Desulfovibrio piger species for prevention or treatment of hepatic steatosis

Abstract

The invention is concerned with a Desulfovibrio species, preferably Desulfovibrio piger or relative thereof for use in preventing and / or treating hepatic steatosis, particularly metabolic dysfunction-associated steatotic liver disease (MASLD) and / or metabolic dysfunction-associated steatohepatitis (MASH). Said Desulfovibrio species may be combined with at least one Eubacterium species or Anaerobutyricum species, preferably Anaerobutyricum soehngenii or relative thereof.

Description

[0001] Desulfovibrio species for prevention or treatment of hepatic steatosis

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of preventing and / or treating hepatic steatosis.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Metabolic dysfunction-associated steatotic liver disease (MASLD) is recognized as the most prevalent chronic liver disease worldwide, and its spectrum ranges from simple steatosis (non-alcoholic fatty liver) to metabolic dysfunction-associated steatohepatitis (MASH), MASH- fibrosis, cirrhosis and hepatocellular carcinoma. The current estimated global prevalence of MASLD is 25%-30% in the general population, and up to 80% in individuals with metabolic syndrome and Type 2 Diabetes mellitus. By definition, excessive alcohol use precludes a diagnosis of MASLD.

[0006] MASLD refers to a spectrum of disease in which excess fat accumulates in the liver in patients who drink little or no alcohol. The most common form of MASLD is also referred to as non-alcoholic fatty liver. As the occurrence and progression of MASLD are strongly driven by insulin resistance, multiple therapeutic strategies in clinical development for MASLD aim at reducing insulin resistance.

[0007] MASLD refers to liver inflammation triggered by lipotoxicity in the setting of hepatic steatosis. MASH gives a markedly increased risk of developing cirrhosis and hepatocellular carcinoma (HCC) and it is associated with increased atherosclerotic cardiovascular disease. Since the association between MASLD / MASH and insulin resistance is well-known, strategies to lower insulin resistance may decrease disease progression or symptoms in MASLD / MASH.

[0008] The gut microbiota has been linked to the development and prevalence of MASLD and MASH. Disease occurrence is significantly lower in individuals taking a plant-based, low- animal-protein diet, which is thought to be mediated by gut microbiota. Hence, Witjes at al (Hepatology Communications, Vol. 4, no. 11, 2020) proposed transplantation of fecal microbiota from lean vegan donors as a potential treatment. Instead, WO2023 / 099579 proposes to administer specific probiotic formulation, i.e. comprising Anaerobutyricum soehngenii or relative thereof, optionally in combination with Bifidobacterium species, Akkermansia species and / or Lactobacillus species. However, there is a need in the art for new and better interventions in the prevention and treatment of MASLD and MASH.

[0009] It is an object of the present disclosure, amongst other objects, to address the above need in the art to provide a new and / or better strategies for preventing and / or treating MASLD and MASH.

[0010] SUMMARY OF THE DISCLOSURE

[0011] The present inventors surprisingly found that administration of a Desulfovibrio species, in particular Desulfovibrio piger or relative thereof, to subjects having hepatic steatosis, may decrease in situ (endogenously produced intestinal) ethanol level and may convert in situ ethanol into short-chain fatty acid(s), particularly acetate. Accordingly, administration of Desulfovibrio species may be applied in a strategy for prevention and / or treatment of hepatic steatosis.

[0012] In addition, it was found that combining a Desulfovibrio species with an Eubacterium / Anaerobutyricum species, in particular Anaerobutyricum soehngenii or relative thereof provides a synergistic therapeutic effect in the prevention or treatment of hepatic steatosis, in particular in Metabolic dysfunction-associated steatotic liver disease (MASLD) and / or metabolic dysfunction-associated steatohepatitis (MASH), and may decrease in situ ethanol level and / or may convert in situ ethanol into short-chain fatty acid(s), particularly acetate and / or butyrate.

[0013] Further, the present disclosure provides for a (gut) bacterium that (over) expresses NADH dehydrogenase (e.g. relative to a natural occurring (gut) bacterium of the same species / strain). The NADH dehydrogenase enzyme scavenges (endogenous) ethanol produced from dietary sugars in the gut. In contrast, overexpression of the human adh genes in bacterial strains would drive increased production of acetaldehyde, which is toxic due to production of reactive oxygen species. The (gut) bacterium that (over) expresses NADH dehydrogenase may also be used in preventing and / or treating hepatic steatosis, MASLD, and / or MASH, optionally alone or together with said Desulfovibrio species and / or Eubacterium / Anaerobutyricum species according to the present disclosure.

