Microbiome

Abstract

Described herein are compositions that include two or more bacterial strains belonging to at least two of the following genera: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, or Anaerostipes. The embodiments described herein can be used to treat liver diseases or conditions, symptoms thereof.

Description

[Technical field]

[0001] Aspects and embodiments described herein relate to the field of microbial compositions for human and animal health. [Background technology]

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with a global prevalence estimated at approximately 25%, with the Middle East and South America reporting most cases and Africa reporting the lowest prevalence. (1) NAFLD is the hepatic manifestation of metabolic syndrome, with common comorbidities including obesity, type 2 diabetes, hyperlipidemia, and hypertension. (2) The prevalence of NAFLD and associated comorbidities is increasing (1,3), and as a result, NAFLD is rapidly becoming a major economic and social burden. (4) NAFLD can be further classified into nonalcoholic fatty liver (NAFL), also known as simple steatosis, and nonalcoholic steatohepatitis (NASH), which is characterized by the presence of hepatic inflammation. NASH is an aggressive form of the disease that can progress to liver fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC). (2) Metabolic dysregulation is a hallmark of NAFLD. Because insulin resistance, hyperlipidemia, or dyslipidemia are common features, lipid-regulating agents such as thiazolidinediones (TZDs) have been successfully tested in clinical trials for the treatment of NAFLD. However, significant side effects such as weight gain, fluid retention, and risk of myocardial infarction have been reported. (6-8) In addition, to date, there is no approved pharmacological therapy for NASH.

[0003] Transplantation of fecal material from lean rats into NASH rats fed a high-fat glucose / fructose diet (HFGFD) has been reported (9). However, no significant histological changes were observed. The fact that fecal transplantation or fecal microbial transplantation (FMT) is a poorly characterized procedure, with the associated risk of infectious disease transmission, raises doubts about its applicability to less acute and life-threatening pathologies. Indeed, FMT is unlikely to be a long-term treatment option, especially considering the chronic and progressive nature of the disease.

[0004] WO 2017 / 134240 describes a consortium consisting essentially of Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum, and Anaerostipes caccae and its use in gastrointestinal disorders. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above, there remains a need for improved means and methods that can be used to treat, delay, ameliorate, prevent or cure liver pathology or related conditions and symptoms, which do not suffer from all of the drawbacks of existing therapies. [Means for solving the problem]

[0006] Surprisingly, it has been found that certain combinations of bacteria provide special and surprising beneficial effects on animal and human health. In particular, as detailed elsewhere herein and in the experimental part, the compositions described herein have been found to exert at least the following beneficial effects: in particular, reduced weight gain, reduced insulin levels, reduced glucose levels, reduced insulin resistance, reduced portal pressure and intrahepatic vascular resistance, alleviation of endothelial dysfunction, reduced hepatic inflammation, reduced steatosis, reduced hepatocyte ballooning, and reduced fibrosis.

[0007] Thus, the aspects and embodiments described herein solve at least some of the problems and needs discussed herein.

[0008] In one aspect, a composition is provided comprising two or more bacterial strains of at least two of the following genera: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, for use in a method of treating, delaying, ameliorating, preventing, or curing a liver disease or condition or a symptom thereof. In some embodiments, the composition for use is a composition comprising two or more bacterial strains of at least two of the following species: Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and Anaerostipes caccae. In some embodiments, the composition for use is a composition comprising at least one bacterial strain capable of producing butyrate and at least one bacterial strain capable of producing propionate or a propionate precursor. In some embodiments, the composition for use is a composition further comprising at least one, preferably at least two, more preferably three additional bacterial strains capable of producing propionate or a propionate precursor.In some embodiments, the one or more additional bacterial strains capable of producing propionate or a propionate precursor belong to the genus Phocaeicola, Veillonella, or Blautia, preferably the species Phocaeicola vulgatus, Veillonella parvula, Veillonella atypica, Blautia obeum, Blautia wexlerae, or Blautia luti. In some embodiments, the composition for use comprises: -Phocaeicola vulgatus; - Veillonella parvula or Veillonella atypica; and - A composition further comprising Blautia obeum, Blautia wexlerae, or Blautia luti.

[0009] In some embodiments, the composition is for use in a method of treating, delaying, ameliorating, preventing, or curing non-alcoholic fatty liver disease (NAFLD), liver fibrosis, cirrhosis, and / or liver neoplasms, and the liver neoplasm is preferably hepatocellular carcinoma (HCC) or a symptom thereof. Preferably, the symptoms include weight gain, insulin resistance, liver inflammation, steatosis, hepatocyte ballooning, intrahepatic vascular resistance (IHVR), portal hypertension, endothelial dysfunction, and / or fibrosis. In some embodiments, the composition is for use in a method of delaying or preventing disease progression of NAFL, preferably in a method of delaying the progression of NAFL to NASH, the progression of NASH to liver fibrosis, the progression of liver fibrosis to cirrhosis, and / or the progression of cirrhosis to HCC.

[0010] In another embodiment, the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus and Anaerostipes are preferred, preferably Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum and Anaerostipes caccae. In some embodiments, the composition further comprises at least two additional bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, the composition further comprises three additional bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, the two or more additional bacterial strains capable of producing propionate or a propionate precursor belong to the genus Phocaeicola, the genus Veillonella, or the genus Blautia, preferably the species Phocaeicola vulgatus, the species Veillonella parvula, the species Veillonella atypica, the species Blautia obeum, the species Blautia wexlerae, or the species Blautia luti. In some embodiments, the composition comprises: -Phocaeicola vulgatus; - Veillonella parvula or Veillonella atypica; and -Further including Blautia obeum, Blautia wexlerae or Blautia luti.

[0011] Deposit of biological material Purified cultures of the microbial strains LMG P-29362, LMG P29360, LMG P-29365, LMG P-29361, LMG P-29366, and LMG P29359 described in this application have been deposited by the depositor Prodigest (Technologiepark 3, 9052 Zwijnaarde, Belgium; current address: Technologiepark 82, 9052 Zwijnaarde, Belgium) with the BCCM / LMG Laboratorium voor Microbiologie, Universiteit Gent (UGent), KLLedeganckstraat 35, B-9000 Gent, Belgium, which is recognized as an international depositary institution by the Budapest Treaty and the World Intellectual Property Organization. The initial deposit was made on January 18, 2016.

[0012] explanation composition In one embodiment, a composition is provided that comprises two or more bacterial strains from at least two of the following genera: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes. In other words, this means that the composition comprises at least two bacterial strains from different genera, both selected from Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes. Thus, for example, a composition comprising three bacterial strains may include two strains from one genus and a third strain from a different genus, or three strains from different genera.

[0013] Preferably, the composition is for use in a method of treating, delaying, ameliorating, preventing or curing a liver disease or condition or a symptom thereof, which are described in more detail elsewhere herein (see the "Methods and Uses" section).

[0014] The genus Lactiplantibacillus was previously included in the genus Lactobacillus (Zheng et al. Int J Syst Evol Microbiol 2020;70(4):2782-2858).

[0015] In some embodiments, the compositions described herein may comprise three or more, preferably four or more, or more preferably five or more bacterial strains of at least three, at least four, or at least five of the following genera: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes.

[0016] In a preferred embodiment, a composition is provided that comprises six or more bacterial strains of the following genera: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes. Thus, such a composition comprises at least one bacterial strain of each of the six genera. In some embodiments, a composition may comprise or consist essentially of one bacterial strain of each of the six genera.

[0017] In some embodiments, the compositions provided herein are compositions that comprise at least one bacterial strain belonging to the genus Faecalibacterium and at least one bacterial strain belonging to the genus Anaerostipes.

[0018] It will be understood that bacterial strains, as used herein, refer to genetic variants or subtypes of a microorganism. Several different strains may belong to the same bacterial species. Throughout this disclosure, the term "bacterial strain" can also be replaced with the term "bacteria" or "bacterial member". In any of the compositions provided herein, the bacterial strain can be isolated or purified from a source, such as, for example, a culture or a microbiome sample (e.g., fecal material). As used herein, the terms "isolated" and "purified" refer to a bacterium or bacterial strain or bacterial member, or a composition thereof, that is separated from one or more associated substances found in the source material or any material associated with the bacterium in any process used to produce the preparation, such as another bacterium or bacterial strain, one or more components of a growth medium, and / or one or more components of a sample, such as a fecal sample. In some aspects, the bacteria are "substantially isolated" or "substantially purified" from a source, such as, for example, such that other components of the source are not detectable.

[0019] As mentioned above, the compositions provided herein may include two, three, four, five, six, or more bacterial strains belonging to the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes. In some embodiments, the two, three, four, five, six, or more bacterial strains are selected from specific species within the genera. A preferred species of Faecalibacterium is Faecalibacterium prausnitzii. A preferred species of Butyricicocccus is Butyricicocccus pullicaecorum. A preferred species of Roseburia is Roseburia inulinivorans. A preferred species of Akkermansia is Akkermansia muciniphila. A preferred species of Lactiplantibacillus is Lactiplantibacillus plantarum. A preferred species of Anaerostipes is Anaerostipes caccae.

[0020] The above mentioned species are well known to those skilled in the art, in particular the bacterial species Faecalibacterium prausnitzii (Duncan et al. Int J Syst Evol Microbiol. 2002; 52: 2141-2146), Butyricicoccus pullicaecorum (Eeckhaut et al. Int J Syst Evol Microbiol. 2008; 58: 2799-2802), Roseburia inulinivorans (Duncan et al. Int J Syst Evol Microbiol. 2006; 56: 2437-2441), Akkermansia muciniphila (Derrien et al. Int J Syst Evol Microbiol. 2004;54:1469-1476), Lactiplantibacillus plantarum (Walter.Appl Environ Microbiol 2008;74:4985-4996) and Anaerostipes caccae (Schwiertz et al.Syst Appl Microbiol 2002;25:46-51) are bacterial species well known to those skilled in the art. The species Lactiplantibacillus plantarum was previously known as the species Lactobacillus plantarum (Zheng et al.Int J Syst Evol Microbiol 2020;70(4):2782-2858).

[0021] Thus, in some embodiments, compositions are provided that include two or more bacterial strains of at least two of the following species: Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and Anaerostipes caccae.

[0022] As mentioned above, the compositions provided herein, in some embodiments, may comprise at least one bacterial strain belonging to the genus Faecalibacterium and at least one bacterial strain belonging to the genus Anaerostipes. Thus, in some embodiments, the compositions provided herein are compositions comprising at least one bacterial strain belonging to the species Faecalibacterium prausnitzii and at least one bacterial strain belonging to the genus Anaerostipes caccae.

[0023] In some embodiments, the compositions described herein may comprise three or more, preferably four or more, or more preferably five or more bacterial strains belonging to at least three, at least four, or at least five of the following species: Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and Anaerostipes caccae.

[0024] In a preferred embodiment, a composition is provided comprising six or more bacterial strains belonging to the following species: Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum and Anaerostipes caccae.

[0025] Such compositions therefore comprise at least one bacterial strain of each of the six species, hi some embodiments, the compositions may comprise or consist essentially of one strain of each of the six species.

[0026] As mentioned above, the compositions provided herein may include two, three, four, five, six, or more bacterial strains belonging to the species Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and Anaerostipes caccae. In some embodiments, the two, three, four, five, six, or more bacterial strains are selected from specific bacterial strains within the species.

[0027] Preferred strains of Faecalibacterium prausnitzii are Faecalibacterium prausnitzii LMG P-29362 and Faecalibacterium prausnitzii DSMZ 17677, as well as strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of at least one of the above strains, e.g., SEQ ID NO:4.

[0028] Preferred strains of Butyricicoccus pullicaecorum are Butyricicoccus pullicaecorum LMG P-29360 and Butyricicoccus pullicaecorum LMG24109, as well as strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of at least one of said strains, such as SEQ ID NO:6.

[0029] Preferred strains of Roseburia inulinivorans are Roseburia inulinivorans LMG P-29365 and Roseburia inulinivorans DSMZ 16841, and strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of at least one of said strains, e.g., SEQ ID NO:1.

[0030] Preferred strains of Akkermansia muciniphila are Akkermansia muciniphila LMG P-29361 and Akkermansia muciniphila DSMZ 22959, and strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of at least one of said strains, e.g., SEQ ID NO:2.

[0031] Preferred strains of Lactiplantibacillus plantarum are Lactiplantibacillus plantarum LMG P-29366 and Lactiplantibacillus plantarum ZJ316, and strains having a 16S rRNA or rDNA sequence exhibiting at least 97% sequence identity to the 16S rRNA or rDNA sequence of at least one of said strains, e.g., SEQ ID NO:5.

[0032] Preferred strains of Anaerostipes caccae are Anaerostipes caccae LMG P-29359 and Anaerostipes caccae DSMZ 14662, and strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of at least one of said strains, e.g., SEQ ID NO:3.

[0033] In some embodiments, the above-mentioned strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to a particular 16S rRNA or rDNA sequence (e.g., SEQ ID NOs: 1-6) may exhibit 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to said 16S rRNA or rDNA (e.g., SEQ ID NOs: 1-6).

[0034] As used herein, the term "16S ribosomal RNA" or "16S rRNA" refers to a nucleic acid sequence that is a component of the small prokaryotic ribosomal subunit (30S). 16S rRNA is known to function as a scaffold that defines the position of ribosomal proteins. The gene that encodes it is called the 16S rRNA gene or 16S rDNA, and is commonly used in phylogenetic studies because it is known to be a highly conserved sequence.

[0035] Those skilled in the art are well aware that 16S rRNA or rDNA sequences can be deposited in online sequence databases, such as GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ), and that 16S rRNA or rDNA sequences can be retrieved based on their unique accession numbers for use as reference 16S rRNA or rDNA sequences in assessing sequence homology, for example, as described in Eeckhaut et al. Int J Syst Evol Microbiol 2008;58:2799-2802. The GenBank accession numbers of the 16S rRNA sequences of some bacterial strains provided herein are listed below. These accession numbers can be used to retrieve the respective 16S rRNA sequences from http: / / www.ncbi.nlm.nih.qov / genbank / to assess sequence homology: Roseburia inulinivorans DSMZ 16841:AJ270473.3 (SEQ ID NO: 1) Akkermansia muciniphila DSMZ 22959:AY271254.1 (SEQ ID NO:2) Anaerostipes caccae DSMZ 14662:AJ270487.2 (SEQ ID NO:3) Faecalibacterium prausnitzii DSMZ 17677:AJ270469.2 (SEQ ID NO: 4) Lactiplantibacillus plantarum ZJ316: JN126052.1 (SEQ ID NO: 5) Butyricicoccus pullicaecorum LMG 24109 HH793440.1 (sequence number 6).

[0036] The above mentioned strains with accession numbers LMG P-29362, LMG P29360, LMG P-29365, LMG P-29361, LMG P-29366 and LMG P29359 have been deposited at the BCCM / LMG Laboratorium voor Microbiologie, Universiteit Gent (UGent), KLLedeganckstraat 35, B-9000 Gent, Belgium.

[0037] The above mentioned strains having accession numbers DSMZ 17677, LMG24109, DSMZ 16841, DSMZ 22959, ZJ316 and DSMZ 14662 have been deposited in public collections, are well described and are readily available to those skilled in the art worldwide.

