide similarity, at least 99% nucleotide similarity, at least 99.5% nucleotide similarity, at least 99.9% nucleotide similarity, or 100% nucleotide similarity) to any of the glutamic acid decarboxylase versions provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12]

[0105] Representative examples of putrescine aminotransferases (EC 2.6.1.82) are provided below in Table 5 and are identified by their EMBL / GenBank / DDBJ ID numbers. Any of the bacteria provided in Table 10 can be engineered with any version of putrescine aminotransferase (EC 2.6.1.82) shown in Tables 3 and 5. For example, a bacterium can be engineered with a version of a putrescine aminotransferase enzyme that has at least 50% nucleotide similarity (e.g., at least 60% nucleotide similarity, at least 70% nucleotide similarity, at least 80% nucleotide similarity, at least 90% nucleotide similarity, at least 91% nucleotide similarity, at least 92% nucleotide similarity, at least 93% nucleotide similarity, at least 94% nucleotide similarity, at least 95% nucleotide similarity, at least 96% nucleotide similarity, at least 97% nucleotide similarity, at least 98% nucleotide similarity, at least 99% nucleotide similarity, at least 99.5% nucleotide similarity, at least 99.9% nucleotide similarity, or 100% nucleotide similarity) to any of the putrescine aminotransferase versions provided in Table 5. [Table 5-1] [Table 5-2]

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

[0106] Representative examples of gamma-aminobutyraldehyde dehydrogenases (EC 1.2.1.19) are provided below in Table 6 and are identified by their EMBL / GENBANK / DDBJ ID numbers. Any of the bacteria provided in Table 10 can be engineered with any version of gamma-aminobutyraldehyde dehydrogenase (EC 1.2.1.19) shown in Tables 3 and 6. For example, a bacterium can be engineered with a version of the gamma-aminobutyraldehyde dehydrogenase enzyme that has at least 50% nucleotide similarity (e.g., at least 60% nucleotide similarity, at least 70% nucleotide similarity, at least 80% nucleotide similarity, at least 90% nucleotide similarity, at least 91% nucleotide similarity, at least 92% nucleotide similarity, at least 93% nucleotide similarity, at least 94% nucleotide similarity, at least 95% nucleotide similarity, at least 96% nucleotide similarity, at least 97% nucleotide similarity, at least 98% nucleotide similarity, at least 99% nucleotide similarity, at least 99.5% nucleotide similarity, at least 99.9% nucleotide similarity, or 100% nucleotide similarity) to any of the gamma-aminobutyraldehyde dehydrogenase versions provided in Table 6. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10]

[0107] Representative examples of arginine decarboxylases (EC 4.1.1.19) are provided below in Table 7 and are identified by their EMBL / GENBANK / DDBJ ID numbers. Any of the bacteria provided in Table 10 can be engineered with any version of arginine decarboxylases (EC 4.1.1.19) shown in Tables 3 and 7. For example, a bacterium can be engineered with a version of an arginine decarboxylase enzyme that has at least 50% nucleotide similarity (e.g., at least 60% nucleotide similarity, at least 70% nucleotide similarity, at least 80% nucleotide similarity, at least 90% nucleotide similarity, at least 91% nucleotide similarity, at least 92% nucleotide similarity, at least 93% nucleotide similarity, at least 94% nucleotide similarity, at least 95% nucleotide similarity, at least 96% nucleotide similarity, at least 97% nucleotide similarity, at least 98% nucleotide similarity, at least 99% nucleotide similarity, at least 99.5% nucleotide similarity, at least 99.9% nucleotide similarity, or 100% nucleotide similarity) to any of the arginine decarboxylase versions provided in Table 7. [Table 7-1] [Table 7-2] [Table 7-3]

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

[0108] Representative examples of agmatinases (EC 3.5.3.11) are provided below in Table 8 and are identified by their EMBL / GENBANK / DDBJ ID numbers. Any of the bacteria provided in Table 10 can be engineered with any version of agmatinase (EC 3.5.3.11) shown in Tables 3 and 8. For example, a bacterium can be engineered with a version of the agmatinase enzyme that has at least 50% nucleotide similarity (e.g., at least 60% nucleotide similarity, at least 70% nucleotide similarity, at least 80% nucleotide similarity, at least 90% nucleotide similarity, at least 91% nucleotide similarity, at least 92% nucleotide similarity, at least 93% nucleotide similarity, at least 94% nucleotide similarity, at least 95% nucleotide similarity, at least 96% nucleotide similarity, at least 97% nucleotide similarity, at least 98% nucleotide similarity, at least 99% nucleotide similarity, at least 99.5% nucleotide similarity, at least 99.9% nucleotide similarity, or 100% nucleotide similarity) to any of the agmatinase versions provided in Table 8. [Table 8-1]

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

[0109] Representative examples of ornithine decarboxylases (EC 4.1.1.17) are provided below in Table 9 and are identified by their EMBL / GENBANK / DDBJ ID numbers. Any of the bacteria provided in Table 10 can be engineered with any version of ornithine decarboxylases (EC 4.1.1.17) shown in Tables 3 and 9. For example, a bacterium can be engineered with a version of an ornithine decarboxylase enzyme that has at least 50% nucleotide similarity (e.g., at least 60% nucleotide similarity, at least 70% nucleotide similarity, at least 80% nucleotide similarity, at least 90% nucleotide similarity, at least 91% nucleotide similarity, at least 92% nucleotide similarity, at least 93% nucleotide similarity, at least 94% nucleotide similarity, at least 95% nucleotide similarity, at least 96% nucleotide similarity, at least 97% nucleotide similarity, at least 98% nucleotide similarity, at least 99% nucleotide similarity, at least 99.5% nucleotide similarity, at least 99.9% nucleotide similarity, or 100% nucleotide similarity) to any of the ornithine decarboxylase versions provided in Table 9. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7]

[0110] Various microorganisms (e.g., bacteria) can be engineered to produce GABA (e.g., by engineering one or more of the enzymes listed in Table 2). For example, any of the bacteria listed in Table 10 can be engineered to produce GABA. That is, bacteria having a 16S rDNA nucleotide sequence at least 50% similar to that listed in Table 10 below can be engineered to produce GABA (e.g., using one of the enzymes listed in Tables 3-9). The bacteria may have a 16S rDNA sequence that is at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 91% similar, at least 92% similar, at least 93% similar, at least 94% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar, at least 99% similar, at least 99.5% similar, or 100% similar to the 16S rDNA nucleotide sequence listed in Table 10.