[0014] The present disclosure provides a new and improved strategy for preventing and / or treating hepatic steatosis, MASLD, and / or MASH. Accordingly, the said Desulfovibrio species, Eubacterium / Anaerobutyricum species, and / or NADH dehydrogenase overexpressing (gut) bacterium according to the present disclosure can be administered to a subject in order to avoid onset of any of said diseases, for example in subjects wherein risk markers associated with pre-stage or early stage of the respective disease have been detected (before diagnosis of the respective disease). Such primary or secondary prevention strategy may prevent the development of the disease.

[0015] DETAILED DESCRIPTION OF THE DISCLOSURE

[0016] The present disclosure relates to a Desulfovibrio species, particularly for use in preventing and / or treating hepatic steatosis, and / or for increasing production of acetate, propionic acid / propionate and / or butyric acid / butyrate or a derivative of any thereof in the intestine.

[0017] The Desulfovibrio species is preferably chosen from the group consisting of Desulfovibrio piger, Desulfovibrio fairfieldensis, Desulfovibrio desulfuricans, desulfovibrio indonensis, Desulfovibrio alaskensis, Desulfovibrio vulgaris, Desulfovibrio vietnamensis and Desulfovibrio gigas, most preferably Desulfovibrio piger or relative thereof having a 16S rRNA gene sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:1 or SEQ ID NO: 16, preferably SEQ ID NO: 16. Such a relative can be any bacterium with a 16S rRNA gene sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:16, preferably SEQ ID NO:16. 16S rRNA sequence identity is a well-accepted criterion to define functional relatives of a species, see for example Hold et al (FEMS Microbiology Ecology 39 (2002) 33-39), EP1523320B1 and EP1838837B1.

[0018] In accordance with the foregoing, the present disclosure relates to a method for preventing and / or treating hepatic steatosis, e.g. in a subject in need thereof, involving administration, e.g. to said subject, of said Desulfovibrio species.

[0019] Hepatic steatosis is a condition where excess fat builds up in the liver. There are two stages of fatty liver disease: Metabolic dysfunction-associated steatotic liver disease (MASLD) and alcoholic liver disease. MASLD is made up of simple fatty liver and metabolic dysfunction- associated steatohepatitis (MASH). MASLD has previously been referred to as Nonalcoholic fatty liver disease (NAFLD), while MASH has previously been referred to as nonalcoholic steatohepatitis (NASH).

[0020] In the present disclosure, the hepatic steatosis may in a particular be chosen from Metabolic dysfunction-associated steatotic liver disease (MASLD) and / or metabolic dysfunction- associated steatohepatitis (MASH). The term ‘metabolic dysfunction-associated steatotic liver disease’ (MASLD) refers to a group of conditions where there is accumulation of excess fat in the liver of people who drink little or no alcohol. The most common stage of MASLD is called fatty liver. MASLD is strongly associated with insulin resistance and type 2 diabetes mellitus, therefore treatments of MASLD may aim at lowering insulin resistance.

[0021] The term 'metabolic dysfunction-associated steatohepatitis’ (MASH) refers to liver inflammation and damage caused by a buildup of fat in the liver. MASH is associated with a markedly increased risk of developing cirrhosis and hepatocellular carcinoma as well as other diseases not directly associated with liver damage, including increased risk of cardiovascular disease. An association between insulin resistance and the development of MASH ( / MASLD) is well-known, and strategies to lower insulin resistance may decrease disease progression or symptoms in MASH ( / MASLD).

[0022] The use according to the disclosure reduce liver inflammation (e.g. as determined by (sum of) lobular inflammation score 0-3, microgranulomas score 0-1, large lipogranulomas score 0-1, and / or portal inflammation score 0-1 as shown below); or as determined by necroinflammatory activity score (NAS). Hence, the use according to the disclosure can reduce liver inflammation (e.g. as determined by (sum of) lobular inflammation score 0-3, microgranulomas score 0-1 , large lipogranulomas score 0-1 , and / or portal inflammation score 0-1 as shown below); or as determined by necroinflammatory activity score.