[0038] Butyrate production In some embodiments, the compositions described herein are compositions comprising at least one bacterial strain capable of producing butyrate, hi some embodiments, the compositions described herein are compositions comprising at least two, at least three, or at least four bacterial strains capable of producing butyrate.

[0039] In some embodiments, any of two, three, four, five, six, or more bacterial strains belonging to the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, as described elsewhere herein, can be bacterial strains capable of producing butyrate. In some embodiments, at least one, two, three, four, five, six, or more of the two, three, four, five, six, or more bacterial strains of the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, as described elsewhere herein, may be bacterial strains capable of producing butyrate. In some embodiments, all of the two, three, four, five, six, or more bacterial strains belonging to the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, as described elsewhere herein, can be bacterial strains capable of producing butyrate.

[0040] Bacterial strains belonging to the genera Faecalibacterium, Butyricicocccus, Roseburia, and Anaerostipes are known to be capable of producing butyrate. In particular, Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, and Anaerostipes caccae are species known to be capable of producing butyrate.

[0041] Roseburia inulinivorans is known to produce both butyrate and propionate.

[0042] In some embodiments, provided herein is a composition as described above to which an additional bacterial strain capable of producing butyrate is added. In this context, commensal gut bacteria capable of producing butyrate are preferred.

[0043] As used herein, a bacterial strain capable of producing butyrate may be understood to refer to a bacterial strain that has at least one pathway for the production of butyrate in its genome. There are four major pathways for the production of butyrate in bacteria (see, for example, Figure 1 in Vital et al. mBio 2014;5(2):e00889-14). These pathways utilize one of four substrates: pyruvate, glutarate, 4-aminobutyrate, and lysine. All pathways are integrated with a central energy-generating step in which crotonyl-CoA is converted to butyryl-CoA, catalyzed by the butyryl-CoA dehydrogenase electron transfer flavoprotein complex (Bcd-Etfαβ). The final production of butyrate is catalyzed by either butyryl-CoA:acetate-CoA transferase (But) or butyrate kinase (Buk).

[0044] One of skill in the art will understand that in any given experimental setup, a bacterial strain capable of producing butyrate as described herein may or may not be capable of producing butyrate depending on the test conditions, including, for example, the growth medium.

[0045] The presence of these pathways in bacteria can be evaluated by genetic methods known to those skilled in the art, such as those described in Reichardt et al. The ISME Journal 2014;8:1323-1335. By way of example, the presence of marker genes can be detected by PCR or sequencing-based methods, or using in silico phylogenetic tools. A suitable marker gene for the presence of the butyrate kinase pathway is butyrate kinase (Buk). A suitable marker gene for the presence of the butyryl-CoA:acetate CoA-transferase pathway is butyryl-CoA:acetate CoA-transferase (BCoAT or But).

[0046] Alternatively, or in addition, butyrate production can be assessed by chemical analysis of in vitro, in vivo, or in situ samples using methods known to those of skill in the art, including gas chromatography (GC), liquid chromatography (LC), nuclear magnetic resonance (NMR), and capillary electrophoresis (CE).

[0047] Propionate production As shown in the experimental part, it has surprisingly been found that the addition of at least one additional bacterial strain capable of producing propionate or a propionate precursor to the compositions already described herein is associated with particular advantages.

[0048] Thus, in some embodiments, the compositions described herein are compositions comprising at least one bacterial strain capable of producing propionate or a propionate precursor, hi some embodiments, the compositions described herein are compositions comprising at least two, preferably at least three, more preferably at least four, or more preferably at least five bacterial strains capable of producing propionate or a propionate precursor.

[0049] In some embodiments, any of two, three, four, five, six, or more bacterial strains belonging to the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, as described elsewhere herein, can be bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, at least one, two, three, four, five, six, or more of the two, three, four, five, six, or more bacterial strains of the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, as described elsewhere herein, can be bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, all of the two, three, four, five, six, or more bacterial strains belonging to the genera Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, as described elsewhere herein, can be bacterial strains capable of producing propionate or a propionate precursor.

[0050] Bacterial strains belonging to the genera Roseburia and Akkermansia are known to be capable of producing propionic acid. In particular, Roseburia inulinivorans and Akkermansia muciniphila are species known to be capable of producing propionic acid.

[0051] Roseburia inulinivorans is known to produce both butyrate and propionate.

[0052] In some embodiments, the compositions described herein are compositions comprising at least one bacterial strain capable of producing butyrate and at least one bacterial strain capable of producing propionate or a propionate precursor. In some embodiments, the compositions described herein are compositions comprising at least two bacterial strains capable of producing butyrate and at least two bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, the compositions described herein are compositions comprising at least three bacterial strains capable of producing butyrate and at least three bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, the compositions described herein are compositions comprising at least four bacterial strains capable of producing butyrate and at least four bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, the compositions described herein are compositions comprising at least four bacterial strains capable of producing butyrate and at least five bacterial strains capable of producing propionate or a propionate precursor.

[0053] A preferred composition comprises at least four bacteria capable of producing butyrate and at least two bacteria capable of producing propionate or a propionate precursor, and a more preferred composition comprises four bacteria capable of producing butyrate and three, preferably four, and more preferably five bacteria capable of producing propionate or a propionate precursor.

[0054] In general, the ability to produce propionate or butyrate from sugar residues has been observed to exist in different species, although some exceptions are known. Notable exceptions that can produce both butyrate and propionate are, for example, bacterial strains of Roseburia inulinivorans.

[0055] In some embodiments, any composition comprising two or more bacterial strains of at least two of the following genera, as described elsewhere herein: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, or Anaerostipes, further comprises at least one additional bacterial strain capable of producing propionate or a propionate precursor. In some embodiments, the compositions described herein further comprise at least two additional bacterial strains capable of producing propionate or a propionate precursor. In some embodiments, the compositions described herein further comprise at least three additional bacterial strains capable of producing propionate or a propionate precursor. In a preferred embodiment, the compositions described herein further comprise three additional bacterial strains capable of producing propionate or a propionate precursor.

[0056] In this context, commensal gut bacteria capable of producing propionate or propionate precursors are preferred.

[0057] In some embodiments, the additional bacterial strain capable of producing propionate or a propionate precursor belongs to the family Veillonellaceae, Akkermansiaceae, Lachnospiraceae, or Bacteroidetes, preferably Veillonellaceae, Lachnospiraceae, or Bacteroidetes.

[0058] A preferred genus of the family Veillonellaceae is Veillonella. Preferred species are Veillonella parvula and Veillonella atypica.

[0059] A preferred genus of the family Akkermansiaceae is Akkermansia. A preferred species is Akkermansia muciniphila.

[0060] In the family Lachnospiraceae, the preferred genera are Blautia and Roseburia. Within Blautia, the preferred species are Blautia obeum, Blautia wexlerae, and Blautia luti. Within Roseburia, the preferred species is Roseburia inulinivorans.

[0061] A preferred genus of the family Bacteroidetes is Phocaeicola.

[0062] A preferred species within Phocaeicola is Phocaeicola vulgatus.

[0063] Thus, in some embodiments, additional bacterial strains capable of producing propionate or propionate precursors as described herein are bacterial strains of the genus Veillonella, Akkermansia, Blautia, Roseburia, or Phocaeicola.

[0064] In a preferred embodiment, the additional bacterial strain capable of producing propionate or a propionate precursor as described herein is a bacterial strain of the genus Phocaeicola, Veillonella, or Blautia.

[0065] In a preferred embodiment, the additional bacterial strain capable of producing propionate or a propionate precursor as described herein is a bacterial strain of the species Phocaeicola vulgatus, Veillonella parvula, Veillonella atypica, Blautia obeum, Blautia wexlerae, or Blautia luti.

[0066] Blautia wexlerae and Blautia luti strains are known to produce the propionate precursor succinate.

[0067] In a more preferred embodiment, a composition comprising two or more bacterial strains of at least two of the following genera: Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, or Anaerostipes, as described elsewhere herein, comprises: -Phocaeicola vulgatus; - Veillonella parvula or Veillonella atypica; and -Further including Blautia obeum, Blautia wexlerae or Blautia luti.

[0068] In some embodiments, the compositions described herein are selected from the group consisting of bacterium species belonging to the genus Faecalibacterium, Butyricicoccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, and preferably selected from the species Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and the like. and Anaerostipes caccae, and further comprising at least two additional bacterial strains capable of producing propionate or a propionate precursor. It will be appreciated that such compositions may include a total of eight strains (where one strain of each genus / species is included) or may include more than a total of eight strains (e.g., where multiple strains of a genus / species are further included).

[0069] In some embodiments, the at least two additional bacterial strains capable of producing propionate or a propionate precursor can be three additional bacterial strains capable of producing propionate or a propionate precursor.

[0070] In some embodiments, the two or more additional bacterial strains capable of producing propionate or a propionate precursor may belong to the genus Phocaeicola, Veillonella, or Blautia, preferably the species Phocaeicola vulgatus, Veillonella parvula, Veillonella atypica, Blautia obeum, Blautia wexlerae, or Blautia luti.

[0071] In some embodiments, the compositions described herein are selected from the group consisting of Faecalibacterium, Butyricicocccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, preferably Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and the like. plantarum species and Anaerostipes caccae, the composition comprising: -Phocaeicola vulgatus; - Veillonella parvula or Veillonella atypica; and -Further including Blautia obeum, Blautia wexlerae or Blautia luti.

[0072] In some embodiments, the compositions described herein are compositions comprising a bacterial strain belonging to at least one of the genera Faecalibacterium, Butyricicoccus, Roseburia, Akkermansia, Lactiplantibacillus, Anaerostipes, and Phocaeicola, as well as Veillonella or Blautia. Preferred are the species Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, Anaerostipes caccae, and Phocaeicola vulgatus, as well as the species Veillonella parvula, Veillonella atypica, Blautia obeum, and the like. The composition includes a bacterial strain belonging to at least one of the species Blautia obeum, Blautia wexlerae, or Blautia luti. In some cases, the bacterial member may include a Veillonella, preferably Veillonella parvula or Veillonella atypica.In other examples, the bacterial members may include Blautia, preferably Blautia obeum, Blautia wexlerae, or Blautia luti. In yet other examples, the bacterial members may include both Veillonella and Blautia, preferably Veillonella parvula or Veillonella atypica; and Blautia obeum, Blautia wexlerae, or Blautia luti.

[0073] In some embodiments, additional bacterial strains capable of producing propionate or propionate precursors as described herein are selected from specific strains of the above-mentioned species. Preferred strains of Phocaeicola vulgatus are Phocaeicola vulgatus LMG17767 and strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of said strain (SEQ ID NO: 7).

[0074] Preferred strains of Veillonella parvula are Veillonella parvula DSM 2007 and strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of said strain (SEQ ID NO:8).

[0075] Preferred strains of Blautia obeum are Blautia obeum DSM 25238 and strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to the 16S rRNA or rDNA sequence of said strain (SEQ ID NO:9).

[0076] In some embodiments, the above-mentioned strains having a 16S rRNA or rDNA sequence that exhibits at least 97% sequence identity to a particular 16S rRNA or rDNA sequence (e.g., SEQ ID NOs: 7-9) may exhibit 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to said 16S rRNA or rDNA (e.g., SEQ ID NOs: 7-9).

[0077] The above mentioned strains having the accession numbers LMG17767, DSM 2007 and DSM 25238 have been deposited in public collections, are well described and are readily available to anyone skilled in the art worldwide.

[0078] As used herein, a bacterial strain capable of producing propionate may be understood to refer to a bacterial strain that has in its genome a pathway for propionate production, such as at least one of an acrylic acid pathway, a propanediol pathway, and a succinic acid pathway (see Figure 1 in Reichardt et al. The ISME Journal 2014;8:1323-1335).

[0079] As used herein, a bacterial strain capable of producing a propionate precursor may be understood to refer to a bacterial strain that has at least one of the pathways for producing a propionate precursor in its genome. The propionate precursor may be any precursor or metabolic intermediate in any one of the various pathways for propionate production. In some embodiments, the propionate precursor described herein may be selected from the group consisting of acrylate, aspartate, fumarate, malate, 2-oxoglutarate, malate, oxaloacetate, and succinate. In a preferred embodiment, the propionate precursor described herein is succinate.

[0080] One skilled in the art will understand that in any given experimental setup, a strain capable of producing propionate or a propionate precursor as described herein may or may not produce propionate or a propionate precursor depending on the test conditions, including, for example, the growth medium. This may be because, for example, some propionate production pathways require specific substrates. For example, some Blautia strains, such as Blautia obeum, produce propionate from fucose.

[0081] The presence of the propionate pathway in bacteria can be evaluated by genetic methods known to those skilled in the art, such as those described in Reichardt et al. The ISME Journal 2014;8:1323-1335. By way of example, the presence of marker genes can be detected by PCR or sequencing-based methods, or using in silico phylogenetic tools. A suitable marker gene for the presence of the acrylic acid pathway is lactoyl-CoA dehydratase (lcdA). A suitable marker gene for the presence of the succinate pathway is methylmalonyl-CoA decarboxylase (mmdA). A suitable marker gene for the presence of the propanediol pathway is propionaldehyde dehydrogenase or propanol dehydrogenase (pduP, pduQ).

[0082] Alternatively, or in addition, production of propionate (or propionate precursors) can be assessed by chemical analysis of in vitro, in vivo, or in situ samples using methods known to those of skill in the art, including gas chromatography (GC), liquid chromatography (LC), nuclear magnetic resonance (NMR), and capillary electrophoresis (CE).

[0083] Generally, any bacterial strain capable of producing butyrate as described herein can be selected from the following genera: Faecalibacterium, Butyricicocccus, Roseburia, and Anaerostipes; and the following species: Faecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburia inulinivorans, and Anaerostipes caccae.

[0084] Similarly, any bacterial strain capable of producing propionate or a propionate precursor as described herein may be selected from the following families: Veillonellaceae, Akkermansiaceae, Lachnospiraceae, and Bacteroidetes; the following genera: Roseburia, Akkermansia, Veillonella, Blautia, and Phocaeicola; and the following species: Roseburia inulinivorans, Akkermansia muciniphila, Phocaeicola vulgatus, Veillonella parvula, and the following species: parvula, Veillonella atypica, Blautia obeum, Blautia wexlerae, and Blautia luti.

[0085] Further ingredients In a further aspect, the compositions described herein may further comprise one or more prebiotics. The term "prebiotic" refers to chemicals that can induce the growth or activity of microorganisms (e.g., bacteria) that contribute to the health of the host. Thus, prebiotics can affect or change the composition of organisms in the intestinal flora. However, in principle, prebiotics is a more general term that can also refer to other areas of the body. Typically, but not exclusively, prebiotics are non-digestible fiber compounds that pass at least partially undigested through the upper part of the digestive tract and stimulate the growth or activity of beneficial bacteria that colonize the large intestine by acting as their substrate.

[0086] In further aspects, the compositions described herein may further comprise one or more therapeutic agents. In some embodiments, the therapeutic agent is a statin (e.g., simvastatin), other lipid-lowering therapies, non-lipid-lowering therapies, GLP-1 receptor agonists (e.g., semaglutide, dulaglutide, liraglutide), SGLT1 / 2 inhibitors (e.g., licoglifozin), SGLT2 inhibitors, DPP-4 inhibitors (e.g., itagliptin, saxagliptin, linagliptin, alogliptin), FXR agonists (e.g., OCA, tropifexor, cilofexor), apoptosis inhibitors (e.g., selonsertib, emricasan), anti-inflammatory drugs (e.g., pentoxifylline), antihypertensive drugs such as angiotensin receptor blockers, CCR2 / 5 antagonists (e.g., cenicriviroc), ACC inhibitors (e.g., filsocostat), antioxidant stress drugs (e.g., vitamin E, UDCA), and PPAR agonists such as thiazolidinediones (TZDs) (e.g., pioglitazone).