[0111] As shown in Example 8, E. coli was engineered to overexpress glutamic acid decarboxylase, and without wishing to be bound by theory, this led to the expression of GABA by the engineered E. coli. As shown in Figure 6, the engineered E. coli was able to induce the growth of E. gabavorous KLE1738. GABA can be produced by intestinal epithelial cells and some bacteria, such as Escherichia coli and Listeria monocytogenes, by the decarboxylation of glutamate. In E. coli, GABA production generally occurs at low pH, in some embodiments, because the decarboxylation of glutamate can act as a mechanism to lower intracellular pH.

[0112] As shown in Example 8, expression, e.g., overexpression, of an E. coli glutamate decarboxylase (e.g., gadA or gadB) induces E. gabavorous KLE1738 growth to levels seen for B. fragilis KLE1758. For example, Figure 6A shows that E. gabavorous KLE1738 is induced in the presence of E. coli engineered to express glutamate decarboxylase gadA. Similarly, Figure 6B shows that E. gabavorous KLE1738 is induced in the presence of E. coli engineered to express glutamate decarboxylase gadB. As shown in Figure 6C, growth of E. gabavorous KLE1738 was qualitatively similar to that seen in the presence of B. fragilis KLE1758. In contrast, as shown in Figure 6D, when E. coli was engineered to express the GABA antiporter gadC, no growth of E. gabavorous KLE1738 was observed. Similarly, no growth of E. gabavorous KLE1738 was observed in the presence of empty vehicle (Figure 6E). Without wishing to be bound by theory, the results of Example 8 demonstrate that bacteria can be engineered to produce GABA (e.g., via expression or overexpression of glutamic acid decarboxylase). In some embodiments, bacteria (e.g., E. coli) can be engineered to produce GABA inside the human intestine.

[0113] In some embodiments, the present disclosure also provides for disrupting one or more repressors of GABA production (e.g., gadX or gadW). In some embodiments, these repressors can regulate the pH limitation of GABA production in E. coli, a method for increasing native GABA production. This can be accomplished, for example, by gene deletion, insertion, or substitution, as known by those skilled in the art of molecular biology.

[0114] Altering the pH of the growth medium for KLE1738 did not alter the GABA-dependent phenotype. Without wishing to be bound by theory, this suggests that engineering bacteria to overexpress glutamic acid decarboxylase is an effective way to produce GABA and induce growth of E. gabavorous.

[0115] In addition to E. coli, other bacteria can be engineered to produce GABA (e.g., at a physiologically relevant pH, e.g., between 4.5 and 7.5). For example, any of the bacteria listed in Table 10 can be engineered to produce GABA (e.g., at a physiologically relevant pH, e.g., between 4.5 and 7.5). For example, bacteria can be engineered to contain DNA encoding one or more of the enzymes listed in Tables 3-9. Sequence ID numbers for the 16S nucleotide sequences of the listed bacteria are also provided in Table 10. In some embodiments, a bacterium that has been engineered to produce GABA may have at least 90% 16S sequence similarity to a 16S sequence provided in Table 10 (e.g., at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, at least 99% similarity, at least 99.5% similarity, at least 99.9% similarity, or 100% similarity). [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5]

Table 10-6

Table 10-7

Table 10-8

Table 10-9

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 10-17

Table 10-18

Table 10-19

Table 10-20

Table 10-21

Table 10-22

[0116] Any of the GABA-producing bacteria described herein (e.g., naturally occurring or engineered bacteria), or any combination thereof (including combinations of naturally occurring and engineered bacteria), can be added to a therapeutic composition. For example, the therapeutic composition can be administered to a patient in need thereof to treat or alleviate symptoms of a psychiatric illness or a disease of the central nervous system. Strain purification

[0117] In some embodiments, the bacteria are purified before being added to the therapeutic composition. For example, the bacteria can be purified so that the population of bacteria is substantially free of other bacteria (e.g., contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99% of the specific bacterial strain(s) desired in the composition).

[0118] In some embodiments, the therapeutic composition is a probiotic or medical food containing at least one GABA-producing bacterial strain. The bacterial strain can be administered, for example, as a probiotic, in the form of capsules, tablets, caplets, pills, troches, lozenges, powders, and / or granules. The bacterial strain can also be formulated as a medical food. The GABA-producing bacteria can also be administered as a fecal transplant or suppository.

[0119] In some embodiments, the dose of the therapeutic agent is 1×10 4 , 1×10 5 , 1×10 6 , 1×107 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 or 1×10 11 It can contain more colony forming units (CFU) of a desired bacterial species. For example, the desired bacterial species can be a GABA-producing bacterium, a bacterium capable of inhibiting the growth of a GABA-consuming bacterium, or a combination thereof.