[0023] In a particularly preferred embodiment, the use according to the present disclosure is for reducing hepatic necro-inflammatory activity score.

[0024] The term hepatic necro-inflammatory activity score may be interchangeable with the terms MASLD score and / or MASH score.

[0025] To determine the hepatic necro-inflammatory activity score, the MASH Clinical Research Network (MASH-CRN) classification may be used as described by Kleiner et al Volume 41, Issue 6 June 2005), e.g. with use of hematoxylin and eosin-stained slides for steatosis, inflammation and ballooning, and with a sirius red-stained slide for evaluation of fibrosis. The score preferably is the unweighted sum of steatosis grade (0-3), lobular inflammation (0-3), and hepatocellular ballooning (0-2), see below:

[0026]

[0027] * Ballooning classification: few indicates rare but definite ballooned hepatocytes as well as case that are diagnostically borderline. t The “None to rare” category is meant to alleviate the need for time-consuming searches for rare examples or deliberation over diagnostically borderline changes. If the feature is identified after a reasonable search, it should be coded as “many.” t Diagnostic classification may not be available on adult biopsy observations.

[0028] The use according to the disclosure can also decrease steatosis grade score, particularly as defined above (score 1 , 2, 3); and / or fibrosis stage score, particularly as defined above (score 1, 1A, 1 B, 1C, 2, 3, or 4).

[0029] The Desulfovibrio species as referred to herein is preferably chosen from the group consisting of Desulfovibrio piger ( TCC 29098), Desulfovibrio fairfieldensis (ATCC700045), Desulfovibrio desulfuricans (Essex 6 ATCC 29577), D. desulfuricans (MB ATCC 27774), Desulfovibrio indonensis (NCIMB 13468), Desulfovibrio alaskensis (NCIMB 13491), Desulfovibrio vietnamensis (DSM 10520), Desulfovibrio gigas (DSM 1382), Desulfovibrio intestinalis (DSM 11275), Desulfovibrio longreachensis (ACM 3958), Desulfovibrio termitidis (DSM 5308), Desulfovibrio vulgaris subsp. vulgaris (DSM 644), and Desulfovibrio vulgaris subsp. oxamicus (DSM 1925). It is also foreseen that the Desulfovibrio species as referred to herein is not Desulfovibrio piger (ATCC 29098). ATCC stands for American Type Culture Collection, a major biological resource center that stores and distributes reference strains of microorganisms.

[0030] Most preferably the Desulfovibrio species is a Desulfovibrio piger from the GOR group (GOR cluster), i.e. Desulfovibrio piger GOR (including for example Desulfovibrio piger GOR1 and Desulfovibrio piger CH-104), preferably Desulfovibrio p / ger CH- 104, or a relative thereof having at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9% sequence identity with the 16S rDNA sequence of Desulfovibrio p / gerCH-104 (e.g. SEQ ID NO:16). Such a relative can be any bacterium, preferably a Desulfovibrio piger strain, with a 16S rRNA gene sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:16. Such cut-off value based on 16S rDNA similarity can define species with similar characteristics and / or functionality. The Desulfovibrio piger strain GOR1 may have Taxonomy ID: 613189. Most preferably the Desulfovibrio species according to the present disclosure (e.g. Desulfovibrio piger GOR) has a genome comprising a sequence which has at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, or 100% sequence identity with SEQ ID NO:17. Accordingly, the Desulfovibrio species according to the present disclosure (e.g. Desulfovibrio piger GOR) may have a genome having at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, or 100% sequence identity with the genome of Desulfovibrio piger CH-104 (or Desulfovibrio piger GOR 1). In a further aspect, the Desulfovibrio species, more particularly Desulfovibrio piger, or Desulfovibrio piger GOR1 (or Desulfovibrio piger CH-104) according to the disclosure may be a Desulfovibrio species, more particularly Desulfovibrio piger, or Desulfovibrio piger GOR1 or particularly Desulfovibrio piger CH-104 as deposited by Stichting Amsterdam UMC on 25 April, 2025 at the Westerdijk Fungal Biodiversity Institute located at Uppsalalaan 8, 3584CT Utrecht, the Netherlands, assigned the deposit number CBS 153622.