[0087] In a preferred embodiment, the therapeutic agent is selected from statins (e.g., simvastatin), GLP-1 receptor agonists (e.g., semaglutide, dulaglutide, liraglutide), SGLT2 inhibitors, and PPAR agonists such as thiazolidinediones (e.g., pioglitazone). A preferred example of a GLP-1 receptor agonist is liraglutide. In a preferred embodiment, the therapeutic agent is a PPAR agonist such as a thiazolidinedione (also known as a glitazone) (e.g., pioglitazone).

[0088] Specific Compositions and Derived Products In some embodiments, the compositions described herein may be pharmaceutical compositions, i.e., compositions that further comprise a pharma- ceutically acceptable ingredient.

[0089] As used herein, "a pharma- ceutically acceptable ingredient" includes a pharma- ceutically acceptable carrier, a filler, a preservative, a solubilizer, a vehicle, a diluent, and / or an excipient. Thus, one or more pharma- ceutically acceptable ingredients can be selected from the group consisting of a pharma- ceutically acceptable carrier, a filler, a preservative, a solubilizer, a vehicle, a diluent (e.g., a starch, a cellulose derivative, or a sugar derivative), and / or an excipient. Such pharma- ceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents, and / or excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, 23rd edition. Elsevier (2020), which is incorporated herein by reference.

[0090] The compositions described herein can be formulated to provide rapid and efficient therapeutic effect.In some embodiments, the compositions described herein can be formulated for intestinal delivery, for example, as a composition for rectal administration or oral administration, preferably oral administration.

[0091] In some embodiments, the compositions described herein can be formulated as capsules, microcapsules, tablets, granules, powders, lozenges, pills, dietary supplements, suspensions, suppositories, or syrups, preferably as powders, suspensions, tablets, or dietary supplements. In some embodiments, the compositions described herein can be provided in suspension form.

[0092] In some embodiments, the (pharmaceutical) compositions described herein can be formulated as compositions for rectal administration. In this regard, any of the compositions described herein can be provided as a (pharmaceutical) dosage form for rectal administration, such as a suppository.

[0093] In some embodiments, the (pharmaceutical) compositions described herein can be formulated as compositions for oral administration such as capsules, microcapsules, tablets, granules, powders, troches, pills, dietary supplements, suspensions, or syrups.

[0094] In some embodiments, the compositions described herein may comprise lyophilized bacteria or bacteria provided in active culture form, hi a preferred embodiment, the bacteria may be lyophilized.

[0095] In some embodiments, the compositions described herein can be included in food, beverages, dietary supplements, or nutraceuticals.Thus, aspects of the present disclosure also provide foods, beverages, dietary supplements, and nutraceuticals that include any of the compositions described herein.Foods, beverages, dietary supplements, and nutraceuticals that include any (combination) of the bacterial strains described herein are also included within the scope of the present disclosure.

[0096] A "food" is an edible material that typically consists primarily of one or more proteins, carbohydrates, and fats. Foods may also include one or more micronutrients, such as vitamins and minerals. Examples of foods that may incorporate the composition include snack bars, cereals, breads, muffins, biscuits, cakes, pastries, processed vegetables, sweet foods, probiotic preparations, including yogurt, beverages, vegetable oil-based liquids, animal fat-based liquids, frozen confections, and cheese. Preferred foods include yogurt, cheese, and other dairy products.

[0097] Examples of beverages include soft drinks, syrups, squash, dry drink mixes, and energy drinks.

[0098] A nutraceutical is a food ingredient, dietary supplement, or food that is believed to provide a medical or health benefit, including the prevention and treatment of disease. Nutraceuticals are also sometimes called "functional foods," i.e., foods that are generally marketed to provide health benefits to the consumer beyond providing pure nutrition.

[0099] The present disclosure also provides probiotics comprising the composition discussed herein.Probiotics are typically live supplements that can enhance gut flora.Such probiotics can be administered to humans, but also to farm animals, livestock, and aquatic organisms.Probiotics can additionally comprise one or more acceptable excipients or flavorings that are suitable for human or animal consumption.

[0100] In some embodiments, the compositions described herein comprise at least 10 2 , preferably at least 10 3 , more preferably at least 10 4 , and even more preferably at least 10 5 In some embodiments, the compositions described herein contain 10 CFU of bacteria. 2 ~10 14 , preferably 10 3 ~10 13 , more preferably 10 4 ~10 12 , and even more preferably 10 5 ~10 11 In some embodiments, the compositions described herein contain at least 10 5 of bacterial colony forming units, e.g. 10 5 ~10 11 containing bacterial colony forming units.

[0101] With respect to any of the compositions provided throughout this disclosure, it will be understood that the term "comprising" can be replaced with the terms "consisting essentially of" or "consisting of." In other words, in some embodiments, the compositions provided herein consist essentially of or consist of the provided bacterial strains.

[0102] In addition to the compositions described herein, preferred aspects of the present disclosure also relate to the same compositions for use in any of the methods described herein below (see the "Methods and Uses" section).

[0103] Method for obtaining the composition; and the composition obtained thereby In some embodiments, the compositions provided herein can be obtained by the methods described in this section. In some embodiments, the compositions provided herein can be obtainable by the methods described in this section. In other words, in some embodiments, the bacterial strains included in the compositions provided herein have been previously subjected to the methods described in this section. In this context, the bacterial strains can also be said to be "pre-adapted" or "conditioned" by any of the methods described in this section.

[0104] The methods provided in this section impart certain advantageous properties to the compositions of the present disclosure: in particular, the onset of the composition's effect on the host may be more rapid and / or the efficacy may be greater, i.e., the composition provides a rapid and efficient therapeutic effect.

[0105] More specifically, the "pre-adapted" or "adjusted" composition can significantly shorten the time of biopharmaceutical expression, i.e., by pre-adaptation, it is meant that the set of microorganisms can exert their function at least 5% faster (on a time scale), preferably at least 10% faster, more preferably at least 20% faster, and most preferably at least 30% faster, compared to a set of the same strains collected in a rough manner. The "pre-adapted" or "adjusted" composition can also significantly increase the efficacy of the treatment, i.e., by pre-adaptation, it is meant that the set of microorganisms can exert their function with at least 5% higher efficacy, preferably at least 10% higher efficacy, more preferably at least 20% and most preferably at least 30% higher efficacy. The efficacy depends on the endpoint for which the set of microorganisms was designed. Possible functionalities include, but are not limited to, in particular, reduced weight gain, improved insulin levels, reduced glucose levels, reduced insulin resistance, reduced portal vein pressure and intrahepatic vascular resistance, alleviated endothelial dysfunction, reduced liver inflammation, reduced steatosis, reduced hepatocyte ballooning, and reduced fibrosis.

[0106] In one aspect, a method is provided for obtaining a composition described herein, comprising growing bacterial strains together in a reactor.

[0107] In some embodiments, the reactor is under standardized conditions representative of the gastrointestinal tract.

[0108] Parameters characterizing the standardized conditions include, but are not limited to, pH (in the range of 1.5 to 8); availability of a carbon source (carbohydrate or protein, or a combination thereof); retention time in a specific reactor (in the range of 10 minutes to 200 hours); availability of oxygen (in the range of 0 to 8 g / L); availability of micronutrients; presence or absence of antibiotics; concentration of bile salts (in the range of 0 to 20 mM); presence of heavy metals; presence of host factors as immune molecules. In a preferred embodiment, parameters characterizing the standardized conditions include pH, retention time in a specific reactor, and concentration of bile salts, all as previously defined herein. Depending on the complexity of the population, a period of 1 to 15 days may be suitable to obtain a functionally stable population. On average, to obtain a population composed of 7 to 14 members, a period of 3 to 10 days is suitable to obtain a functionally stable population (depending on the environmental conditions). Thus, the compositions defined herein can be obtained after training or culturing for a period of 3 to 10 days under conditions in which pH, ​​retention time in a specific reactor, and concentration of bile salts are set as defined herein. Such a process allows for the production of functionally stable compositions or populations.

[0109] As used herein, a "functionally stable population" can be a composition described herein that contains the original distinct number of bacterial species even after at least 3 days, or 5 days, or 10 days of culture.

[0110] In a further aspect, a reactor is provided that operates under standardized conditions representative of the GI tract, including a pH range of 1.5-8; availability of a carbon source; retention time of 10 min-200 h; availability of oxygen of 0-8 g / L; availability of micronutrients; presence or absence of antibiotics; concentration of bile salts of 0-20 mM; presence of heavy metals; presence of host factors as immune molecules. In one embodiment, said reactor is such that the parameters characterizing the standardized conditions include pH, retention time in the particular reactor, and concentration of bile salts as defined in the previous paragraph. In one embodiment, such reactor comprises a composition of 5-20 different bacterial members, or 6-14 different bacterial members, or 5-15 different bacterial members. In a preferred embodiment, such composition is present in such reactor for 3-14 days or 3-10 days to obtain a functionally stable population.

[0111] Thus, in the context of the compositions, methods and uses for use described herein, treatment may result in a more rapid onset of biological effect and / or increased efficacy, i.e., a rapid and efficient therapeutic effect, as described herein.

[0112] Methods and Uses As detailed in the experimental part, it is surprisingly found that the above composition exerts several direct and indirect beneficial effects on liver.Specifically, the composition described herein exerts at least the following beneficial effects: in particular, reducing weight gain, reducing insulin level, reducing glucose level, reducing insulin resistance, reducing portal vein pressure and intrahepatic vascular resistance, alleviating endothelial dysfunction, reducing liver inflammation, reducing steatosis, reducing hepatocyte ballooning, and reducing fibrosis.Therefore, the composition provided herein has broad applicability in treating, delaying, improving, preventing, or curing various diseases and conditions of the liver, as well as symptoms, underlying diseases or conditions, and extrahepatic symptoms thereof.

[0113] Thus, in a further aspect, the compositions described herein are provided for use in the medical field.Preferably, the compositions described herein are used in a method for treating, delaying, improving, preventing, or curing a liver disease or condition. Also encompassed herein is treating symptoms of a liver disease or condition, underlying diseases or conditions of a liver disease or condition, and extrahepatic manifestations of a liver disease or condition, each of which aspects are described in more detail elsewhere herein.Thus, in some embodiments, the compositions described herein are provided for use in a method for treating, delaying, improving, preventing, or curing a liver disease or condition or a symptom thereof.In some embodiments, the compositions described herein are provided for use in a method for treating, delaying, improving, preventing, or curing a liver disease or condition or a symptom thereof, underlying disease or condition, or extrahepatic manifestations.

[0114] In a further aspect, a method is provided for treating, delaying, ameliorating, preventing, or curing a liver disease or condition comprising administering a composition as described herein. In some embodiments, a method is provided for treating, delaying, ameliorating, preventing, or curing a liver disease or condition or a symptom thereof comprising administering a composition as described herein. In some embodiments, a method is provided for treating, delaying, ameliorating, preventing, or curing a liver disease or condition, a symptom thereof, an underlying disease or condition, or an extrahepatic symptom comprising administering a composition as described herein. In some embodiments, administering a composition means administering the composition to a subject, such as a subject in need thereof. In a preferred embodiment, an effective amount or a therapeutically effective amount of the composition is administered.

[0115] In a further aspect, there is provided a use of the compositions described herein for the manufacture of a medicament for treating, delaying, ameliorating, preventing, or curing a liver disease or condition. In some embodiments, there is provided a use of the compositions described herein for the manufacture of a medicament for treating, delaying, ameliorating, preventing, or curing a liver disease or condition or a symptom thereof. In some embodiments, there is provided a use of the compositions described herein for the manufacture of a medicament for treating, delaying, ameliorating, preventing, or curing a liver disease or condition, a symptom thereof, an underlying disease or condition, or an extrahepatic symptom.

[0116] In a further aspect, there is provided a use of the compositions described herein for treating, delaying, improving, preventing, or curing a liver disease or condition. In some embodiments, there is provided a use of the compositions described herein for treating, delaying, improving, preventing, or curing a liver disease or condition or a symptom thereof. In some embodiments, there is provided a use of the compositions described herein for treating, delaying, improving, preventing, or curing a liver disease or condition, a symptom thereof, an underlying disease or condition, or an extrahepatic symptom.

[0117] In the context of the compositions, methods, and uses for which they are provided herein, an effective amount or a therapeutically effective amount of the compositions described herein can be administered.

[0118] As used herein, an "effective amount" is an amount sufficient to exert a beneficial or desired result. Thus, a "therapeutically effective amount" is an amount that, when administered to a subject in need thereof, is sufficient to exert any of the therapeutic or prophylactic effects described herein, including, but not limited to, reduced weight gain, improved insulin levels, reduced glucose levels, reduced insulin resistance, reduced portal vein pressure and intrahepatic vascular resistance, alleviation of endothelial dysfunction, reduced hepatic inflammation, reduced steatosis, reduced hepatocyte ballooning, and reduced fibrosis, compared to an untreated subject. A "therapeutically effective" amount may vary from subject to subject, depending, for example, on the age, progression of the disease, or overall general condition of the individual. The appropriate "therapeutically effective" amount in any individual case can be determined by one of skill in the art using routine experimentation, such as the methods described below in this specification and / or the methods in the experimental part of this specification. It should be understood that "effective amount" and "therapeutically effective amount" refer to the combined total amount of the bacterial strains in the composition. Depending on the end application, the combined total may be the result of equal amounts of each bacterial strain or unequal amounts of each bacterial strain, with each single strain having a minimum abundance of 0.0001% of the combined total, preferably a minimum abundance of 0.001% of the combined total, and more preferably a minimum abundance of 0.01% of the combined total.

[0119] Depending on the end application, the effective amount or therapeutically effective amount (understood to refer to the combined total amount of bacteria) is at least 10 2 , preferably at least 10 3 , more preferably at least 10 4 , and even more preferably at least 10 5 In some embodiments, the effective amount or therapeutically effective amount (which is understood to refer to the combined total amount of bacteria) may be 10 2 ~10 14 , preferably 10 3 ~10 13 , more preferably 10 4 ~10 12 , and even more preferably 10 5 ~10 11In some embodiments, the amount is a daily dose, i.e., the amount administered every day.

[0120] The compositions described herein can be administered according to any dosing schedule. In some embodiments, the compositions can be administered as a single dose. In preferred embodiments, the compositions can be administered to a subject on a regular or periodic basis, for example, the compositions can be administered to a subject once every 12 hours, 24 hours, or 48 hours, i.e., twice a day, once a day, or once every two days. In preferred embodiments, the compositions can be administered daily. In some embodiments, the compositions described herein are administered for at least 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 1 year, or more. In some embodiments, the compositions described herein are administered for life.

[0121] As used herein, a "subject" or a "subject in need" may be a human (preferred) or a non-human animal. In some embodiments, a subject (in need) may be a healthy subject, particularly in the context of preventing a liver disease or condition or a symptom thereof as described herein. In some embodiments, a subject (in need) may also be suffering from or at risk of developing any of the diseases and conditions described herein. In some embodiments, a subject (in need) may be a subject suffering from NASH.