[0120] In some embodiments, the therapeutic composition or dosage unit is provided in a pharmaceutically acceptable formulation comprising an enteric coating or the like that allows it to survive the acidity of the stomach and be delivered to the small or large intestine, prebiotics (such as, but not limited to, amino acids (including arginine, glutamate, and ornithine), biotin, fructooligosaccharides, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, The therapeutic composition may comprise tagatose, resistant maltodextrin (e.g., resistant starch), transgalactooligosaccharides, pectins (e.g., xylogalacturonan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soybean fiber, sugar beet fiber, pea fiber, corn bran, and oat fiber) and xylooligosaccharides, polyamines (e.g., but not limited to, spermidine and putrescine), an effective amount of an antibacterial, antifungal, antiviral, or antiparasitic agent, or any combination of the above. For example, the therapeutic composition may be in the form of yogurt containing one or more purified strains of GABA-producing bacteria. Disease indication

[0121] In one or more embodiments of any of the above aspects, the psychiatric or central nervous system disease that can be treated by administration of a therapeutic composition described herein is selected from depression, bipolar disorder, schizophrenia, anxiety, anxiety disorders, addiction, social phobia, major depressive disorder, treatment-resistant major depressive disorder (TR-MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), neurodegenerative amyloid disorders (Parkinson's, Alzheimer's, and Huntington's disease), orthostatic tremor, Lafora's disease, restless legs syndrome, neuropathic pain, pain disorders, dementia, epilepsy, stiff-person syndrome, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention-deficit hyperactivity disorder (ADHD).

[0122] In some embodiments, the method further comprises reducing at least one symptom of the psychiatric disorder or disease of the central nervous system in the subject selected from the group consisting of fatigue, insomnia, motor dysfunction, stress, persistent anxiety, persistent sadness, social withdrawal, substance withdrawal, irritability, suicidal ideation, self-harm ideation, restlessness, decreased libido, lack of concentration, loss of appetite, convulsions, memory loss, anger, fits of emotional response, confusion, pain, and muscle spasms. Treatment method

[0123] The therapeutic compositions described herein can be administered to a patient in need thereof, for example, for the treatment of a psychiatric illness or a disease of the central nervous system. In some embodiments, the treatment method can include first diagnosing a patient who could benefit from treatment with a therapeutic composition described herein. In some embodiments, the method further includes administering to the patient a therapeutic composition described herein. Patient diagnosis

[0124] In some embodiments, the process of identifying a subject with a psychiatric illness or central nervous system disorder can be performed by a trained psychologist, psychiatrist, or neurologist. For example, a psychiatrist, psychologist, or neurologist can assess a subject's behavior for symptoms of a psychiatric illness or central nervous system disorder to diagnose a subject with a psychiatric illness or central nervous system disorder. Those skilled in the art will appreciate the diagnostic and statistical manual of mental disorders. It will also be appreciated that the subject's psychiatric illness may be identified with the aid of the Statistical Manual of Mental Disorders (DSM-5), (American Psychiatric Association).

[0125] In one or more embodiments, the process of identifying a subject with a psychiatric illness or a central nervous system disorder can also include diagnosing the subject with a psychiatric illness or a central nervous system disorder. In some embodiments, the psychiatric illness or central nervous system disorder is identified or diagnosed using fMRI. In some embodiments, the psychiatric illness or central nervous system disorder can be identified using standard psychological and neurological investigations or by other methods known to those skilled in the art.

[0126] In some embodiments, a subject in need of treatment with a therapeutic composition described herein can be identified by identifying low GABA levels in the subject's blood, serum, stool, or other bodily fluids. In some embodiments, the amount of GABA in the subject's stool (e.g., the initial amount of GABA in the subject's stool) is less than about 8 μg GABA per gram of stool. The amount of GABA can be measured using wet or dry weight of stool by LC / MS or another technique known in the art. In some embodiments, the amount of GABA in the subject's blood or serum (e.g., the initial amount of GABA in the subject's blood or serum) is less than about 10 μg / L + / - 5 μg / L GABA per gram of blood or serum (e.g., as measured by LC / MS). In some embodiments, the amount of GABA in the prefrontal cortex or other areas of the brain is less than about 1.0 mM / kg as measured by proton magnetic resonance (PMR) or another similar technique.

[0127] In some embodiments, the proportion (e.g., initial amount) of GABA-producing bacteria in the subject's intestine represents about 10% of all 16S sequences, as measured by sequencing using methods such as 16S rDNA gene Illumina sequencing or quantitative PCR. In some embodiments, the proportion of GABA-producing bacteria in the subject's intestine represents about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than about 1% of all 16S sequences measured in the subject's intestine.

[0128] Determining the initial amount of GABA in a subject's blood, serum, brain region, or stool can help identify subjects who can benefit from treatment with GABA-producing bacteria.In some embodiments, subjects whose initial amount of GABA in serum or blood is less than 10 μg GABA per L can benefit from the administration of GABA-producing bacteria.In some embodiments, subjects whose initial amount of GABA in serum or blood is less than 100 μg, less than 50 μg, less than 25 μg, less than 20 μg, less than 15 μg, less than 10 μg, less than 9 μg, less than 8 μg, less than 7 μg, less than 6 μg, less than 5 μg, less than 4 μg, less than 3 μg, less than 2 μg, less than 1 μg, less than 0.5 μg, less than 0.1 μg, less than 0.01 μg, less than 10 ng, or less than 1 ng, or less than 0.1 ng can benefit from the administration of GABA-producing bacteria.

[0129] In some embodiments, subjects with initial brain GABA levels of about 1.0 mM / kg in regions such as the prefrontal cortex (or other areas of the brain) can benefit from treatment with GABA-producing bacteria. In some embodiments, subjects with initial brain GABA levels of less than 100 mM, less than 50 mM, less than 25 mM, less than 20 mM, less than 15 mM, less than 10 mM, less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, less than 0.5 mM, less than 0.1 mM, less than 0.01 mM, or less than 0.001 mM in regions such as the prefrontal cortex (or other areas of the brain) can benefit from treatment with GABA-producing bacteria.

[0130] In some embodiments, subjects with an initial stool GABA level of less than 8 μg GABA per gram of stool (wet or dry weight) can benefit from the administration of GABA-producing bacteria. In some embodiments, subjects with an initial stool GABA level of less than 100 μg, less than 50 μg, less than 25 μg, less than 20 μg, less than 15 μg, less than 10 μg, less than 9 μg, less than 8 μg, less than 7 μg, less than 6 μg, less than 5 μg, less than 4 μg, less than 3 μg, less than 2 μg, less than 1 μg, less than 0.5 μg, less than 0.1 μg, less than 0.01 μg, less than 10 ng, or less than 1 ng, or less than 0.1 ng can benefit from the administration of GABA-producing bacteria.