[0031] Also encompassed is any bacterial strain derived from the deposited bacterium, which can be strains obtained by using the deposited strain as taught herein as starting material. The strain that derives therefrom may be a mutant strain, which may be derived from a strain of the invention by means of, for instance, genetic engineering, radiation, UV light, chemical treatment. Alternatively, such derivative or mutant strain may be a strain derived from the deposited strain as taught herein that has been subjected to growth adaptation to particular conditions resulting in an additional benefit to the derivative strain, such as more rapid growth, better survival in the gut, enhanced ethanol conversion, using methods that are well- known to the skilled person. It is preferred that the derivative or mutant is functionally equivalent to the deposited strain as taught herein. A preferred derivative or mutant as taught herein has substantially the same activity or function as the deposited strain as taught herein, (e.g. has enhanced ethanol conversion). The derivative or mutant advantageously provides substantially the same benefits to a mammal (e.g. humans or other mammals) administered with said derivative or mutant as would be the case upon administration of the deposited strain. The derivative or mutant strain may also be a spontaneous derivative or mutant strain having the same characteristics as described herein for the deposited strain.

[0032] The Desulfovibrio piger or relative thereof (for use) according to the disclosure is preferably for use for decreasing in situ ethanol level (e.g. in the gut), preferably for in situ converting ethanol into short-chain fatty acid(s), particularly acetate. Preferably relative to non using of the same, or relative to using placebo.

[0033] Preferably, a (daily) amount of at least 104, 105, 106, 107, 108Desulfovibrio cells may be used, e.g. in a composition wherein the Desulfovibrio species is comprised, for example per ml or per g of said composition. Alternatively or additionally, a total of between 104and 1016, 104and 1015, 104and 1014, 104and 1012, 106and 1012, preferably between 108and 1010, Desulfovibrio cells may preferably be used, e.g. per ml or per g composition wherein the Desulfovibrio species is comprised.

[0034] Alternatively or additionally, the Desulfovibrio cells may be viable, but it is also envisaged that (only) attenuated or dead cells are used, e.g. obtained after pasteurization, or for example obtained after incubation at 50-100, 60-80, 65-75, or 70 degrees Celsius, preferably for a period of at least 5, 10, 15, 20 , 25, 30, 40, 50 minutes, or obtained exposure to UV or gamma irradiation, preferably for a period of at least 1 , 5 , 10, 20 30 seconds, or 1 , 5, 10, 15, 20 , 25, 30, 40, 50 minutes, or obtained after incubation with oxygen, e.g. gas comprising at least 15, 20, 25, 30, 35, 40, 45, 50, 60 ,70, 80, 90, 99, 100 vol. % oxygen, preferably for a period of at least 1 , 5 , 10, 20 30 seconds, or 1 , 5, 10, 15, 20 , 25, 30, 40, 50 minutes. Preferably, the Desulfovibrio species is the first, second, third, fourth, or fifth most dominant bacterial species in the composition, i.e. has the highest cell count in comparison to other bacterial species contained in the composition, or is at least in the top 5.

[0035] The Desulfovibrio species according to the present disclosure is preferably not comprised in fecal matter, or, if it is comprised in fecal matter (e.g. as an alternative to the above- mentioned composition), it is preferably enriched, i.e. the number of Desulfovibrio cells is higher than in prior art fecal matter, for example Desulfovibrio cells have been added to the fecal matter, or the fecal matter has been exposed to conditions favoring growth of said Desulfovibrio species. If the Desulfovibrio species according to the present disclosure is comprised in fecal matter, preferably at least at least 104, 105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 108, 109, 1O10, 1011, 1012, 1013Desulfovibrio cells are comprised in said fecal matter, for example per ml or per g fecal matter. Preferably, the Desulfovibrio species is the first, second, third, fourth, or fifth most dominant bacterial species in the fecal matter, i.e. has the highest cell count in comparison to other bacterial species contained in the fecal matter, or is at least in the top 5.

[0036] Additionally or alternatively, the Desulfovibrio species may be comprised in a composition comprising not more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8,7 ,6, 5, 4, 3, 2, 1 bacterial species.