[0122] In the context of the compositions for use, methods and uses provided herein, the compositions can be administered to a subject in need thereof as described herein.

[0123] In the context of the compositions, methods and uses for use provided herein, the compositions can be administered by different modes of administration. In some embodiments, the mode of administration can be rectal or oral. Oral administration is preferred.

[0124] The compositions provided herein can be administered directly or indirectly using suitable means known in the art.Thus, in view of the progress already achieved so far, it is expected that the means for providing the compositions described herein to individuals will be improved.Of course, such future improvements can be incorporated to achieve the desired effects described herein.

[0125] In the context of the compositions for use, methods, and uses provided herein, the compositions described herein can be used in combination with one or more therapeutic agents. In some embodiments, the therapeutic agent is a statin (e.g., simvastatin), other lipid-lowering therapies, non-lipid-lowering therapies, GLP-1 receptor agonists (e.g., semaglutide, dulaglutide, liraglutide), SGLT1 / 2 inhibitors (e.g., licoglifozin), SGLT2 inhibitors, DPP-4 inhibitors (e.g., itagliptin, saxagliptin, linagliptin, alogliptin), FXR agonists (e.g., OCA, tropifexor, cilofexor), apoptosis inhibitors (e.g., selonsertib, emricasan), anti-inflammatory drugs (e.g., pentoxifylline), antihypertensive drugs such as angiotensin receptor blockers, CCR2 / 5 antagonists (e.g., cenicriviroc), ACC inhibitors (e.g., filsocostat), antioxidant stress drugs (e.g., vitamin E, UDCA), and PPAR agonists such as thiazolidinediones (TZDs) (e.g., pioglitazone).

[0126] In a preferred embodiment, the therapeutic agent is selected from statins (e.g., simvastatin), GLP-1 receptor agonists (e.g., semaglutide, dulaglutide, liraglutide), SGLT2 inhibitors, and PPAR agonists such as thiazolidinediones (e.g., pioglitazone). A preferred example of a GLP-1 receptor agonist is liraglutide. In a preferred embodiment, the therapeutic agent is a PPAR agonist such as a thiazolidinedione (also known as a glitazone) (e.g., pioglitazone).

[0127] The compositions provided herein and the one or more therapeutic agents may be administered in a single composition or may be administered separately.

[0128] Liver Diseases and Conditions In some embodiments, in the context of the compositions, methods, and uses for use provided herein, the liver disease or condition may be a liver disease or condition associated with dysbiosis.

[0129] In some embodiments, in the context of the compositions, methods and uses provided herein, liver disease or condition can be alcohol-related liver disease (ARLD), cholestatic liver disease, non-alcoholic fatty liver disease (NAFLD), liver fibrosis, liver cirrhosis, and / or liver neoplasm or its symptoms.Alcohol-related liver disease (ARLD) includes alcoholic fatty liver disease, alcoholic hepatitis, and (alcohol-related) liver cirrhosis.Non-limiting examples of cholestatic liver disease are primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0130] In some embodiments, in the context of the compositions, methods, and uses for use provided herein, the liver disease or condition can be non-alcoholic fatty liver disease (NAFLD), liver fibrosis, cirrhosis, and / or liver neoplasms or symptoms thereof.

[0131] NAFLD is now recognized as a hepatic manifestation of metabolic syndrome. Therefore, "NAFLD" as used in this disclosure can be replaced with terms such as "hepatic part of metabolic syndrome", "hepatic manifestation of metabolic syndrome", or "metabolic (dysfunction)-associated fatty liver disease" (MAFLD) (as proposed in Eslam M et al. A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement. J Hepatol. 2020; 73: 202-209).

[0132] NAFLD is considered a general term for a spectrum of liver diseases that can be divided into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) (see Lackner C. (2018) Nonalcoholic Fatty Liver Disease. Practical Hepatic Pathology: a Diagnostic Approach, 167-187, incorporated herein by reference).

[0133] Thus, in some embodiments, in the context of the compositions, methods, and uses for use provided herein, a liver disease or condition can be non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and / or liver neoplasms, symptoms thereof, or an underlying disease or condition.

[0134] As used herein, the term "liver neoplasm" may be understood to refer to both liver neoplasms and intrahepatic bile duct tumors. Thus, in some embodiments, terms such as "liver neoplasm" may be replaced with terms such as "liver or intrahepatic bile duct tumors."

[0135] In some embodiments, the liver neoplasm described herein may be liver cancer. In a preferred embodiment, the liver cancer is hepatocellular carcinoma (HCC).

[0136] In the context of the compositions, methods for use, and uses provided herein, a method for treating, delaying, ameliorating, preventing, or curing a disease or condition of the liver can be a method of delaying or preventing disease progression of NAFL, preferably the progression of NAFL to NASH, the progression of NASH to liver fibrosis, the progression of liver fibrosis to cirrhosis, and / or the progression of cirrhosis to HCC, more preferably the progression of NAFL to NASH or the progression of NASH to liver fibrosis.

[0137] In the context of the compositions, methods, and uses for use provided herein, "treating, delaying, ameliorating, preventing, or curing" as used herein may be understood to mean treating, delaying, ameliorating, preventing, or curing said disease, condition, or symptom compared to the disease, condition, or symptom prior to administration of the composition. In some embodiments, "treating, delaying, ameliorating, preventing, or curing" as used herein may be understood to mean treating, delaying, ameliorating, preventing, or curing said disease, condition, or symptom compared to the same disease, condition, or symptom in a subject suffering from the disease, condition, or symptom administered a placebo. Similarly, the occurrence of any of the effects described elsewhere herein may be compared in the same manner.

[0138] Symptoms In some embodiments, symptoms of a liver disease or condition described herein include weight gain, insulin resistance, hepatitis, steatosis, hepatocellular ballooning, intrahepatic vascular resistance (IHVR), portal hypertension, endothelial dysfunction, and / or fibrosis.

[0139] In the context of the compositions, methods, and uses for use provided herein, the compositions and / or medicaments described herein preferably exhibit at least one, at least two, at least three, or all of the following effects: Reduced weight gain: Improves insulin levels: Lowering Glucose Levels: Reduces insulin resistance: ·Reducing portal pressure: ·Decreased intrahepatic vascular resistance: Reduces endothelial dysfunction: Reduce liver inflammation: Reduces adiposity: Reduce ballooning: and -Reduction of fibrosis.

[0140] Means and methods for measuring these symptoms and effects are well known in the art. Non-limiting examples of methods for measuring these symptoms and effects are provided in the Experimental Section.

[0141] In this context, "reduce" (respectively "improve") and the like refer to at least a detectable decrease (respectively a detectable improvement) using an assay known to one skilled in the art, such as the assay performed in the experimental part. The decrease can be at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% decrease. In some embodiments, administering a composition described herein can result in a reduction in weight gain, an improvement in portal hypertension, or an improvement in insulin sensitivity, compared to a subject with a comparable liver disease or condition, optionally administered a placebo. In some embodiments, administering a composition described herein can result in about a 40% decrease in fasting blood insulin levels within 2 weeks of treatment, compared to a subject optionally administered a placebo. In some embodiments, administering a composition described herein can result in about an 80% decrease in intrahepatic vascular resistance (IHVR) within 10 weeks of treatment, compared to a subject optionally administered a placebo.

[0142] The Nonalcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) has been defined by the Pathology Committee of the NASH Clinical Research Network as the unweighted sum of the score values ​​for steatosis (range 0-3), lobular inflammation (range 0-3), and ballooning (range 0-2), thus providing a NAS score ranging from 0 to 8 (see Kleinen et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321, incorporated herein by reference, see especially Table 1 therein). A fibrosis score ranging from 0 to 4 has also been defined by Kleinen et al., see especially Table 1 therein.

[0143] In the context of the compositions, methods and uses for use provided herein, the compositions and / or medicaments described herein may result in a reduction in NAFLD activity score (NAS), steatosis score, lobular inflammation score, ballooning score or fibrosis score, preferably NAS score, steatosis score, ballooning score or fibrosis score, more preferably fibrosis score. The reduction is preferably at least 1 score level, or at least 2 or 3 levels.

[0144] In some embodiments, any of the described effects are observed after at least 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 1 year or more of treatment, optionally including the amounts and dosing regimens described elsewhere herein.

[0145] Underlying diseases and conditions The liver diseases and conditions or symptoms thereof described herein may be found in a number of diseases and conditions that may be referred to as underlying diseases and conditions. Therefore, it should be understood that the present disclosure also encompasses that the compositions provided herein can be used to treat, delay, ameliorate, prevent, or cure any underlying disease or condition.

[0146] For example, NAFLD is found in a number of diseases and conditions that may be referred to as underlying diseases and conditions, as summarized in Table 1.

[0147] [Table 1]

[0148] Additionally, portal hypertension can result from gastrointestinal disorders including primary sclerosing cholangitis (PSC) and chronic pancreatitis.

[0149] Accordingly, it should be understood that the present disclosure also encompasses that the compositions provided herein can be used to treat, delay, ameliorate, prevent, or cure any of the diseases or conditions set forth in Table 1. Thus, the present disclosure encompasses any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing obesity, genetic or metabolic diseases, drug or toxin induced conditions, extrahepatic conditions, nutritional disorders, viral infections, hepatic ischemia, or aging.

[0150] Thus, the disclosure encompasses any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing metabolic disorders of insulin resistance (such as metabolic syndrome, syndrome X, insulin resistance syndrome, type 2 diabetes, Alström syndrome, Bardet-Biedl syndrome, lipodystrophy syndrome, PPARγ mutations, etc.), leptin deficiency or resistance, hypothalamic obesity, or Prader-Willi syndrome. The disclosure also encompasses any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing Wilson's disease, Weber-Christian disease, Wolman disease, cholesterol ester storage disease, abetalipoproteinemia, familial hypobetalipoproteinemia, Dorman-Chanarin syndrome, pseudoneonatal adrenoleukodystrophy, galactosemia, tyrosinemia, hereditary fructose intolerance, cystinuria, Sandhoff disease, pituitary insufficiency, or hypothyroidism. The disclosure also includes any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing toxicity or overdose associated with corticosteroids, estrogens, NSAIDs, calcium antagonists, amiodarone, tamoxifen, tetracycline, chloroquine, perhexiline maleate, antiretroviral drugs, the industrial solvent dimethylformamide, paint thinners and solvents, petrochemical exposure, rapeseed foods, or cocaine. The disclosure also includes any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing heart failure, inflammatory bowel disease and other intestinal diseases, intestinal bacterial overload syndrome, cystic fibrosis, craniopharyngioma, or pregnancy-related liver disease. The disclosure also includes any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing liver-related conditions of jejunoileal bypass, gastric bypass, gastroplasty, biliopancreatic bypass, small bowel resection, total parenteral nutrition, prolonged starvation, rapid weight loss, protein malnutrition, high carbohydrate diet, or Schwachmann's syndrome. The disclosure also includes any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing infection with Hepatitis C, B, or D. The disclosure also includes any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing hepatic ischemia or senescence.The disclosure also encompasses any of the compositions provided herein for use in treating, delaying, ameliorating, preventing, or curing gastrointestinal disorders, including primary sclerosing cholangitis (PSC) and chronic pancreatitis.

[0151] In some embodiments, the compositions provided herein are for use in a method for managing liver symptoms in any of the above conditions in Table 1. In some embodiments, the compositions provided herein are for use in a method for improving liver function in any of the above conditions in Table 1. In some embodiments, the compositions provided herein are for use in slowing liver detoriation in any of the above conditions in Table 1.

[0152] extrahepatic symptoms Extrahepatic manifestations may be understood to refer to extrahepatic disorders and / or symptoms that are the result of the liver diseases or conditions described herein. Therefore, it should be understood that the present disclosure also encompasses that the compositions provided herein can be used to treat, delay, ameliorate, prevent, or cure any extrahepatic manifestations of the liver diseases or conditions described herein.

[0153] Extrahepatic symptoms may be due to effects on organs, tissues, or cells other than the liver. Extrahepatic symptoms may be due to effects on the brain, vasculature, or extravascular effects, for example. In some embodiments, the extrahepatic symptoms described herein may be hepatic encephalopathy, varices, or ascites, and preferably hepatic encephalopathy.

[0154] Without wishing to be bound by any particular theory, it is believed that the above compositions and their uses and methods may be based on the following mechanism: (a) stimulating the growth and / or activity of one or a limited number of beneficial bacteria in the intestinal tract; (b) inhibiting the growth and / or activity of one or a limited number of pathogenic bacteria in the intestinal tract; (c) increasing the relative adhesion of non-pathogenic bacteria to the mucosal surface of the gastrointestinal tract; (d) reducing unregulated uptake of antigens, proinflammatory bacteria or bacterial products by the intestine; (e) providing anti-inflammatory activity to the intestinal surface; (f) Increase intestinal barrier function (g) producing bacterial metabolic products; (h) reducing the production of harmful intestinal metabolites; (i) induce wound repair; or (j) any combination of (a) through (i).

[0155] Without wishing to be bound by any particular theory, alternatively or additionally, the above compositions and their uses and methods may be based on the following liver-related mechanisms: (a) restores hepatic insulin signaling; (b) reducing pro-fibrotic markers in the liver; (c) reducing lipid accumulation in the liver; or (d) any combination of (a) to (c).

[0156] Some specific aspects and embodiments of the present disclosure are listed below.

[0157] In one aspect, the disclosure provides a composition comprising purified bacterial members, including Faecalibacterium prausnitzii, Butyricococcus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, Anaerostipes caccae, and Phocaeicola vulgatus, as well as Veillonella parvula, Veillonella atypica, Blautia obeum, and the like. In some instances, the bacterial members may include Veillonella parvula or Veillonella atypica. In other instances, the bacterial members may include Blautia obeum, Blautia wexlerae, or Blautia luti. In yet other instances, the bacterial members may include; - Veillonella parvula or Veillonella atypica; and - Blautia obeum, Blautia wexlerae or Blautia luti.

[0158] Advantageously, compositions of bacterial members may be useful in treating or preventing certain undesirable conditions of the liver, such as liver inflammation.

[0159] In some embodiments, the composition may include a particular strain of a bacterial member. For example, in some cases, the bacterial member may include Faecalibacterium prausnitzii strain LMG P-29362. In some cases, the bacterial member may include Butyricococcus pullicaecorum strain LMG P-29360. In some cases, the bacterial member may include Roseburia inulinivorans strain LMG P-29365. In some cases, the bacterial member may include Akkermansia muciniphila strain LMG P-29361. In some cases, the bacterial member may include Lactiplantibacillus plantarum strain LMG P-29366. In some cases, the bacterial members may include Anaerostipes caccae strain LMG P-29359. In some cases, the bacterial members may include Phocaeicola vulgatus strain LMG17767. In some cases, the bacterial members may include Veillonella parvula strain DSM 2007. In some cases, the bacterial members may include Blautia obeum strain DSM 25238.