[0131] In some embodiments of any of the above aspects, the amount of GABA in the subject's stool, e.g., as measured in step (b) of any of the above aspects, is increased by 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000 percent or more relative to the initial amount of GABA in the subject's stool. In some embodiments, the amount of GABA in the subject's blood or serum, e.g., as measured in step (b) of any of the above aspects, is increased by 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000 percent or more relative to the initial amount of GABA in the subject's blood or serum. In some embodiments, the amount of GABA in a region of the subject's brain, for example, but not limited to, the prefrontal cortex, is increased by 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000 percent or more relative to the initial amount of GABA in the subject's brain, e.g., as measured in step (b) of any of the above aspects. In some embodiments, at least one GABA-producing bacterium in the subject's stool is increased by 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000 percent or more relative to the initial amount of GABA-producing bacteria in the subject's stool, e.g., as measured in step (b) of any of the above aspects. In some embodiments, the level of expression of at least one GABA-producing enzyme in the subject's stool is increased by 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000 percent or more relative to the initial level of GABA-producing enzyme expression in the subject's stool, as measured by qPCR or some other suitable method known to those skilled in the art.

[0132] In some embodiments of the present disclosure, the amount of GABA-consuming bacteria may be reduced, e.g., reduced in a subject's stool, serum, etc. The GABA-consuming bacteria may be, for example, Evtepia gabavorous or Firmicutes bacterium MGS:114. In some embodiments, the GABA-consuming bacteria may be reduced by 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000 percent, or more.

[0133] Thus, the present disclosure provides a treatment for a psychiatric illness or a disease of the central nervous system comprising administering to a subject GABA-producing bacteria, or prebiotics, to stimulate the growth or GABA-producing ability of GABA-producing bacteria. How to Cultivate GABA-Consuming Bacteria

[0134] In some embodiments, the present disclosure provides a method for culturing bacteria that require GABA for survival and replication. In some cases, these bacteria have not been cultured before or could not be cultured. In some cases, the bacteria are cultured by supplying endogenous GABA to the growth medium. In some embodiments, the bacteria are cultured by co-cultivating the bacteria with different bacteria that can produce GABA (for example, the bacteria described above).

[0135] In some embodiments, the previously uncultured bacterium is E. gabavorous. E. gabavorous can be cultured on a suitable substrate, such as agar. In some embodiments, the agar can contain added GABA. In some embodiments, the disclosure provides a method of culturing E. gabavorous, the method comprising co-culturing E. gabavorous with another bacterial strain capable of producing GABA, e.g., at physiologically relevant conditions found in the human gastrointestinal tract (e.g., a pH below about 4.5 to about 7.5).

[0136] Without wishing to be bound by theory, some previously unculturable bacteria (e.g., E. gabavorous) may be able to grow near culturable organisms that produce growth factors the bacteria require to survive or grow. Accordingly, the present disclosure teaches the discovery and cultivation of E. gabavorous in the presence of GABA as a necessary growth factor.

[0137] E. gabavorous was identified as a slow-growing colony closely spaced with Bacteroides fragilis KLE1758. Growth of E. gabavorous KLE1738 was found to be induced in the presence of supernatant derived from Bacteroides fragilis KLE1758. Chemical analysis of the supernatant of Bacteroides fragilis KLE1738 by HPLC and NMR revealed GABA as a growth factor required for E. gabavorous.

[0138] As shown in Figure 1 and Example 2, E. gabavorous was initially discovered because it grows in the presence of Bacteroides fragilis KLE1758. It was proposed that Bacteroides fragilis KLE1758 produces growth factors necessary for E. gabavorous growth and survival. Figure 1A shows a photograph of an agar plate containing bacterial colonies after treatment with human stool. The inset in the upper right corner shows a close-up photograph of a KLE1758 colony with a 1738 colony growing nearby. Figure 1B shows a KLE1758 colony capable of supporting multiple KLE1738 colonies on an agar plate containing no other bacteria. Without wishing to be bound by theory, it is believed that Bacteroides fragilis KLE1758 can support the growth of E. gabavorous KLE1738. Without wishing to be bound by theory, Bacteroides fragilis KLE1758 and E. gabavorous KLE1738 can coexist in a symbiotic relationship in which E. gabavorous KLE1738 can consume the GABA produced by Bacteroides fragilis KLE1758.

[0139] As shown in Figure 2 and Examples 3-4, the supernatant from a 48-hour culture of Bacteroides fragilis KLE1758 was found to support the growth of E. gabavorous KLE1738, whereas standard agar did not. After a series of purification and isolation steps of the KLE1758 supernatant, GABA was discovered to be a factor in the growth of E. gabavorous KLE1738. Figure 2A shows that E. gabavorous KLE1738 grew in the presence of Bacteroides fragilis supernatant. However, Figure 2B shows that E. gabavorous KLE1738 did not grow in the presence of sterile vehicle on standard agar. After initial fractionation of spent medium from Bacteroides fragilis KLE1758, it was found that the most polar fraction was able to induce growth of E. gabavorous KLE1738 (Figure 2C), whereas the less polar fraction was unable to induce growth (Figure 2D). Figures 2E and 2F show close-up views demonstrating that the most polar fraction of the supernatant of Bacteroides fragilis KLE1758 was able to induce growth of E. gabavorous KLE1738 (Figure 2E), whereas the less polar fractions were not (Figure 2F). As shown in Figure 2G, only GABA was identified as being able to induce growth of E. gabavorous KLE1738.

[0140] The 16S nucleotide sequence of E. gabavorous KLE1738 is given in SEQ ID NO:2286.