[0037] In a particularly preferred embodiment, the Desulfovibrio species according the disclosure is combined with an Anaerobutyricum species or Eubacterium species, preferably Anaerobutyricum soehngenii (e.g. DSM17630 / KCTC15707) and / or Anaerobutyricum hallii (DSM3353 / ATCC27751).

[0038] In a study by Shetty et al (Int J Syst Evol Microbiol. 2018 Dec;68(12):3741-3746), the species formerly known as Eubacterium hallii has been reclassified into two groups: Anaerobutyricum hallii and Anaerobutyricum soehngenii. Both Anaerobutyricum soehngenii and / or Anaerobutyricum hallii are considered as an anaerobic Gram-positive, catalase-negative bacterium belonging to the clostridial cluster XlVa (also known as Lachnospiracaea) of the phylum Firmicutes.

[0039] The Anaerobutyricum species or Eubacterium species, or relative thereof, (for use) according to the disclosure is preferably for use for decreasing in situ ethanol level (e.g. in the gut), preferably for in situ converting ethanol into short-chain fatty acid(s), particularly butyrate, or most preferably for converting acetate into butyrate. Preferably relative to non using of the same, or relative to using placebo.

[0040] Most preferably the at least one Anaerobutyricum species according to the present disclosure is Anaerobutyricum soehngenii (e.g. DSM17630 / KCTC15707), or a relative thereof having a 16S rRNA gene sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with the 16S rDNA sequence of Anaerobutyricum soehngenii (SEQ ID NO:2). Such cut-off value based on 16S rDNA similarity can define species with similar characteristics and / or functionality.

[0041] In addition or alternatively, the Anaerobutyricum species according to the present disclosure is Anaerobutyricum hallii (e.g. DSM3353 / ATCC27751), or a relative thereof having a 16S rRNA gene sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with the 16S rDNA sequence of Anaerobutyricum hallii (SEQ ID NO:3). Such cut-off value based on 16S rDNA similarity can define species with similar characteristics and / or functionality.

[0042] It was found that this combination is synergistic, leading to an unexpected reduction in hepatic necro-inflammatory activity score.

[0043] The Anaerobutyricum species or Eubacterium species may be administered separately, sequentially or simultaneously with the Desulfovibrio species. Accordingly, said Anaerobutyricum species or Eubacterium species may be comprised in the same or in a separate composition with respect to said Desulfovibrio species.

[0044] Preferably, an amount of at least 104, 105, 106, 107, 108Anaerobutyricum species or Eubacterium species cells may be used, e.g. in a composition wherein the Anaerobutyricum species or Eubacterium species is comprised, for example per ml or per g of said composition. Alternatively or additionally, a total of between 104and 1016, 104and 1015, 104and 1014, 104and 1012, 106and 1012, preferably between 108and 1010, Anaerobutyricum species or Eubacterium species cells may preferably be used, e.g. per ml or per g composition wherein the Anaerobutyricum species or Eubacterium species is comprised.

[0045] Alternatively or additionally, the Anaerobutyricum species or Eubacterium species cells may be viable, but it is also envisaged that (only) attenuated or dead cells are used, e.g. obtained after pasteurization, or for example obtained after incubation at 50-100, 60-80, 65-75, or 70 degrees Celsius, preferably for a period of at least 5, 10, 15, 20 , 25, 30, 40, 50 minutes, or obtained exposure to UV or gamma irradiation, preferably for a period of at least 1 , 5 , 10, 20 30 seconds, or 1 , 5, 10, 15, 20 , 25, 30, 40, 50 minutes, or obtained after incubation with oxygen, e.g. gas comprising at least 15, 20, 25, 30, 35, 40, 45, 50, 60 ,70, 80, 90, 99, 100 vol % oxygen, preferably for a period of at least 1 , 5 , 10, 20 30 seconds, or 1 , 5, 10, 15, 20 , 25, 30, 40, 50 minutes. Preferably, the Anaerobutyricum species or Eubacterium species is the first, second, third, fourth, or fifth most dominant bacterial species in the composition, i.e. has the highest cell count in comparison to other bacterial species contained in the composition, or is at least in the top 5.