[0160] The composition can be formulated to provide a rapid and efficient therapeutic effect. In some cases, the composition can be formulated for delivery to the intestine. For example, the composition can be formulated as a powder, suspension, tablet, dietary supplement, and the like. In some cases, for example, the bacterial members can be lyophilized. For example, the bacterial members can be freeze-dried. In some cases, the bacterial members composition can be provided in a suspension form. The composition can be administered in a concentration of at least 100 mg / mL. 5 For example, the composition may contain 10 colony forming units of bacteria. 5 ~10 11 As described herein, the bacteria may be provided in a lyophilized or active culture form.

[0161] In another aspect, the present disclosure provides a method for improving a liver condition, the method comprising administering to a subject in need thereof a composition comprising a bacterial member, the bacterial member including, but not limited to, Faecalibacterium prausnitzii, Butyricococcus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, Anaerostipes caccae, and Phocaeicola vulgatus, as well as Veillonella parvula, Veillonella atypica, and the like. atypica, Blautia obeum, Blautia wexlerae, or Blautia luti, and administration of the composition optionally improves liver status as compared to liver status prior to administration of the composition. In some cases, the bacterial members may include Veillonella parvula or Veillonella atypica. In some cases, the bacterial members may include Blautia obeum, Blautia wexlerae, or Blautia luti. In other examples, the bacterial members may include: - Veillonella parvula or Veillonella atypica; and - Blautia obeum, Blautia wexlerae or Blautia luti.

[0162] The method may be useful for treating a liver condition. The condition may be, for example, any one of liver inflammation, obesity, genetic or metabolic disease, drug or toxin-induced condition, extrahepatic condition, nutritional disorder, viral infection, hepatic ischemia, or aging. For example, in some cases, the condition may include liver inflammation, and administering the composition may improve liver inflammation as evidenced by a decrease in the subject's NAFLD activity score (NAS). NAS is a scoring method based on the histological lesion characteristics of NAFLD that is accepted in clinical and experimental settings. In some cases, for example, the subject's NAS may be reduced by a value of about 1.0 within 9 weeks of administering the composition, compared to a subject that is optionally administered a placebo. In some cases, the condition may include NASH. For example, the composition may be administered to a subject with NASH, thereby delaying the progression of NASH, compared to a subject suffering from NASH that is optionally administered a placebo instead of the composition.

[0163] In some cases, administering the composition to a subject may result in weight gain, improvement of portal hypertension, or improvement of insulin sensitivity, compared to a subject with comparable liver status, optionally administered a placebo. In some cases, administering the composition may result in about a 40% reduction in fasting blood insulin levels within 2 weeks of treatment, compared to a subject optionally administered a placebo. In a further example, administering the composition may result in about an 80% reduction in intrahepatic vascular resistance (IHVR) within 10 weeks of treatment, compared to a subject optionally administered a placebo. The composition may be administered according to any dosing schedule, for example, the composition may be administered as a single dose, or the composition may be administered to the subject on a regular or cyclical basis, for example, the composition may be administered to the subject once every 12 hours, 24 hours, or 48 hours for at least 7 days, 8 days, 9 days, or 10 days or more. In some cases, the composition is formulated for delivery to the intestine. In some cases, the bacterial members may be lyophilized. In some cases, the composition may be provided in a suspension form.

[0164] In some cases, the composition comprises Faecalibacterium prausnitzii strain LMG P-29362, Butyricococcus pullicaecorum strain LMG P-29360, Roseburia inulinivorans strain LMG P-29365, Akkermansia muciniphila strain LMG P-29361, Lactiplantibacillus plantarum strain LMG P-29366, Anaerostipes caccae strain LMG P-29359, Phocaeicola vulgatus strain LMG P-29362, vulgatus strain LMG 17767, Veillonella parvula strain DSM 2007, and Blautia obeum strain DSM 25238. 5 ~10 11 The bacterial colony forming units may include

[0165] General information Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly and commonly understood by one of ordinary skill in the art to which this invention belongs and as interpreted in light of the present disclosure.

[0166] Sequence identity In the context of the present disclosure, a nucleic acid molecule, such as a nucleic acid molecule encoding a 16S rRNA gene, is represented by a nucleic acid or nucleotide sequence that encodes the 16s rRNA.

[0167] It is to be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct identified herein by a given sequence identification number (SEQ ID NO:) is not limited to this particular sequence disclosed. Each coding sequence identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct, or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.

[0168] Throughout this application, whenever a sequence number of a particular nucleotide sequence encoding a given protein fragment or polypeptide or peptide or derived peptide or rRNA is mentioned (take as an example SEQ ID NO:X), this may be replaced by: i. a nucleotide sequence comprising a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:X; ii. a nucleotide sequence, the sequence of which differs from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code; or iii. A nucleotide sequence encoding an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% amino acid identity or similarity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:X.

[0169] Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0170] In the context of the 16S rRNA gene, due to its high conservation, preferred levels of sequence identity are generally high. Thus, preferred levels of sequence identity for the 16S rRNA gene are 97%, 97.5%, 98%, 98.5%, 99% or 99.5%.

[0171] Throughout this application, whenever a particular amino acid sequence SEQ ID NO (take SEQ ID NO: Y as an example), is mentioned, this can be replaced by: a polypeptide represented by an amino acid sequence comprising a sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0172] Each nucleotide sequence or amino acid sequence described herein by a percentage identity or similarity to a given nucleotide sequence or amino acid sequence, respectively, is in a further preferred embodiment at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74% with the given nucleotide sequence or amino acid sequence, respectively. , at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity or similarity.

[0173] Each non-coding nucleotide sequence (i.e., the sequence of a promoter or another regulatory region) can be replaced by a nucleotide sequence that contains a nucleotide sequence having at least 60% sequence identity or similarity with the sequence number of a specific nucleic acid sequence (take sequence number A as an example). Preferred nucleotide sequences have at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:A. In a preferred embodiment, such a non-coding nucleotide sequence, such as a promoter, exhibits or exerts at least an activity of such a non-coding nucleotide sequence, such as an activity of a promoter, known to those of skill in the art.

[0174] Terms such as "homology" and "sequence identity" are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or between two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of the sequences of two given SEQ ID NOs, or a portion thereof. The portion preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of the sequences of both SEQ ID NOs. In the art, "identity" also refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as the case may be, as determined by the match between strings of amino acid or nucleic acid sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each of which is incorporated herein by reference in its entirety.

[0175] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide sequences or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Sequences can then be called "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (described below) (e.g., when optimally aligned by the programs EMBOSS needle or EMBOSS water using default parameters).

[0176] Global alignment is appropriately used to determine sequence identity when two sequences have similar lengths. When sequences have substantially different overall lengths, local alignment, such as alignment using the Smith-Waterman algorithm, is preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, EMBOSS needle and EMBOSS water are used with default parameters of gap opening penalty=10(nucleotide sequence) / 10(protein) and gap extension penalty=0.5(nucleotide sequence) / 0.5(protein). For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

[0177] Alternatively, the similarity or identity percentage can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present disclosure can further be used as "query sequences" to perform searches of public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, which are incorporated herein by reference. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the BLASTx program, using score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, incorporated herein by reference. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information, accessible on the World Wide Web at www.ncbi.nlm.nih.gov / .

[0178] Optionally, when determining the degree of amino acid similarity, those skilled in the art can also take into account so-called conservative amino acid substitutions. As used herein, "conservative" amino acid substitution refers to the interchangeability of residues with similar side chains. Examples of classes of amino acid residues for conservative substitution are shown in the table below.

[0179] [Table 2]

[0180] [Table 3]

[0181] [Table 4]

[0182] For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are: Ala and Ser; Arg and Lys; Asn and Gln or His; Asp and Glu; Cys and Ser or Ala; Gln and Asn; Glu and Asp; Gly and Pro; His and Asn or Gln; Ile and Leu or Val; Leu and Ile or Val; Lys and Arg; Gln and Glu; Met and Leu or Ile; Phe and Met, Leu, or Tyr; Ser and Thr; Thr and Ser; Trp and Tyr; Tyr and Trp or Phe; and Val and Ile or Leu.

[0183] Genes or coding sequences The term "gene" refers to a DNA fragment that contains a region (transcribed region) that is transcribed in a cell into an RNA molecule (e.g., mRNA) and is operably linked to appropriate regulatory regions (e.g., a promoter). A gene usually contains several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3'-untranslated sequence (3' end), including polyadenylation and / or transcription termination sites. Chimeric or recombinant genes are genes that are not normally found in nature, such as genes in which the promoter is not essentially associated with some or all of the transcribed DNA region. "Expression of a gene" refers to the process by which a DNA region operably linked to appropriate regulatory regions, in particular a promoter, is transcribed into RNA that is biologically active, i.e. that can be translated into a biologically active protein or peptide.

[0184] Proteins and Amino Acids The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably and refer to a molecule consisting of a chain of amino acids, without regard to a particular mechanism of action, size, three-dimensional structure, or origin. In the amino acid sequences described herein, the amino acids or "residues" are designated by their three letter symbols. These three letter symbols and their corresponding one letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. The residues can be any proteinogenic amino acid, any non-proteinogenic amino acid, such as D-amino acids and modified amino acids formed by post-translational modifications, and any unnatural amino acid.

[0185] In this specification and the claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included and that items not specifically mentioned are not excluded. In addition, the verb "consisting of" can be replaced with "consisting essentially of" to mean that the compositions described herein may include components other than those specifically disclosed, and the additional components do not change the inherent characteristics of the present disclosure. In addition, the verb "consisting of" can be replaced with "consisting essentially of" to mean that the methods described herein may include steps other than those specifically disclosed, and the additional steps do not change the inherent characteristics of the present disclosure.

[0186] The reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is only one of the element. Thus, the indefinite article "a" or "an" normally means "at least one".

[0187] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ..., etc.

[0188] Moreover, in this specification and claims, terms such as first, second, and third are used to distinguish between similar elements and are not necessarily used to describe an order or chronology. It will be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein can operate in orders other than those described or illustrated herein.

[0189] The terms "about" or "approximately" when used in connection with a numerical value (e.g., about 10), preferably mean that the value can be plus or minus 1% (of 10) of the given value.

[0190] As used herein, the term "and / or" indicates that one or more stated instances may occur alone or in combination with at least one of the stated instances up to all of the stated instances.

[0191] Various embodiments are described herein. Each embodiment disclosed herein can be combined together unless otherwise specified. Titles, subtitles, headings, and the like are used for ease of reading only and are not intended to limit or restrict the disclosure in any way.

[0192] All patent applications, patents, and publications cited herein are incorporated herein by reference in their entirety, except for any definitions, disclaimers or disclaimers of subject matter, and except to the extent that the incorporated material contradicts the explicit disclosure of this specification, in which case the language of the present disclosure shall control.

[0193] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used to practice the present invention. Indeed, the present invention is in no way limited to the methods and materials described. It should also be understood that the present disclosure encompasses generalizations of aspects of the following examples to the preceding disclosure.

[0194] This invention is further illustrated by the following examples which should not be construed as limiting its scope. [Brief description of the drawings]