[0141] The gene sequence of E. gabavorous KLE1738 was identified as shown in Example 5. The annotated genome of E. gabavorous (2,500,009 bp) is provided in the accompanying sequence listing, including SEQ ID NOs: 1-2288, and SEQ ID NOs: 2218-2285. Without wishing to be bound by theory, the genome did not reveal any obvious entry points for the metabolism of general sugars or other carbon sources.

[0142] Without wishing to be bound by theory, transport systems for common sugars or other carbon sources were also found to be incomplete. Without wishing to be bound by theory, their absence suggests a recent loss of function. E. gabavorus is predicted to have a limited set of transporters, including those for methionine, branched-chain amino acids, dipeptides, oligopeptides, and choline / betaine, as predicted in Table 10.

[0143] [Table 10A]

[0144] Without wishing to be bound by theory, these amino acids, unlike serine, threonine, glutamate, etc., are not always able to support bacterial growth as a sole carbon source, as evidenced by the inability of E. gabavorous to grow on the amino acids tested.

[0145] Without wishing to be bound by theory, it is proposed that the metabolic pathway of E. gabavorous is similar to that of Clostridium aminobutyricum, as all enzymes in this pathway were identified in the E. gabavorous genome (Table 11), as shown in Figure 3. [Table 11]

[0146] The pH dependence of B. fragilis KLE1758's ability to produce GABA was investigated. As shown in Example 6, B. fragilis KLE1758 was grown at various pH values, and supernatants from the growth were analyzed using LCMS. As shown in Figure 4A, GABA is primarily produced at relatively low pH (e.g., about 5.5 and lower) compared to glutamate.

[0147] Thus, B. fragilis KLE1758 was found to be capable of producing GABA at low pH, while at relatively high pH, ​​it was found to produce primarily glutamate. As shown in Example 6 and Figure 4A, at pHs of about 5 and about 5.5, B. fragilis KLE1758 produced significantly more GABA than glutamate. However, at pHs of about 6 and about 6.5, B. fragilis KLE1758 was found to produce primarily glutamate and relatively little GABA. Biological screening for GABA-producing strains

[0148] The present disclosure also teaches methods for identifying bacteria capable of producing GABA. Given the strict requirement for GABA for E. gabavorous growth, the present disclosure provides a method for screening for bacteria capable of producing GABA, for example, using the growth of E. gabavorous and / or other GABA-dependent bacteria as a bioassay. Importantly, by using a buffered medium (e.g., buffered agar), the assay techniques presented herein can be used to identify bacteria capable of producing GABA at various pH values ​​(e.g., between about 5.5 and about 7.5).

[0149] As shown in Example 7, a sample suspected of containing GABA-producing bacteria, such as a human stool sample, can be mixed with molten agar. A diluted solution of E. gabavorous can then be streaked onto the agar containing the bacterial sample. As shown herein, E. gabavorous cannot grow without GABA, and therefore, any E. gabavorous colonies that form will necessarily grow in close proximity to GABA-producing strains.

[0150] Because GABA production by some bacteria, including E. coli, occurs only at very low pH (e.g., at a pH that is not appropriate for the human intestine), the assay method presented herein was adapted to control the pH of the medium, allowing for the identification of organisms capable of producing GABA at a pH between about 4.5 and about 7.5. Without wishing to be bound by theory, a pH between about 4.5 and about 7.5 is the appropriate pH in the human intestine. Thus, in some embodiments, bacteria capable of producing GABA at these pH values ​​may be able to produce GABA in the human intestine.

[0151] In other words, by controlling the pH of the growth medium (e.g., by buffering the dissolved agar), the present disclosure can distinguish GABA-producing strains that can produce GABA at a physiologically relevant pH (e.g., between about 4.5 and about 7.5) from bacteria that cannot produce GABA at a physiologically relevant pH (e.g., bacteria that can only produce substantial amounts of GABA at a pH below about 4.5).

[0152] Using this method, several representatives from multiple genera were identified, including, but not limited to, Bacteroides, Bifidobacterium, Blautia, Coprococcus, Gordonibacter, Dorea, and Clostridium. Figure 4B shows a representative agar plate showing the growth of E. gabavorous in the presence of GABA-producing bacteria. Figure 4C shows a phylogenetic tree of GABA-producing bacteria identified using this method.

[0153] Figure 5 shows the GABA production capacity of certain strains of GABA-producing strains identified using the techniques described herein. As shown in Figure 5, eight strains of GABA producers were grown in buffered media (e.g., between about pH 4.5 and about pH 5.0, and between about pH 6.5 and about pH 7.0). Using the method described in Example 6, the GABA production capacity of GABA-producing bacteria at various pH values ​​was investigated. As shown in Figure 5, certain bacteria (e.g., B. dorei KLE1912) produced relatively small amounts of GABA at low pH (e.g., between about 4.5 and about 5.0). In contrast, certain bacteria produced different amounts of GABA depending on the pH (e.g., B. vulgatus KLE1910 and B. ovatus KLE1770). Of note, some bacteria were found to produce relatively more GABA at lower pH than at higher pH, as shown for B. vulgatus KLE1910 and B. ovatus KLE1770, while some bacteria were found to produce relatively more GABA at higher pH than at lower pH.

[0154] In some embodiments of the method for identifying bacteria capable of producing GABA, the substrate is agar. In some embodiments, the step of contacting the substrate with E. gabavorous includes streaking a diluted solution of E. gabavorous onto the agar. GABA-producing colonies are then identified by inducing growth of E. gabavorous. As described above, grown E. gabavorous is used to identify whether a bacterial strain produces GABA. However, those skilled in the art will understand that any bacteria that strictly require GABA for growth and survival can similarly be used to identify bacteria capable of producing GABA, as described above. [Example]

[0155] The present disclosure is further illustrated by the following examples and synthetic examples, which should not be construed as limiting the scope or spirit of the disclosure to the specific procedures described herein. It should be understood that these examples are provided to illustrate certain embodiments, and no limitations on the scope of the disclosure are intended thereby. It should be further understood that various other embodiments, modifications, and equivalents thereof that may be suggested to those skilled in the art may be employed without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0156] Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Anhydrous solvents were obtained from Sigma-Aldrich (Milwaukee, WI) and used directly.