[0046] The Anaerobutyricum species or Eubacterium species according to the present disclosure is preferably not comprised in fecal matter, or, if it is comprised in fecal matter (e.g. as an alternative to the above-mentioned composition), it is preferably enriched, i.e. the number of Anaerobutyricum species cells or Eubacterium species cells is higher than in prior art fecal matter, for example Anaerobutyricum species cells or Eubacterium species cells have been added to the fecal matter, or the fecal matter has been exposed to conditions favoring growth of said Anaerobutyricum species or Eubacterium species. If the Anaerobutyricum species or Eubacterium species according to the present disclosure is comprised in fecal matter, preferably at least at least 104, 105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 108, 109, 1010, 1011, 1012, 1013Anaerobutyricum species cells or Eubacterium species cells are comprised in said fecal matter, for example per ml or per g fecal matter. Preferably, the Anaerobutyricum species or Eubacterium species is the first, second, third, fourth, or fifth most dominant bacterial species in the fecal matter, i.e. has the highest cell count in comparison to other bacterial species contained in the fecal matter, or is at least in the top 5.

[0047] Additionally or alternatively, the Anaerobutyricum species or Eubacterium species may be comprised in a composition comprising not more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8,7 ,6, 5, 4, 3, 2, 1 bacterial species. The present disclosure further provides for a (gut) bacterium that (over) expresses (at least one) Nicotinamide Adenine Dinucleotide (NAD) + Hydrogen (H) dehydrogenas, i.e. NADH dehydrogenase (e.g. relative to a natural occurring (gut) bacterium of the same species / strain). In addition or alternatively, said (gut) bacterium (over) expresses (at least one) ethanol dehydrogenase (alcohol dehydrogenase) and / or (at least one) acetaldehyde dehydrogenase The (gut) bacterium may provide a (strong) therapeutic effect in the prevention or treatment of hepatic steatosis, MASLD and / or MASH. Expression level may be determined via qRT-PCR. The expression level may be such that the (gut) bacterium comprises (or is able to comprise after 48 hrs optimal growth conditions) at least 104, 105, 106,

[0048] 107, 108, 109, or 1010NADH dehydrogenase copies, or at least 104, 105, 106, 107, 108, 109, or 1010NADH dehydrogenase encoding mRNA molecules. The NADH dehydrogenase enzyme can scavenge any ethanol produced in the gut. In addition or alternatively, the said (gut) bacterium is capable of oxidizing NADH to NAD+ (Nicotinamide Adenine Dinucleotide), while ubiquinone is reduced to ubiquinol. The transfer of electrons from NADH to ubiquinone via NADH dehydrogenase is coupled with the translocation of protons across the inner mitochondrial membrane or the plasma membrane. This results in the generation of a proton gradient. In addition or alternatively, the (gut) bacterium (over) expresses NADH dehydrogenase at least to an extent that 106, 107, 108, 109, or 1O10cells of said (gut)bacterium would decrease gut pH by at least 1 , 2, 3, or 4 (e.g. in a (male) human of 70-80 kg). In addition or alternatively, the (gut) bacterium (over) expresses NADH dehydrogenase at least to an extent that 106, 107, 108, 109, or 1010cells of said (gut)bacterium would decrease pH in a tank of 1 , 5, 10, 100, 1000 L water by at least 1 , 2, 3, or 4. In addition or alternatively, the (gut) bacterium (over) expresses NADH dehydrogenase at least to an extent that 106, 107,

[0049] 108, 109, or 1010cells of said (gut)bacterium would decrease intestinal ethanol level by at least 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 40% (e.g. in a (male) human of 70-80 kg). Preferably, said human is characterized by having (1 , 2, 3, 4, 5% or more) elevated (endogenously produced) ethanol level in the gut, e.g. as compared to healthy reference or average in humans.