[0195] [Figure 1] Experimental design. HFGFD-V: rats receiving sham gavage (vehicle); HFGFD-C: rats receiving daily bacterial consortium; HFGFD-Tr: rats receiving intestinal microbiota (IM) taken from lean control rats (one transplant followed by sham gavage). Abbreviations: HFGFD, high-fat high-glucose / fructose diet. [Diagram 2] Assessment of weight gain and insulin resistance. (A) Weight gain after 2 weeks of intervention. (B) Fasting blood insulin levels measured after 2 weeks of intervention. (C) Calculation of the homeostasis model assessment of insulin resistance index. Abbreviations: HFGFD, high fat high glucose / fructose diet. [Diagram 3] Relative mRNA expression of (A) α-SMA and (B) COL1A1 by quantitative RT-PCR, expressed as log2 ratio. β-actin was used as an endogenous control, and results were normalized to the HFGFD-V group. Abbreviations: α-SMA, α-smooth muscle actin; COL1A1, collagen type I α1 chain; HFGFD, high-fat high-glucose / fructose diet. [Figure 4-1]Hepatic hemodynamic parameters and intrahepatic markers of endothelial dysfunction. (A) Portal vein pressure (PP). (B) Intrahepatic vascular resistance (IHVR). (C-E) Western blot analysis of intrahepatic markers of endothelial dysfunction. Bar graphs show quantification of phosphorylated (P)-Akt, P-eNOS, and KLF2 normalized to the HFGFD-V group using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Abbreviations: Akt, protein kinase B; eNOS, endothelial nitric oxide synthase; HFGFD, high-fat high-glucose / fructose diet; KLF2, Krüppel-like factor 2. [Figure 4-2] Hepatic hemodynamic parameters and intrahepatic markers of endothelial dysfunction. (A) Portal vein pressure (PP). (B) Intrahepatic vascular resistance (IHVR). (C-E) Western blot analysis of intrahepatic markers of endothelial dysfunction. Bar graphs show quantification of phosphorylated (P)-Akt, P-eNOS, and KLF2 normalized to the HFGFD-V group using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Abbreviations: Akt, protein kinase B; eNOS, endothelial nitric oxide synthase; HFGFD, high-fat high-glucose / fructose diet; KLF2, Krüppel-like factor 2. [Diagram 5] Inverse Simpson diversity index. Values ​​indicate p-values ​​of t-tests calculated without including the outlier R72. [Figure 6] Multidimensional Scaling (MDS). MDS is based on the Bray-Curtis distance. [Figure 7]sPLS-DA classification. (A) BER for contrast. (B) Top 10 discriminatory taxa. (C) Euler diagram of contrast-specific taxa. Discriminant taxa: Strain identifiers correspond to hits confirmed by WMS taxonomic composition analysis performed by CosmosID and reflect the composition of the database used for the analysis. These should be considered as a proxy for strains actually present in each treatment group. Euler diagram-numbers should be interpreted as follows: For HFGFD-C, 47 derived taxa were discriminants for differentiation from HFGFD-V or HFGFD-Tr, or both. 13 derived taxa were discriminants for HFGFD-V in both V-Tr and VC contrasts. 12 HFGFD-V and HFGFD-Tr associated taxa were suppressed by HFGFD-C. Ninety-one HFGFD-V-related taxa were suppressed by HFGFD-C or HFGFD-Tr, or both. [Figure 8] Effects of the 6-strain and 9-strain populations on (A) fasting blood insulin levels, (B) HOMA-IR index, and (C-D) hepatic hemodynamics. HFGFD-V: rats receiving sham gavage (vehicle); HFGFD-C1: rats receiving the 6-strain population daily; HFGFD-C2: rats receiving the 9-strain population daily; HFGFD-Tr: rats receiving intestinal flora (IM) from control rats (one transplantation followed by sham gavage). [Figure 9] Effect of the 6-strain and 9-strain populations on markers of endothelial dysfunction. (A) Hepatic AKT phosphorylation, (B) eNOS phosphorylation, and (C) KLF2. HFGFD-V: rats receiving sham gavage (vehicle); HFGFD-C1: rats receiving the 6-strain population daily. HFGFD-C2: rats receiving the 9-strain population daily. HFGFD-Tr: rats receiving intestinal microbiota (IM) from control rats (one transplant followed by sham gavage). Legend: AKT, AKT serine / threonine kinase; eNOS, endothelial nitric oxide synthase; KLF2, Krüppel-like factor 2. [Figure 10] STAM™ Study Design. C57BL / 6J male mice were subcutaneously injected with 200 μg of STZ, a beta cell toxin, on day 2 of life. Four-week-old animals were fed a high-fat diet (HFD) or a normal control diet (CD). Treatment began at 5 weeks of age, with animals on the control diet and STAM mice being sham-gavaged (CD+VEH and STAM+VEH, respectively) for a total of 4 weeks. In the two test groups, STAM animals were also administered either a 9-strain population (109 CFU / day) (STAM+CON) or telmisartan (10 mg / kg / day) (STAM+TLM) for a total of 4 weeks. At 9 weeks of age, animals were sacrificed (euthanasia 1). Two groups of STAM animals, either in the vehicle control group or the population group, were followed for an additional 3 weeks until sacrifice at 12 weeks of age (euthanasia 2). [Figure 11] The 9-strain population improves weight gain at late disease stages in STAM™ mice. Weight gain is shown relative to the weight recorded at 5 weeks of age when the 9-strain population and telmisartan (TLM) treatment was started (i.e., 1 week after HFD initiation). Animals were divided into groups and sacrificed at different time points (i.e., 7 weeks, 9 weeks, and 12 weeks of age to target steatosis / steatohepatitis, early fibrosis, and late fibrosis, respectively). Treatment with TLM or the 9-strain population was started at 5 weeks of age and lasted for 2 weeks (until euthanasia 1) or 4 weeks (until euthanasia 2). Some animals of the 9-strain population and vehicle-treated groups were followed up until 12 weeks of age (euthanasia 3). Legend: CD, control diet group; Euth, euthanasia; TLM, telmisartan; VEH, vehicle; CON, 9-strain population. [Figure 12] The 9-strain population improves whole blood HbA1c. Whole blood HbA1c (%) was measured in the STAM™ study. Whole blood was collected in heparinized tubes at 9 and 12 weeks of age (euthanasia 1 and 2, respectively) and used for analysis of glycated hemoglobin, a marker that indicates the presence of excess glucose in the bloodstream. Legend: CD, control diet group; TLM, telmisartan; VEH, vehicle; CON, 9-strain population. [Figure 13-1] Representative images of H&E stained liver sections taken in the STAM™ study. Liver sections were taken at both 9 and 12 weeks of age (euthanasia 1 and 2, respectively). [Figure 13-2] Representative images of H&E stained liver sections taken in the STAM™ study. Liver sections were taken at both 9 and 12 weeks of age (euthanasia 1 and 2, respectively). [Figure 14] NAFLD Activity Score (NAS) in the STAM™ Study. Histopathology was evaluated on H&E stained liver sections and NAS scores were calculated according to the Kleiner criteria. (22) Components (A) inflammation, (B) steatosis, (C) ballooning, and (D) composite score were evaluated at both 9 and 12 weeks of age (euthanasia 1 and 2, respectively). ND: Not determined in CD+VEH and STAM+TLM groups at 12 weeks of age. [Figure 15-1] Representative images of (A) Picro-Sirius Red stained and (B) F4 / 80 immunostained liver sections taken during the STAM™ study. Liver sections were taken at both 9 and 12 weeks of age (euthanasia 1 and 2, respectively). [Figure 15-2] Representative images of (A) Picro-Sirius Red stained and (B) F4 / 80 immunostained liver sections taken during the STAM™ study. Liver sections were taken at both 9 and 12 weeks of age (euthanasia 1 and 2, respectively). [Figure 16] Liver fibrosis area, F4 / 80 positive area, and serum CK-18 levels in the STAM™ study. (A) Fibrosis assessed on Sirius Red stained liver sections. (B) Immunohistochemistry of macrophages assessed on F4 / 80 immunostained sections. (C) Serum levels of CK-18, a marker of apoptotic hepatocytes. All markers were assessed at both 9 and 12 weeks of age (euthanasia 1 and 2, respectively). ND: Not determined in CD+VEH and STAM+TLM groups at 12 weeks of age. [Figure 17-1]Short-chain fatty acid (SCFA) profiles in HV5 medium. (A) Veillonella parvula and Veillonella atypica strains. (B) Phocaeicola vulgatus strain. (C) Blautia obeum, Blautia wexlerae, and Blautia luti strains. [Figure 17-2] Short-chain fatty acid (SCFA) profiles in HV5 medium. (A) Veillonella parvula and Veillonella atypica strains. (B) Phocaeicola vulgatus strain. (C) Blautia obeum, Blautia wexlerae, and Blautia luti strains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0196] Working Example In Example 1, the efficacy of a bacterial consortium consisting of nine human gut commensal lineages was tested in a relevant in vivo animal disease model, namely the rat NASH model of portal hypertension (PHT). Results were compared to animals receiving a single fecal transplant from lean rats. The bacterial consortium was shown to improve weight gain and portal hypertension, and also improved endothelial dysfunction markers by restoring insulin sensitivity. In addition, liver transcriptomics identified multiple pro-fibrotic pathways that were upregulated.

[0197] In Example 2, the 9-strain population from Example 1 was compared to the 6-strain population in a rat NASH model. The 9-strain population includes the 6 strains of the 6-strain population, plus three additional species included to increase propionate production. Both populations were effective: the 9-strain population showed consistent results across multiple endpoints, and the 6-strain population induced a protective effect on several endpoints, including fasting insulin levels, HOMA-IR index, and liver hemodynamics (PP, IHVR).

[0198] In Example 3, the efficacy of the 9-strain group was further tested in another relevant in vivo animal disease model, namely the STAM™ mouse model of NASH. Administration of the 9-strain group confirmed anti-fibrotic effects up to 12 weeks of age. In addition, F4 / 80 immunohistochemistry was accompanied by a reduction in liver inflammation, and serum levels of cytokeratin-18 (CK-18), a blood marker of apoptotic hepatocytes that correlates with disease severity, were significantly reduced after 5 weeks of treatment.

[0199] Altogether, these results indicate that microbe-based approaches can be used to manage liver conditions such as NAFLD, and that gut commensal populations offer patients a safe, convenient, and long-term solution to slow disease progression.

[0200] The six-strain panel tested included the following bacterial species: Faecalibacterium prausnitzii (strain LMG P-29362) Butyricococcus pullicaecorum (LMG P-29360 strain) Roseburia inulinivorans (strain LMG P-29365) Akkermansia muciniphila (strain LMG P-29361) Lactiplantibacillus plantarum (LMG P-29366 strain) Anaerostipes caccae (strain LMG P-29359).

[0201] The nine-strain panel tested included the following bacterial species: Faecalibacterium prausnitzii (strain LMG P-29362) Butyricococcus pullicaecorum (LMG P-29360 strain) Roseburia inulinivorans (strain LMG P-29365) Akkermansia muciniphila (strain LMG P-29361) Lactiplantibacillus plantarum (LMG P-29366 strain) Anaerostipes caccae (strain LMG P-29359) Phocaeicola vulgatus (strain LMG17767) Veillonella parvula (DSM 2007 strain) Blautia obeum (strain DSM 25238).

[0202] The six-strain population is also described in WO 2017 / 134240. In WO 2017 / 134 / 240, the population was shown to be robust and independent of the exact strains contained in each species. Thus, one skilled in the art will understand that the results presented herein can be obtained using strains other than those exemplified.

[0203] Example 1: Evaluation of a six-strain panel in a rat NASH model of portal hypertension (PHT) 1.1. Materials and methods Experimental design 1.1.2. For model development, Sprague-Dawley rats were fed a HFGFD (high-fat diet and high glucose / fructose syrup) for 8 weeks (Figure 1). Rats were then randomly assigned into three groups: HFGFD-vehicle (HFGFD-V, n = 13), a HFGFD population of nine human commensal bacterial strains (HFGFD-C, n = 11), and a HFGFD transplanted with fecal material from lean rats (HFGFD-Tr, n = 11). HFGFD-V individuals received oral probing vehicle (sterile PBS), whereas individuals in the HFGFD-C and HFGFD-Tr groups received different microbiota-based treatments consisting of oral administration of bacterial commensals and intestinal microbiota (IM) transplantation, respectively. Each treatment is described in detail in the following sections. The treatment period lasted for 2 weeks, during which the animals were maintained on their original diet (Figure 1). At week 10, liver hemodynamics and blood biochemistry were performed and liver tissue samples were taken for histological and molecular analysis. Additionally, cecal contents were collected for shotgun sequencing of gut microbes.

[0204] Animal models and diets Male Sprague-Dawley OFA rats (Charles River Laboratories, L'Arbresle, France) weighing 200–220 g at the start of the experiment were used for the diet-induced NASH model, as already described previously in (9). The rats were housed under a 12-h light-dark cycle at constant temperature (24 ± 1 °C) and relative humidity (55 ± 10%). The animals were fed ad libitum a high-fat high-glucose / fructose diet (HFGFD) consisting of 30% fat (butter, coconut oil, palm oil, beef tallow) containing mainly saturated fatty acids (5.73 kcal / g; Ssniff Spezialdiaeten GmbH, Soest, Germany), supplemented with cholesterol (1 g / kg), and a beverage with a high glucose / fructose (42 g / L, 45% glucose, and 55% fructose) content providing 157.7 kcal / L. Body weight and food and beverage intake were monitored once a week.

[0205] All procedures were performed in the animal facilities of the Vall d'Hebron Research Institute in accordance with the European Union Guidelines for Ethical Care of Experimental Animals (EC Directive 86 / 609 / EEC on animal experimentation), approved by the Animal Care Committee of the Vall d'Hebron Research Institute (Barcelona, ​​Spain).

[0206] Bacterial population treatment The group treatment started after 8 weeks of dietary intervention. Lyophilized vials containing the nine bacterial strains were incubated in anaerobic conditions (N 2 :CO 2 :H 2After decontamination of the anaerobic chamber and vial lids, the concentrated strains obtained were gently resuspended in 20 ml of previously filtered anaerobic DPBS (Dulbecco's phosphate buffered saline without calcium chloride and magnesium chloride, Sigma Aldrich). The contents of the vials were poured into sterile 50 ml Falcon tubes and divided and removed from the anaerobic chamber. The 50 ml Falcons were centrifuged at 5,340 g for 10 min at room temperature. The Falcons were returned to the anaerobic chamber and the supernatant was then poured off and discarded. Each pellet was resuspended in 3 ml of anaerobic DPBS and then 3.5 ml of DPBS was added to each tube to obtain a final volume of 6.5 ml per vial. All samples were finally transferred to glass sealed sterile vials in an anaerobic atmosphere. Once the populations were reconstituted, 1 ml of suspension per individual (2 × 10 9 The rats were immediately administered 100 mg of 1000 mg of 10000 CFU (equivalent to a daily dose of 10000 CFU) and the procedure was repeated every 24 hours for 10 consecutive days. For the rats belonging to the vehicle group, the same procedure was followed but they were administered sterile DPBS instead. The rats that received the bacterial consortium were housed individually during the treatment period to avoid cross-contamination.

[0207] Intestinal microbiota (IM) transplantation IM transplantation was performed after 8 weeks of dietary intervention. To achieve a higher homogeneity in the transplantation procedure, feces from three control (lean) rat donors were pooled and used for IM transplantation. Omeprazole was administered orally at a dose of 50 kg / day for 3 days prior to intestinal decontamination, aiming to reduce gastric acidity and increase microbial survival. To facilitate intestinal emptying, rats were subsequently isolated on a high-speed grill and administered two oral doses of 1 ml and 2 ml of CitraFleet (sodium picosulfate 0.16 mg / ml and magnesium oxide 51.2 mg / ml), respectively, 24 h and 12 h prior to transplantation. These doses were each administered with 2 ml of water. Recolonization was performed by a single oral gavage of 100 mg of the fecal pool dissolved in 2 ml of sterile DPBS.

[0208] blood biochemistry Blood samples were collected from the vena cava at the time of sacrifice. Glucose, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, and albumin were measured by standard methods (see Garcia-Lezana, T et al. Restoration of a Healthy Intestinal Microbiota Normalizes Portal Hypertension in a Rat Model of Nonalcoholic Steatohepatitis. HEPATOLOGY 2018;67(4)). Insulin was measured using an ELISA kit according to the manufacturer's instructions (EMD Millipore, Billerica, MA, United States). Insulin resistance was estimated by applying the homeostatic model of insulin resistance index (HOMA-IR): (fasting insulin (ng / mL) × fasting glucose (mg / dL)) / 405.

[0209] Histological analysis For histological analysis, liver samples were extracted, fixed in 4% formalin, embedded in liquid paraffin at 65 °C, and sectioned on 4 μm-thick slides. Samples were then stained with hematoxylin and eosin (H&E) to evaluate the liver parenchyma and with Sirius Red to detect collagen fibers. All stained liver samples were examined by an expert hepatic pathologist who was blinded to the interventions performed on the animals.

[0210] The diagnosis of NASH was established based on the presence of all three characteristic patterns of the disease, including the coexistence of steatosis, lobular inflammation, and hepatocellular ballooning. NASH activity was quantified using the NASH-CRN activity score (NAS). The NAS comprises the unweighted sum of the histologic components: steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2). Fibrosis was also classified into five stages according to the NASH-CRN system, ranging from F0 (no fibrosis) to F4 (cirrhosis).

[0211] Hepatic Hemodynamics Hemodynamic measurements were performed in fasting rats under intraperitoneal anesthesia with ketamine (100 mg / Kg) and midazolam (5 mg / kg) with body temperature maintained at 37°C. Mean arterial pressure (MAP) was measured by catheterization of the femoral artery, and portal vein pressure (PP) was assessed by ileocolic vein catheterization using a highly sensitive pressure transducer (Harvard apparatus, Holliston, MA, USA). Superior mesenteric artery (SMA) blood flow (SMABF, mL / [min×100 g]) and portal vein blood flow (PBF, mL / [min×100 g]) were measured with a perivascular ultrasonic transit-time flow probe (1 mm diameter, Transonic systems Inc, Ithaca, NY, USA). SMA resistance (SMAR) and intrahepatic vascular resistance (IHVR, mmHg / mL×min×100 g) were calculated as ([MAP-PP] / SMABF) and (PP / PBF), respectively.

[0212] Western blot The liver was perfused with saline for exsanguination, and the samples were directly frozen in liquid nitrogen and stored at -80°C until further use. The liver samples were then manually cold-ground to powder without thawing. The liver samples were then homogenized in Triton lysis buffer, sonicated, and centrifuged at 14,000 rpm for 10 min at 4°C. The total protein concentration of the supernatant was quantified by BCA protein assay kit (Thermo Fisher scientific, Waltham, MA, USA). 40 μg of protein was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membrane (Thermo Fisher scientific, Waltham, MA, USA). The membrane was washed several times with TTBS 1X and blocked with PhosphoBLOCKER™ 5% (Cell biolabs, San Diego, CA, USA) for 1 h at RT to detect phosphorylated proteins P-Akt (1 / 500, Cell signaling, Danvers, MA, USA) and P-eNOS (Ser1177, 1 / 250) or with skim milk to detect Krüppel-like factor 2 (KLF2) (1 / 200, Santa Cruz biotechnology, Dallas, TX, USA). GAPDH antibody (1 / 5000, Ambion, Austin, TX, USA) was used as a loading control. Membranes were developed with an ECL kit (GE Healthcare; Little Chalfont, UK) and protein expression was finally determined by densitometric analysis bands using Image Studio Lite (Lincoln, NE, USA).