[0157] Unless otherwise specified, PCR was performed using the general bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') (set forth in SEQ ID NO: 2287) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (set forth in SEQ ID NO: 2288) to amplify the 16S rRNA gene. The PCR reaction mixture consisted of 12.5 μL of GoTaq Master Mix (Promega), 1 μL of 10 μM 27F and 1492R primers, 9.5 μL of nuclease-free water (Promega), and 1 μL of colony resuspended in 100 μL of sterile distilled water. The amplification conditions were: 1 cycle of 95°C for 5 minutes; 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds; 72°C for 90 seconds; and a final cycle of 72°C for 7 minutes. Amplification of the PCR reaction was then confirmed using gel electrophoresis on a 0.8% agarose gel supplemented with ethidium bromide. Successful PCR products were sequenced at Macrogen Corporation using the 27F primer on an Applied Biosystems 3730xl DNA analyzer. Quality control of the sequences was performed using DNA Baser (www.DnaBaser.com), which trimmed ends until there were more than 75% good bases (defined by having a QV score higher than 25) in an 18-base window. Phylogenetic neighbor identification and pairwise sequence similarity calculations were performed using the EzTaxon server. Example 1 Human stool collection

[0158] Stool samples from healthy human donors were collected using commercially available stool collection containers. Within 5 minutes of collection, 1 gram of stool was resuspended in 9 mL of 20% sterile glycerol in PBS and homogenized using a vortex mixer for 30 seconds. 1 mL aliquots of this mixture were filled into cryotubes and stored at -80°C for incubation. Example 2 Culturing helper-uncultivated pairs from human stool samples

[0159] All culture procedures were performed in a Coy anaerobic vinyl chamber with an atmosphere of 5% hydrogen, 10% CO2, and 85% nitrogen. Serial dilutions of thawed stool samples were prepared in PBS and bead-spread onto 1X Fastidious Anaerobic Agar (Accumedia) (FAAy) plates containing 2.5% yeast extract (7–10 beads per plate). Plates were anaerobically incubated at 37°C for 1 week, and colony appearance each day was tracked by spotting the outside of the plates with different colored markers. At the end of the week, serial dilutions of late-forming colonies (appearing after 4–7 days) were prepared in PBS and bead-spread onto FAAy plates. Close (<2 cm), early-forming colonies (appearing after 1–3 days) were then resuspended in PBS at a higher concentration. 5 μL of this suspension was spotted onto each candidate-dependent plate and incubated in the chamber for up to 1 week, with daily observations. Induction of growth of dependent organisms around the spotted helper indicated a positive hit.

[0160] Stool samples from healthy human donors were diluted and plated on rich agar plates, and newly formed colonies were recorded daily for 1 week. Late-forming colonies (days 3–7) were diluted and plated on nutrient agar plates, and a large inoculum of adjacent, early-forming colonies (days 1–2) was spotted, as shown in Figure 1A. Using this method, several helper-uncultivated pairs were identified, in which the plated uncultivated isolates formed a growth gradient around the spotted cultivable helper.

[0161] One isolate, E. gabavorous KLE1738 (93.22% similar to Flavonifractor plautii ATCC 29863 by 16S rRNA gene sequence), showed growth similar to that of Bacteroides fragilis. KLE1758 (100% similar to Bacteroides fragilis ATCC 25852 by 16S rRNA gene sequence) (Fig. 1B). E. gabavorous is a Gram-positive organism in the Clostridia classification. Example 3 Determination of GABA as a growth factor for E. gabavorous

[0162] The supernatant from a 48-hour culture of B. fragilis KLE1758 grown in rich medium induced growth of E. gabavorous as shown in Figures 2A and 2B, enabling the purification of growth factors to drive bioassays. The supernatant was solvent partitioned with ethyl acetate, and the aqueous residue fraction induced growth of E. gabavorous. The aqueous fraction was then purified using HP-20 column chromatography, and the most polar fraction induced growth of E. gabavorous as shown in Figure 2C. This active fraction was then further fractionated by preparative HPLC. HPLC yielded one active fraction, which was shown by NMR to contain 10 compounds, primarily GABA, threonine, lactate, valine, glutamine, malonic acid, succinate, and alanine. All compounds were spotted onto a plate on which E. gabavorous was spread. Only GABA induced growth induction, as shown in Figures 2E-F. To identify the components in the fractions, 1 H, 13 C. 1 H- 1 Compounds were characterized by NMR analysis, including H COSY, TOCY, HSQC, and HMBC NMR experiments. All NMR experiments were performed on a Varian INOVA 600 MHz NMR spectrometer equipped with an indirect detection probe. Example 4 Testing other compounds for induction of E. gabavorous

[0163] As shown in Figure 2G, several compounds were tested for their ability to induce growth of E. gabavorus. Stocks of each compound (except for the ATCC mineral and vitamin mix, which was purchased from Sigma) were prepared according to their solubility in water at the concentrations shown in Figure 2G. Five microliters of the stock was then spotted onto FAAy plates containing E. gabavorus, incubated anaerobically for one week, and observed for any growth. Compounds other than GABA did not induce growth. Example 5 Whole genome sequencing and annotation