[0050] In addition or alternatively, the expression level may be such that the (gut) bacterium comprises (or is able to comprise after 48 hrs optimal growth conditions) at least 104, 105, 106, 107, 108, 109, or 1010ethanol dehydrogenase (alcohol dehydrogenase) copies, or at least 104,

[0051] 105, 106, 107, 108, 109, or 101° ethanol dehydrogenase (alcohol dehydrogenase) encoding mRNA molecules. In addition or alternatively, the expression level may be such that the (gut) bacterium comprises (or is able to comprise after 48 hrs optimal growth conditions) at least

[0052] 104, 105, 106, 107, 108, 109, or 101° acetaldehyde dehydrogenase copies, or at least 104, 105,

[0053] 106, 107, 108, 109, or 101° acetaldehyde dehydrogenase encoding mRNA molecules. In addition or alternatively, the (gut) bacterium (over) expresses ethanol dehydrogenase (alcohol dehydrogenase) and / or acetaldehyde dehydrogenase at least to an extent that 106, 107, 108, 109, or 1010cells of said (gut) bacterium would decrease intestinal ethanol level by at least 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 40% (e.g. in a (male) human of 70-80 kg). Preferably, said human is characterized by having (1 , 2, 3, 4, 5% or more) elevated (endogenously produced) ethanol level in the gut, e.g. as compared to healthy reference or average in humans. The (at least one) ethanol dehydrogenase (alcohol dehydrogenase) and / or the (at least one) acetaldehyde dehydrogenase may allow to convert ethanol to acetyl-CoA.

[0054] Preferably, said (gut) bacterium is chosen from:

[0055] Firmicutes, such as belonging to the genera Eubacterium, Intestinimonas, Faecalibacterium, Christensenella, Anaerostipes, Agathobacter, Roseburia, Coprococcus, Clostridium, Subdoligranulum, Anaerotruncus, Flavinobacter, Ruminococcus, Butyricicoccus, Butyrovibrio, Sporobacter, Papilibacter, Oscillobacter, Oscillospora, Veilonella, Lactobacillus, Streptococcus’,

[0056] Proteobacteria such as belonging to the genera Escherichia or Enterobacter,

[0057] - Actinobacteria such as belonging to the genera Bifidobacterium or Colinsella-, Bacteroidetes such as belonging to the genera Bacteroides, Prevotella or Alistipes; and / or Verrucomicrobia such as belonging to the genus Akkermansia.

[0058] Preferably, the (gut) bacterium (over) expresses an NADH dehydrogenase encoded by a nucleotide sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:4. In addition, or alternatively, the (gut) bacterium preferably (over) expresses an NADH dehydrogenase with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:5.

[0059] Preferably, the (gut) bacterium (over) expresses an ethanol dehydrogenase (alcohol dehydrogenase) encoded by a nucleotide sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:6. In addition, or alternatively, the (gut) bacterium preferably (over) expresses an ethanol dehydrogenase (alcohol dehydrogenase) with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:7.

[0060] Preferably, the (gut) bacterium (over) expresses an ethanol dehydrogenase (alcohol dehydrogenase) encoded by a nucleotide sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:8. In addition, or alternatively, the (gut) bacterium preferably (over) expresses an ethanol dehydrogenase (alcohol dehydrogenase) with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:9.

[0061] Preferably, the (gut) bacterium (over) expresses an ethanol dehydrogenase (alcohol dehydrogenase) encoded by a nucleotide sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:10. In addition, or alternatively, the (gut) bacterium preferably (over) expresses an ethanol dehydrogenase (alcohol dehydrogenase) with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ I D NO: 11.

[0062] Preferably, the (gut) bacterium (over) expresses an acetaldehyde dehydrogenase encoded by a nucleotide sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:12. In addition, or alternatively, the (gut) bacterium preferably (over) expresses an acetaldehyde dehydrogenase with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:13.

[0063] Preferably, the (gut) bacterium (over) expresses an acetaldehyde dehydrogenase encoded by a nucleotide sequence with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:14. In addition, or alternatively, the (gut) bacterium preferably (over) expresses an acetaldehyde dehydrogenase with at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9, 100% sequence identity with SEQ ID NO:15.

[0064] Preferably, the (gut) bacterium is Escherichia coli strain Nisste. (1917), more preferably a colibactin knockout / knockin Escherichia coli strain Nisste. (1917).

[0065] It was found that a combination of species / bacterium as disclosed herein is synergistic, leading to an unexpected reduction in hepatic necroinflammatory activity score.

[0066] The (gut) bacterium may be administered separately, sequentially or simultaneously with the Desulfovibrio species, or with the Anaerobutyricum species or Eubacterium species. Accordingly, said (gut) bacterium) may be comprised in the same or in a separate composition with respect to said Desulfovibrio species or Anaerobutyricum species or Eubacterium species.