[0213] Real-time PCR Liver and ileum samples were kept in RNAlater (Thermo Fisher scientific, Waltham, MA, USA) for at least 24 hours and then stored at -80°C until further analysis. Total RNA was extracted using RNeasy mini kit (QIAGEN, Venlo, Netherlands) and retrotranscribed into complementary DNA (High-capacity cDNA reverse transcription, Thermo Fisher Scientific, Waltham, MA, USA). 20 ng of cDNA was added to Taqman universal PCR master mix and specific Taqman probes for α-SMA (α-smooth muscle actin) and COL1A1 (collagen type I α1 chain) (Life Technologies Ltd, Renfrew, UK) and loaded into a 384-well plate (Thermo Fisher scientific, Waltham, MA, USA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a 7900HT fast real-time PCR system (Thermo Fisher scientific, Waltham, MA, USA). Relative gene expression was normalized to β-actin. Data were analyzed using the Relative Quantitative qPCR Application on Thermofisher Cloud.

[0214] Cecal whole metagenomic shotgun Genomic DNA was extracted from approximately 0.2 g of cecal contents of each rat using a ZymobioMics DNA Miniprep kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions, quantified using a Qubit™ Flex Fluorometer (Thermo Fisher Scientific, Wilmington, DE, USA), and used for whole-metagenomic shotgun sequencing. Library preparation and sequencing were performed by CosmosID (Rockville, MD, USA) to generate 2 × 150 bp Illumina reads. Unassembled sequencing reads (≥ 12M read depth) were analyzed using the CosmosID bioinformatics platform for taxonomic and functional profiling.

[0215] Whole metagenomic shotgun (WMS) sequencing, taxonomic and pathway profiling were performed by CosmosID (Rockville, MD, USA). 2 × 150 bp Illumina reads were mapped to a proprietary microbial genome database for taxonomic profiling (Thoendel M et al. Journal of Clinical Microbiology 2020;58(3):e00981-19) and to UniRef90 and MetaCyc for pathway profiling (Franzosa EA et al. Species-level functional profiling of metagenomes and metatranscriptomes. Nature Methods 2018;15(11):962-8). Treatment-induced modulation of microbial composition was assessed in both unsupervised (multidimensional scaling with UniFrac distances) and supervised (sparse partial least squares discriminant analysis, sPLS-DA) analyses using the R packages phyloseq (McMurdie PJ, Holmes S. PLoS ONE 2013;8(4):e61217) and mixOmics (Rohart F. PLOS Computational Biology 2017;13(11):e1005752), respectively. The performance of the latter classifiers was assessed by the average false positive and false negative rates (BER).

number

[0216] 1.1.3. Liver transcriptomics RNA extraction, RNA library preparation, and sequencing reactions were performed at GENEWIZ, LLC. (South Plainfield, NJ, USA) according to the manufacturer's instructions (Qiagen, Hilden, Germany) as follows: Total RNA was extracted from fresh frozen liver tissue samples using the Qiagen RNeasy Plus Universal mini kit. RNA samples were quantified using a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was measured using an RNA Screen Tape on Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit according to the manufacturer's protocol (Illumina, Cat# RS-122-2101). First, mRNA was enriched with oligo d(T) beads. The enriched mRNA was fragmented at 94°C for 8 min. Subsequently, first- and second-strand cDNA were synthesized. The second strand of cDNA was marked by incorporating dUTP during synthesis. The cDNA fragments were adenylated at the 3' end, and indexed adapters were ligated to the cDNA fragments. Limited cycle PCR was used to enrich the library. The dUTP incorporated into the second-strand cDNA quenched the amplification to maintain strand specificity. The sequencing library was verified using a DNA Analysis Screen Tape on the Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, CA, USA) and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The pooled libraries were clustered into 7 lanes of a flow cell.After clustering, the flow cells were loaded onto an Illumina HiSeq instrument (4000 or equivalent) according to the manufacturer's instructions and sequenced using a 2 × 150 bp paired end (PE) configuration. Image analysis and base calling were performed by HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from the Illumina HiSeq were converted to fastq files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

[0217] Strand-specific gene expression profiling was achieved by mapping 2 × 150 bp Illumina reads to the Ensembl Rattus norvegicus Rnor_6.0 transcriptome and processing the alignment with the subread package featureCounts (Liao Y et al. Bioinformatics 2014;30(7):923-30). Rat Ensembl gene IDs were mapped to human Ensembl gene equivalents and HUGO Human Genome Nomenclature Committee (HGNC) symbols by Ensembl Biomart. The resulting unique mapping fragment counts were entered into differential gene expression analysis using the R package DESeq2 (Anders S, Huber W. Genome Biology 2010;11(10):R106) to obtain differential gene expression with p-values ​​adjusted for the Benjamini & Hochberg false discovery rate (FDR). Outlier analysis was performed with orthogonal partial least squares analysis using the R package ropls (Thevenot EA. Journal of Proteome Research 2015;14(8):3322-35). At the same time, sPLS-DA was performed to estimate variable importance (VIP). Hepatic genes discriminating between treatment and vehicle contrast were retained as having VIP>1 and adjusted p-value<0.05. Functional overexpression analysis (ORA) of these genes was performed with the R package clusterProfiler (Wu T et al. The Innovation 2021;2(3):100141) using the WikiPathways functional annotation database (Martens M et al. Nucleic Acids Research 2021;49(D1):D613-21.). The R package RcisTarget (Aibar S et al. SCENIC: Single-cell regulatory network inference and clustering. Nature Methods 2017;14(11):1083-6) was used to predict transcriptional motifs enriched in low- or overexpressed genes. High-confidence transcription factors associated with these motifs were retained.

[0218] 1.1.4. Biological parameters analysis Continuous variables were reported as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA). Biological parameters of treatment groups were compared by Student's t test or one-way analysis of variance with Dunnett's multiple comparison test. P values ​​<0.05 were considered statistically significant.

[0219] 1.1.5. Results 1.1.6. Mass treatment significantly improved body weight During the 2-week treatment period, animals in the HFGFD-V group gained significantly more body weight (22.08 ± 3.18 g), as seen in Figure 2A. However, HFGFD-C gained only an average of 2.91 ± 3.55 g, whereas HFGFD-Tr lost an average of 5 ± 3.47 g. Compared to HFGFD-V, both groups receiving microbiota-based treatments gained significantly less weight during the 2-week treatment period (Figure 2A).

[0220] Mass treatment significantly improved metabolic profiles As can be seen in Figures 2B and 2C and Table 2, both microbiota-based treatments showed a significant reduction in fasting blood insulin levels and the HOMA-IR index for the HFGFD-V group.

[0221] [Table 5]

[0222] Decreased expression of profibrotic liver marker genes By the time the animals were sacrificed (10 weeks on HFGFD), profibrotic pathways were already activated, and gene expression of the profibrotic hepatic markers α-SMA and COL1A1 was significantly decreased in the cohort-treated group compared to the vehicle group (Figure 3). These results are further supported by genome-wide RNA-Seq analysis results described below.

[0223] Mass treatment significantly reduced portal vein pressure and improved hepatic hemodynamics Consistent with previous findings (9), despite the absence of histological fibrosis, animals with a 10-week HFGFD intervention showed an increase in portal vein pressure (PP) with a value of 10.32 mmHg on average (Table 3 and Figure 4A). Importantly, both bacterial population treatment and IM transplantation significantly reduced PP compared to the vehicle-treated group, even maintaining the HFGFD during the 2-week treatment period (Table 3 and Figure 4A). This reduction in PP observed in the HFGFD-C group was secondary to a significant reduction in intrahepatic vascular resistance (IHVR) compared to the HFGFD-V (80% reduction) and was comparable to that measured in the IM-Tr group (104% reduction) (Figure 4B). With regard to systemic hemodynamics, no relevant changes were observed between the groups (Table 3).

[0224] [Table 6]

[0225] Characteristics of endothelial dysfunction (ED) were improved in animals following group treatment The properties of ED molecules were evaluated to further characterize the changes observed in IHVR and PP induced by microbiota-based treatment relative to the vehicle-treated group. To this end, phosphorylated protein kinase B (P-Akt) and endothelial nitric oxide synthase (P-eNOS) were analyzed. In addition, to evaluate possible transcriptional regulatory mechanisms of e-NOS, KLF2 was evaluated since it is considered essential for the maintenance of a functional endothelial phenotype. As seen in Figure 4C-E, microbiota-based treatment induced a significant increase in P-Akt. P-eNOS showed a tendency to be increased in both HFGFD-C and HFGFD-Tr compared to HFGFD-V. Interestingly, HFGFD-C was unique in showing a prominent and significant increase in KLF2.

[0226] Microbiota-based treatment improves cecal species diversity and promotes significant cecal microbiota compositional changes The microbiota-based treatment improves cecal species diversity, as shown by the inverse Simpson index in the phyloseq package (Figure 5). One outlier in the microbial population treatment group, rat R72 from the second batch, was identified.

[0227] Beta diversity assessed by multidimensional scaling applied without CD groups shows good separation of HGHFD-V, HGFD-C, and HGFD-Tr groups (Figure 6).

[0228] The performance of the classifier, assessed by the BER of the HFGFD-C contrast (0.03), compares favorably with that of the HFGFD-Tr contrast (0.34) (Figure 7A). It can therefore be argued that HFGFD-C induces more distinct changes than HFGFD-Tr, which may be dominated or limited by the addition of producer strains. However, the corresponding feature selection (VIP>1, n=123) shows that the bacterial population treatment induced the suppression of 72 (58.5% of the observable changes) vehicle-associated taxa and promoted the emergence of 48 (41.5%) non-producer treatment-associated taxa, whereas microbiota transplantation with feature selection (n=178) induced the suppression of 135 (75.8%) taxa and their replacement in 43 (24.2%) taxa. Thus, both treatments led to significant replacement of HGHFD-V associated microbial communities, but bacterial populations achieved a clearer and more abundant replacement than transplantation, which was more inhibitory in nature since in this group gut emptying treatment was performed prior to transplantation to increase engraftment rates. Figure 7B provides the top 10 discriminatory taxa according to the classifier's estimated variable importance (VIP) and their associations, while Figure 7C summarizes the three VC, V-Tr, and C-Tr contrasts in a single Euler plot proportional to the number of features selected in each contrast.

[0229] Bacterial consortium treatment induces differential hepatic gene expression and downregulates fibrosis-associated genes The expression of 32,552 hepatic genes was recorded by RNA-Seq analysis. Of these, 732 (2.2%) were discriminants (VIP>1, FDR p-value<0.05) for either or both of the HFHGD-C (n=252) or HFHGD-Tr (n=632) contrasts. In HFHGD-C, there was a trend towards upregulation of treatment-related genes (n=157, 62.3%), whereas in HFHGD-Tr, there was a balance between upregulation of treatment-related genes (n=315, 49.8%) and downregulation of vehicle-related genes (n=317, 50.4%).

[0230] Overrepresentation analysis (ORA) was performed on upregulated genes in either HFHGD-C or HFHGD-Tr or both and did not detect any significant pathway overrepresentation against the WikiPathways annotation database. The same analysis performed on downregulated genes detected several pathways related to fibrosis.

[0231] Example 2: Comparison of 6-strain and 9-strain populations in a rat NASH model of portal hypertension (PHT) Example 2 was carried out using the same materials and methods as described in Example 1. The 9-strain group showed consistent results for multiple endpoints. The 6-strain group also induced significant protective effects, especially on fasting insulin levels, HOMA-IR index, and hepatic hemodynamics (PP, IHVR) (Figure 8).

[0232] In the 6-strain treated group, phosphorylated (p)-AKT and p-eNOS were virtually unchanged in response to treatment (Figures 9A and 9B), suggesting that other mechanisms may play a role in improving insulin resistance and thereby portal hypertension; in addition, the increased expression of KLF2 appears to be specific to the 9-strain population (Figure 9C).

[0233] Thus, despite their similar composition, the effects of these two populations were not completely comparable. In particular, the addition of a strain capable of producing propionate seems advantageous and may activate additional beneficial mechanisms.

[0234] Example 3: STAM™ Mouse Model of NASH 1.2. Materials and Methods Experimental design STAM™ test was performed at SMC Laboratories, Ltd., Japan. Briefly, NASH was induced in C57BL / 6J male mice by a single subcutaneous injection of 200 μg of streptozotocin (STZ, Sigma-Aldrich, USA) solution on postnatal day 2 and introduction of a high-fat diet (HFD, 57 kcal% fat, Cat#HFD32, CLEA Japan, Inc., Japan) at 4 weeks of age.

[0235] The STAM mouse model has the substantial advantage that the development of HCC due to NASH is identical to the known progression in human patients, but the entire process is completed in a relatively short period of time (Fujii, M. et al. Med. Mol. Morphol. 2013;46:141-152). In this model, 2-day-old male C57BL / 6 mice are administered a single dose of streptozotocin (STZ) to reduce insulin secretion capacity. At 4 weeks of age, mice begin to be fed a high-fat diet (HFD). This model can be summarized as follows (Table 4).

[0236] [Table 7]

[0237] On the second day of life, C57BL / 6J mice are injected with the beta cell toxin STZ. This primes the animals so that a second hit, starting at 4 weeks of age, i.e., on a continuous high-fat diet (HFD), will rapidly progress the disease. By 5 weeks of age, hepatic lipid accumulation (steatosis) is evident, and by 6 weeks of age, animals have increased NAFLD activity scores (NAS) in histology and elevated ALT levels. By 7-8 weeks of age, liver inflammation is evident, and by 9-12 weeks, fibrosis is evident. HCC can be observed at approximately 16 weeks of age. This model is consistent with late-stage T2D progressing to fatty liver, NASH, fibrosis, and eventual hepatocellular carcinoma (HCC). Compared to other NASH-HCC models, the disease progresses over a relatively short period of time, and hepatocellular carcinoma develops in all animals by 20 weeks of age. The advantages of this model are the rapid disease onset and progression, and the complete penetrance of the phenotype. Depending on the target stage of disease progression, animals are euthanized at early or late stages of disease. This model shows prominent signs of fibrosis in histopathology.

[0238] Animals were randomly assigned to three treatment groups based on their body weight the day before feeding the HFD. Randomization was performed by weight-stratified random sampling using Excel software. Animals aged 5 weeks (after 1 week on HFD) were administered either sham gavage (STAM+VEH, n=16), a nine-strain bacterial consortium (STAM+CON, n=18), or telmisartan (STAM+TLM, n=8). A control group (CD+VEH, n=8) fed a regular diet and subjected to sham gavage was also included in the study (Figure 10). Animals were maintained in an SPF facility under controlled conditions of temperature (23±3°C), humidity (50±20%), lighting (12-h artificial light-dark cycle; lights on 8:00–20:00), and air exchange. This animal experiment was performed in accordance with the following guidelines: Act on Welfare and Management of Animals (Ministry of the Environment, Act No. 105 of October 1, 1973, Japan; Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notice No. 88 of the Ministry of the Environment, April 28, 2006, Japan)) and Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006).

[0239] Treatment Management Vehicle (sterile PBS) and bacterial populations were administered orally in a volume of 200 μl / mouse: bacterial populations were administered at 10 9 CFU / day for a total of 4 weeks. Product suspensions were prepared as described above. Telmisartan was used as a benchmark and was orally administered at a daily dose of 10 mg / kg in a volume of 10 ml / kg for 4 weeks. For this, one tablet of telmisartan was transferred to a mortar and crushed with a pestle with the addition of purified water until a homogenous suspension of 1 mg / ml was obtained. All treatments were freshly prepared before administration. Body weights were recorded daily throughout the experimental period.