[0164] DNA from E. gabavorous cells grown anaerobically on FAAy plates containing 1.0 mg / mL GABA for 48 hours was isolated for genome sequencing using the PowerSoil® DNA Isolation Kit (Mo Bio, San Diego, CA) according to the manufacturer's specifications, yielding approximately 5.0 μg of high-quality DNA. Genome sequencing and de novo assembly were performed at the Genomic Core at Tufts University in Boston, Massachusetts. The E. gabavorous genome was sequenced on an Illumina MiSeq using 500 cycles of MiSeq V2 chemistry with a paired-end 250-base format. Briefly, 100 ng of genomic DNA was sheared to an average fragment size of approximately 600 bases using a Covaris M220. The sheared DNA was used as input to prepare sequencing libraries using an Illumina TruSeq Nano DNA Sample Preparation Kit according to the manufacturer's instructions. Base calling and demultiplexing were performed on the raw data from MiSeq using CASAVA to generate fastq files. De novo genome assembly was performed using Edena V3.131028 with a customized parameter optimization pipeline. The best-assembled genome, as assessed by contig statistics, was reported. The assembly yielded 68 contigs (n), all of which had a sequence length greater than 200 bases (n:200). Seven contigs had N50 values ​​greater than 119,748, with the smallest contig length being 355 (min). N20, N50, and N20 were 33,403, 119,748, and 204,670, respectively. The longest contig length (max) was 344,080, and the estimated genome size was 2,500,000. Data were collected using the RAST server and KEGG (Kyoto Encyclopedia of Genes and Genomes). The draft genome was annotated using the KAAS (KEGG Automated Annotation Server) analysis tool in the Encyclopedia of Genes and Genomes database. The E. gabavorous genome was annotated using RAST, and the genomes of CAG:113 and E. gabavorous were compared using RAST. Example 6 Quantification of glutamate and GABA production in B. fragilis

[0165] The absolute amounts of glutamate and GABA contained in the supernatant of B. fragilis KLE1758 were determined by HPLC using fluorophores to aid detection. Specifically, free amines were labeled for analysis by reacting with AccQ reagent (Waters) according to the manufacturer's protocol. A calibration curve was generated from a stock solution (10 mg / mL) prepared by dissolving GABA (2.0 mg) in water (200 μL), glutamate (Glu) (10.2 mg) in water (1020 μL), and cysteic acid (CSA) (16.9 mg) in water (1690 μL). These were serially diluted to generate a concentration gradient. Specifically, stocks were made to final concentrations of 0.1 mg / mL, 0.05 mg / mL, 0.01 mg / mL, and 0.001 mg / mL. An aliquot of a given stock solution was added to AccQ reaction buffer (final 25 μL), followed by AccQ reagent (25 μL) dissolved in acetonitrile. This was reacted at 55-60°C for 10 min and then directly transferred to an LCMS vial fitted with a glass insert. The reaction concentrations of amino acids were 20 ng / μL, 10 ng / μL, 5 ng / μL, 2.5 ng / μL, 1 ng / μL, 0.5 ng / μL, 1 ng / μL, and 0 ng / μL (control). These samples were injected (10 μL) into an Agilent LCMS using a gradient of solvents A (water / 0.1% formic acid) and B (acetonitrile / 0.1% formic acid) over the following time courses: 1) a linear gradient from 2% B to 98% B from 0 to 40 min, 2) an isocratic gradient of 98% B from 40 to 45 min, 3) a linear gradient from 98% B to 2% B from 45 to 45.5 min, and 4) an isocratic gradient of 2% B from 45.5 to 55 min. The CSA-AccA derivative eluted at 9.5 min, the Glu-AccQ derivative at 12.1 min, and the GABA-AccQ derivative at 12.5 min. Calibration curves were obtained by plotting the area under the curve in extracted ion (EIC) mode (m / z 274 for GABA-AccQ, 318 for Glu-AccQ, and 340 for CSA-AccQ) versus the amount of original Glu, CSA, or GABA injected (ng). The average of two runs for each test concentration was used to generate the calibration curve.In the case of GABA and Glu, CSA was added to all reactions to a final concentration of 2.5 μg / mL and used as an internal standard.

[0166] Triplicate cultures of B. fragilis KLE1758 were grown anaerobically in BHIych for 48 h, the cells were centrifuged, and the supernatant was filtered through a 0.2 μm filter. Samples were stored at 4°C until analysis. To analyze the samples, an aliquot (2 μL) of each sample was added to AccQ reaction buffer (16 μL), CSA internal standard (2 μL of a 50 μg / mL solution in buffer), followed by AccQ reagent (20 μL). These samples were heated to 55°C for 10 min and then transferred directly to an LCMS vial fitted with a glass insert. An aliquot (10 μL) of each sample was injected into the LCMS and separated according to the same injection program used for the calibration curve. The areas under the curve for all EICs representing GABA, Glu, and CSA were determined using ChemStation software (Agilent). Each injection represented 25% of the original medium concentration; therefore, the total amount of sample (ng) determined was multiplied by a factor of four to determine the original concentration (ng / µL = µg / mL). All areas were normalized to the area under the curve of the internal standard (CSA), which was kept at a constant concentration throughout the experiment. The results are presented in Figure 4A. Example 7 Co-culture screening for GABA-producing strains using E. gabavorous

[0167] Secretion of GABA may enable bacteria to survive acid stress. GABA is produced by decarboxylation of glutamate, and GABA is exported from the cell in a protonated form, alkalizing the cytoplasm. While E. coli and some Lactobacillus and Bifidobacterium strains have been shown to produce GABA, these organisms are typically found at low abundance in the human intestinal tract, and in the case of E. coli, this depends on low pH (e.g., approximately 4.2 and below). Bacteroides fragilis, a helper of E. gabavorous, is a common gut bacterium. However, as shown in Figure 4A, GABA production by Bacteroides fragilis KLE1758, similar to E. coli, was found to be observed only at pHs below approximately 5.5. At each pH value, GABA is shown in the left column, and glutamate is shown in the right column. Without wishing to be bound by theory, it was therefore thought to be useful to identify microorganisms that can produce GABA at a pH that is physiologically relevant to the human large intestine (e.g., a pH of about 5.5 to about 7.5, or about 5.7 to about 7.4).

[0168] To achieve this, we exploited the strict GABA requirement of E. gabavorus to screen for bacteria capable of secreting GABA in highly buffered media. Metabolic byproducts of bacterial growth can lower the pH of the medium in the absence of a buffer. In an anaerobic chamber, stool samples were mixed with molten agar, poured onto Petri plates, and E. gabavorus was spread on top of the solidified agar. By locating growth induction zones in E. gabavorus and measuring the pH of the agar, we identified bacteria that produced GABA at a pH between approximately 6.0 and 7.0, as well as bacteria that produced GABA at a pH between approximately 4.5 and 5.0, as shown in Figure 4B. The entire 16S rRNA gene was amplified and sequenced using the 27F and 1492R universal primers. Annotation using EZTaxon revealed several representatives from multiple genera, including Bacteroides, Bifidobacterium, Blautia, Coprococcus, Gordonibacter, Dorea, and Clostridium (Figure 4C). Of these, only Bifidobacterium adolescentis has previously been reported to produce GABA. Example 8 Using engineered Escherichia coli strains to produce GABA and induce growth of E. gabavorous

[0169] GABA can be produced in intestinal epithelial cells by decarboxylation of glutamate by some bacteria, such as Escherichia coli and Listeria monocytogenes. In E. coli, decarboxylation of glutamate acts as a mechanism to lower intracellular pH, and GABA production normally occurs at low pH. To investigate whether E. coli can be engineered to produce GABA, E. coli colonies harboring native glutamate decarboxylase (gadA, gadB) or the GABA antiporter (gadC) in the pCA24N IPTG-inducible high-copy-number vector were transformed into E. gabavorous. GABA production was tested by coculture assay. Overexpression of glutamate decarboxylase (gadA or gadB) in E. coli induced growth of KLE1738 to levels seen in B. fragilis, whereas expression of the GABA antiporter gadC did not (Figure 6). Altering the pH of the growth medium for KLE1738 did not alter the GABA-dependent phenotype. Without wishing to be bound by theory, this suggests that engineering bacteria to overexpress glutamate decarboxylase or other GABA-producing enzymes is an effective way to produce GABA, either constitutively or inducibly, as well as induce growth in E. gabavorous. equivalent

[0170] While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and other variations thereof will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are intended to be within the spirit and scope of the present disclosure.

Claims

1. A therapeutic composition for use in a method for increasing endogenous GABA in a subject, said method comprising administering to said subject said therapeutic composition comprising at least one purified bacterial population that produces GABA in a pH range of 4.5 to 7.5, wherein said at least one purified bacterial population consists of bacteria comprising a 16s rDNA sequence that is at least 98% identical to a 16s rDNA sequence selected from the group consisting of SEQ ID NOs: 1-4, 8, 10, 12, 16-18, 28-29, and 81.

2. The therapeutic composition described in claim 1, wherein the at least one bacterial population consists of bacteria containing a 16s rDNA sequence that is at least 99% identical to a 16s rDNA sequence selected from the group consisting of SEQ ID NOs: 1-4, 8, 10, 12, 16-18, 28-29, and 81.

3. The at least one purified bacterial population is selected from the group consisting of Bacteroides caccae; Bacteroides clarus; Bacteroides dorei; Bacteroides finegoldii; Bacteroides stercoris; Bacteroides uniformis; Bacteroides xylanisolvens; Butyricimonas virosa; Clostridium perfringens; Clostridium sordellii; Parabacteroides distasonis; Parabacteroides merdae; Bacteroides 10. The therapeutic composition of claim 1, comprising a bacterium selected from the group consisting of: S. salyersiae, S. sallyersiae, and combinations thereof.

4. The therapeutic composition of claim 1, wherein the at least one purified bacterial population consists of bacteria comprising a DNA sequence encoding an enzyme selected from glutamate decarboxylase; putrescine aminotransferase; gamma-aminobutyraldehyde dehydrogenase; arginine decarboxylase; agmatinase; ornithine decarboxylase; or a combination thereof.

5. The therapeutic composition described in claim 4, wherein the glutamate decarboxylase, putrescine aminotransferase, gamma-aminobutyraldehyde dehydrogenase, arginine decarboxylase, agmatinase, ornithine decarboxylase, or a combination thereof, is encoded by a DNA sequence that is at least 90% identical in DNA sequence to any one of SEQ ID NOs: 275 to 304. (a) the glutamic acid decarboxylase is encoded by a DNA sequence that is at least 90% identical in DNA sequence to a sequence selected from SEQ ID NOs:275-279; (b) the putrescine aminotransferase is encoded by a DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:280-284; (c) the gamma-aminobutyraldehyde dehydrogenase is encoded by a DNA sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:285-289; (d) the arginine decarboxylase is encoded by a DNA sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:290-294; (e) the agmatinase is encoded by a DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 295-299; or (f) the ornithine decarboxylase is encoded by a DNA sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 300-304; The therapeutic composition of claim 4.

7. The therapeutic composition of claim 1, wherein the therapeutic composition is in the form of a capsule, tablet, caplet, pill, troche, lozenge, powder, granule, medical food, fecal transplant, or a combination thereof.

8. The therapeutic composition of claim 1, wherein the therapeutic composition further comprises a prebiotic.

9. The method further comprising identifying a subject who would benefit from an increase in endogenous GABA by measuring an initial amount of GABA in the subject's stool, blood, serum, or brain, wherein the subject: (a) the initial amount of GABA in the subject's stool is less than about 8 μg per gram of wet or dry stool; (b) the initial amount of GABA in the subject's blood or serum is less than about 10 μg per liter of blood; or (c) the initial amount of GABA in the subject's brain is less than about 1.0 mM / kg 2. The therapeutic composition of claim 1, wherein the subject is identified as benefiting from an increase in endogenous GABA if:

10. The therapeutic composition of claim 1, wherein the subject is identified as benefiting from an increase in endogenous GABA if the initial amount of GABA-producing bacteria in the subject's stool is less than about 10% of the total bacteria as measured by 16s sequence mapping.

11. The therapeutic composition described in claim 1, wherein the subject has a psychiatric disorder or a disease of the central nervous system.

12. The therapeutic composition described in claim 11, wherein administration of the therapeutic composition improves symptoms of the psychiatric disorder or central nervous system disorder.

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