[0240] Slaughter and sampling Animals belonging to the control group (CD+VEH) and the STAM groups (STAM+VEH, STAM+CON, and STAM+TLM) were sacrificed after 4 weeks of treatment, i.e., at 9 weeks of age, corresponding to the stage of steatohepatitis / early fibrosis. Furthermore, two groups of STAM animals belonging to the vehicle and population groups were followed for another 3 weeks and sacrificed at 12 weeks of age, corresponding to the stage of end-stage fibrosis. This was done to investigate the possibility of bacterial populations slowing disease progression upon treatment cessation (Figure 10). Animals were sacrificed by exsanguination via direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.). Non-fasting blood was collected from the facial vein in polypropylene tubes containing anticoagulant (Novo-heparin, Mochida Pharmaceutical, Japan). 50 μL of blood collection was used for glycated hemoglobin (HbA1c) analysis. Non-fasting blood was also collected for serum preparation in separate tubes without anticoagulant. They were incubated at room temperature for 30 min and then centrifuged at 3,500 × g for 5 min at 4 °C. The remaining serum was collected and stored at -80 °C until further use in serum CK-18 level quantification and biochemistry. Whole livers were harvested and washed with cold saline. Liver specimens were embedded in OCT (Optimal Cutting Temperature) (OCT, Sakura Finetek Japan, Japan) compound for immunohistochemistry at -80 °C or fixed in Bouin's solution (Sigma-Aldrich Japan, Japan) for 24 h. After fixation, the specimens were embedded in paraffin for H&E and Sirius Red staining. Specimens embedded in OCT were used for F4 / 80 immunohistochemistry (see below).

[0241] blood biochemistry HbA1c in whole blood was quantified by DCN2000+ (Siemens Healthcare Diagnostics, USA). Serum CK-18 levels were measured using a mouse cytokeratin 18-M30 ELISA kit (Cusabio Biotech Co., Ltd, China). Serum alanine aminotransferase (ALT) and triglyceride concentrations were measured by FUJI DRI-CHEM 7000 (Fujifilm Corporation). Serum total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels were quantified by HPLC at Skylight Biotech Inc. (Japan).

[0242] Histological analysis Sections were cut from paraffin blocks of liver tissue using a rotary microtome (Leica Microsystems). After sectioning, each slide was coded with a number for blind evaluation. Each number was generated using the RAND function of Excel software, sorted in ascending order, and assigned to a slide. Tissue slides were used for the following stainings and evaluated by experiment: For H&E staining, sections were cut from paraffin blocks of liver tissue prefixed in Bouin's solution and stained with Lillie-Mayer's hematoxylin (Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (FUJIFILM Wako Pure Chemical Corporation, Japan). NAFLD activity scores (NAS) were calculated according to the Kleiner criteria. (22) For scoring of NAS, bright-field images of H&E stained sections were taken at 50x and 200x magnifications using a digital camera (DFC295; Leica, Germany). A steatosis score of one section / mouse (one representative field at 50x magnification), an inflammation score of one section / mouse (one representative field around the central vein at 200x magnification), and a ballooning score of one section / mouse (one representative field around the central vein at 200x magnification) were estimated. Bouin-fixed liver sections were stained with Picro-Sirius Red solution (Waldeck, Germany) to visualize collagen deposition. Briefly, sections were deparaffinized, hydrophilized with xylene, 100-70% alcohol series, and RO water, and then treated with 0.03% Picro-Sirius Red solution (catalog number: 1A-280) for 60 min. After washing with 0.5% acetic acid solution and RO water, the stained sections were dehydrated, cleared with a 70-100% alcohol series and xylene, sealed with Entellan™ (Merck, Germany) and used for observation. For quantitative analysis of fibrosis areas, bright-field images of Sirius Red-stained sections were taken around the central vein at 200x magnification using a digital camera (DFC295; Leica, Germany), and the positive areas in five fields / section were measured using ImageJ software (National Institutes of Health, USA).

[0243] For immunohistochemistry, sections were cut from frozen liver tissue embedded in Tissue-Tek OCT compound and fixed in acetone. Endogenous peroxidase activity was assayed using 0.03% H 2 O 2 After blocking with 100 mM NaCl for 5 min, sections were incubated with Block Ace (Sumitomo Dainippon Pharma Co., Ltd., Japan) for 10 min. Sections were incubated with anti-F4 / 80 antibody (monoclonal antibody, T-2006, BMA Biomedicals) at 4°C overnight. After incubation with secondary antibody (VECTASTAIN ABC KIT, PK-4004, Vector Laboratories), sections were stained with 3,3'-diaminobenzidine / H 2 O 2 The enzyme-substrate reaction was performed using solution (Nichirei Biosciences, Inc., Japan). Bright-field images of F4 / 80 immunostained sections were taken around the central vein at 200x magnification using a digital camera (DFC295; Leica, Germany), and the positive areas of five fields / section were measured using ImageJ software (National Institutes of Health, USA).

[0244] result Liver transcriptomics performed in NASH rats suggested that the tested bacterial populations could favorably modulate profibrotic pathways, prompting us to test the same populations against a rodent model that shows prominent signs of fibrosis in histopathology. To that end, we used the STAM™ mouse model. In this model, male mice were subcutaneously injected shortly after birth with STZ, a beta cell toxin that induces mild pancreatic islet inflammation and destruction. In addition, STZ-primed mice started a continuous HFD at 4 weeks of age. This induces successive histological changes, from fatty liver to NASH, fibrosis, and finally tumor protrusion. The disease progresses rapidly, with a penetrance rate of almost 100%. This allows us to study the protective effect of drugs at well-defined time points, depending on the stage of the disease of interest. For studies targeting fibrosis, animals can be sacrificed between 8 and 12 weeks of age. Therefore, the effect of the 9-strain group was evaluated at the end of the 4-week treatment period, i.e. at the steatohepatitis / early fibrosis stage (9 weeks of age), and also at 12 weeks of age with late fibrosis. The results were compared with the benchmark telmisartan, an angiotensin II receptor blocker that reduces hepatic fat accumulation and inhibits hepatic stellate cell activation, thus suppressing liver fibrogenesis. The effect was evaluated at the histopathological level and blood was collected for biochemical analysis.

[0245] A trend towards improved weight gain was observed in the group treated by the end of the study, i.e., 9-12 weeks of age (Figure 11). However, the STAM™ model is not the most suitable for tracking obesity, as the STAM animals are lean in contrast to other models of diet-induced NASH. As with the NASH rats, no significant changes were observed in blood parameters, i.e., ALT and cholesterol levels (Error! Reference not found).

[0246] Additionally, a decrease in whole blood levels of HbA1cv (glycated hemoglobin) was observed at the end of the study, indicating a beneficial effect on diabetes (Figure 12).

[0247] [Table 8]

[0248] The gut commensal 9 population ameliorated NAS in pathological tissues at 9 weeks of age Liver sections were taken to assess NAFLD features, namely hepatic inflammation, steatosis, and ballooning. Representative images of H&E stained liver sections are shown in FIG. 13 (Error! Reference not found); calculated scores are shown (Error! Reference not found). As expected, liver sections from vehicle-treated STAM group showed microdroplet and macrodroplet fat deposits, hepatocyte ballooning, and inflammatory cell infiltration. Bacterial consortium-treated STAM mice showed a significant reduction in NAS score compared to the diseased group at 9 weeks of age (Error! Reference not found D). This was the result of a reduction in steatosis and ballooning scores (Error! Reference not found B and C). At 12 weeks of age, this was no longer observed.

[0249] Intestinal commensal 9 population improved fibrosis and reduced hepatic expression of F4 / 80 and serum CK-18 levels To assess collagen deposition, liver sections were stained with picro-sirius red. Representative images are shown in Fig. (Error! Reference not found. Error! Reference not found. A). Calculated areas are shown (Error! Reference not found. A). As expected, liver sections from the vehicle-treated STAM group showed increased collagen deposition in the pericentral regions of the liver lobule compared to the control diet group. The bacterial population-treated group showed a significant reduction in the area of ​​fibrosis (Sirius red positive) compared to the disease group at 12 weeks of age, but not at 9 weeks of age. To further assess inflammation, macrophage F4 / 80 immunostaining was used on liver sections. Although no significant reduction in overall inflammation was observed in H&E stained sections (Error! Reference source not found. A and Figure 13), F4 / 80 stained sections showed a significant reduction in macrophage inflammatory infiltration in the mass-treated group at 9 weeks of age, with a trend towards reduction at 12 weeks of age (Error! Reference source not found. B and Figure (Error! Reference source not found. B)). Finally, CK-18 serum levels were also significantly reduced in the mass-treated group at 9 weeks of age, with a trend towards reduction by 12 weeks of age (Error! Reference source not found. C), a result that correlates nicely with the histological activity.

[0250] These results confirm that the nine-strain panel can indeed prevent the progression of NASH disease to fibrosis.

[0251] Example 4 SCFA production from Phocaeicola vulgatus (strain LMG17767), Veillonella parvula (strain DSM 2007), and Blautia obeum (strain DSM 25238) was measured and compared with alternative strains of the same or closely related species.

[0252] [Table 9]

[0253] All strains tested were commensal gut bacteria isolated from fecal material of healthy human donors. SCFA levels were quantified by gas chromatography (GC).

[0254] All strains tested showed very similar SCFA profiles (Figure 17), indicating that the above population results can also be obtained by substituting Phocaeicola vulgatus (strain LMG17767), Veillonella parvula (strain DSM 2007), and / or Blautia obeum (strain DSM 25238) with alternative strains of the same or closely related species.

[0255] References 1. Younossi ZM, Golabi P, de Avila L, Paik JM, Srishord M, Fukui N, et al.The global epidemiology of NAFLD and NASH in patients with type 2 diabetes: A systematic review and meta-analysis.J.Hepatol.2019;71:793-801. 2. Qian Y,Fan JG.Obesity,fatty liver and liver cancer.Springer;2018. 3. Li J,Zou B,Yeo YH,Feng Y,Xie X,Lee DH,et al.Prevalence,incidence,and outcome of non-alcoholic fatty liver disease in Asia,1999-2019:a systematic review and meta-analysis.Lancet Gastroenterol.Hepatol.2019;4:389-398. 4. Younossi ZM,Tampi R,Priyadarshini M,Nader F,Younossi IM,Racila A.Burden of Illness and Economic Model for Patients With Nonalcoholic Steatohepatitis in the United States.Hepatology.2019;69:564-572. 5. Chalasani N,Younossi Z,Lavine JE,Charlton M,Cusi K,Rinella M,et al.The diagnosis and management of nonalcoholic fatty liver disease:Practice guidance from the American Association for the Study of Liver Diseases. Hepatology.2018;67:328-357. 6. Lago RM,Singh PP,Nesto RW.Congestive heart failure and cardiovascular death in patients with prediabetes and type 2 diabetes given thiazolidinediones:a meta-analysis of randomised clinical trials.Lancet (London,England).2007;370:1129-1136. 7. Lincoff AM,Wolski K,Nicholls SJ,Nissen SE.Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus:a meta-analysis of randomized trials. JAMA.2007;298:1180-1188. 8. Nissen SE, Wolski K. Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N. Engl. J. Med. 2007;356:2457 - 2471. 9. Garcia - Lezana T, Raurell I, Bravo M, Torres - Arauz M, Salcedo MT, Santiago A, et al. Restoration of a healthy intestinal microbiota normalizes portal hypertension in a rat model of nonalcoholic steatohepatitis. Hepatology. 2018;67:1485 - 1498.

[0256]

Table 10

Claims

1. A composition comprising two or more bacterial strains of at least two of the following genera: Faecalibacterium, Butyricicoccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes for use in a method for treating, delaying, ameliorating, preventing, or curing a liver disease or condition or a symptom thereof.

2. The composition for use according to claim 1, comprising two or more bacterial strains of at least two of the following species: Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and Anaerostipes caccae.

3. 3. A composition for use according to claim 1 or 2, comprising at least one bacterial strain capable of producing butyrate and at least one bacterial strain capable of producing propionate or a propionate precursor.

4. The composition for use according to any one of claims 1 to 3, further comprising at least one additional bacterial strain capable of producing propionate or a propionate precursor.

5. The composition for use according to any one of claims 1 to 4, further comprising at least two additional bacterial strains capable of producing propionate or a propionate precursor.

6. A composition for use according to any one of claims 1 to 5, further comprising three additional bacterial strains capable of producing propionate or a propionate precursor.

7. The one or more additional bacterial strains capable of producing propionate or a propionate precursor are selected from the group consisting of the genus Phocaeicola, the genus Veillonella, or the genus Blautia, preferably the species Phocaeicola vulgatus, the species Veillonella parvula, the species Veillonella atypica, the species Blautia obeum, the species Blautia wexlerae, or the species Blautia luti. The composition for use according to any one of claims 3 to 6, wherein the composition belongs to the species Azotobacter truncatula luti.

8. - Phocaeicola vulgatus; - Veillonella parvula or Veillonella atypica; and The composition for use according to any one of claims 1 to 7, further comprising Blautia obeum, Blautia wexlerae or Blautia luti.

9. 9. The composition for use according to any one of claims 1 to 8, wherein the composition is for use in a method for treating, delaying, ameliorating, preventing or curing non-alcoholic fatty liver disease (NAFLD), liver fibrosis, cirrhosis and / or liver neoplasm, wherein the liver neoplasm is preferably hepatocellular carcinoma (HCC) or a symptom thereof.

10. 10. The composition for use according to any one of claims 1 to 9, wherein the symptoms comprise weight gain, insulin resistance, liver inflammation, steatosis, hepatocellular ballooning, intrahepatic vascular resistance (IHVR), portal hypertension, endothelial dysfunction, and / or fibrosis.

11. 11. A composition for use according to any one of claims 1 to 10 for use in a method for delaying or preventing disease progression of NAFL, preferably in a method for delaying the progression of NAFL to NASH, of NASH to liver fibrosis, of liver fibrosis to cirrhosis, and / or of cirrhosis to HCC.

12. The bacterium belongs to the genera Faecalibacterium, Butyricicoccus, Roseburia, Akkermansia, Lactiplantibacillus, and Anaerostipes, and preferably the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, and the like. The composition comprises a bacterial strain belonging to the species Lactobacillus inulinivorans, Akkermansia muciniphila, Lactiplantibacillus plantarum, and Anaerostipes caccae, and further comprises at least two additional bacterial strains capable of producing propionate or a propionate precursor.

13. 13. The composition of claim 12, further comprising three additional bacterial strains capable of producing propionate or a propionate precursor.

14. 14. The composition according to claim 12 or 13, wherein the two or more additional bacterial strains capable of producing propionate or a propionate precursor belong to the genus Phocaeicola, Veillonella or Blautia, preferably to the species Phocaeicola vulgatus, Veillonella parvula, Veillonella atypica, Blautia obeum, Blautia wexlerae or Blautia luti.

15. - Phocaeicola vulgatus; - Veillonella parvula or Veillonella atypica; and The composition according to any one of claims 12 to 14, further comprising Blautia obeum, Blautia wexlerae or Blautia luti.

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