[0067] Preferably, an amount of at least 104, 105, 106, 107, 108(gut) bacterium) cells may be used, e.g. in a composition wherein the bacterium is comprised, for example per ml or per g of said composition. Alternatively or additionally, a total of between 104and 1016, 104and 1015, 104and 1014, 104and 1012, 106and 1012, preferably between 108and 1010, (gut) bacterium cells may preferably be used, e.g. per ml or per g composition.

[0068] CLAUSES

[0069] 1. (Gut) bacterium that (over) expresses Nicotinamide Adenine Dinucleotide (NAD) + Hydrogen (H) dehydrogenas (NADH dehydrogenase), wherein preferably the NADH dehydrogenase expression level is higher relative to a natural occurring (gut) bacterium of the same species and / or strain of said (gut) bacterium.

[0070] 2. (Gut) bacterium that (over) expresses (at least one) ethanol dehydrogenase (alcohol dehydrogenase) and / or acetaldehyde dehydrogenase, wherein preferably the ethanol dehydrogenase (alcohol dehydrogenase) expression level and / or acetaldehyde dehydrogenase expression level is higher relative to a natural occurring (gut) bacterium of the same species and / or strain of said (gut) bacterium.

[0071] 3. The (gut) bacterium according to any one of 1 , or 2 for use in the prevention or treatment of hepatic steatosis, MASLD and / or MASH.

[0072] 4. The (gut) bacterium, according to 1 , 2, or 3, wherein the NADH dehydrogenase, ethanol dehydrogenase (alcohol dehydrogenase) and / or acetaldehyde dehydrogenase expression level is at least 104, 105, 106, 107, 108, 109, or 1O10NADH dehydrogenase copies, or at least 104, 105, 106, 107, 108, 109, or 101° NADH dehydrogenase ethanol dehydrogenase (alcohol dehydrogenase) and / or acetaldehyde dehydrogenase encoding mRNA molecules, preferably as determined after 48 hours growth (under optimal conditions).

[0073] 5. The (gut) bacterium according to any one of the above, which is chosen from:

[0074] Firmicutes, such as belonging to the genera Eubacterium, Intestinimonas, Faecalibacterium, Christensenella, Anaerostipes, Agathobacter, Roseburia, Coprococcus, Clostridium, Subdoligranulum, Anaerotruncus, Flavinobacter, Ruminococcus, Butyricicoccus, Butyrovibrio, Sporobacter, Papilibacter, Oscillobacter, Oscillospora, Veilonella, Lactobacillus, Streptococcus’, Proteobacteria such as belonging to the genera Escherichia or Enterobacter,

[0075] - Actinobacteria such as belonging to the genera Bifidobacterium or Colinsella-, Bacteroidetes such as belonging to the genera Bacteroides, Prevotella or Alistipes; and / or

[0076] Verrucomicrobia such as belonging to the genus Akkermansia.

[0077] 6. The (gut) bacterium according to any one of the above, which is Escherichia coli strain Niss / e. (1917), more preferably a colibactin knockout / knockin Escherichia coli strain Niss / e. (1917). In a preferred embodiment, the prevention and / or treatment according to the present disclosure involves at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or at most 10, 20, 30, 40, 50 separate administrations of the species / bacterium, preferably with intervals of at least 1 , 2, 3, 4, 5, 6, 7, 8, 10, and / or at most 10, 20, 30, 40, 50 weeks between said separate administrations. The prevention and / or treatment may also involve daily, weekly, monthly administrations, such as once or twice within every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 days / weeks / months and / or may be during a period of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 weeks (or months or even years).

[0078] It is further envisaged that the species according to the present disclosure is / are present in lyophilized form. The skilled person is capable of lyophilizing based on well-known techniques, wherein oxygen-free conditions may be applied to preserve viability of any bacteria contained in the fecal matter.

[0079] Lyophilisation methods include, without limitation, slow, gradual freezing to -40°C before drying, rapid freezing by placing at -80°C before drying, or ultra rapid freezing by dripping cells with cryoprotectant in liquid nitrogen before drying. Cryoprotectants are often employed to protect compositions during lyophilisation and to enhance shelf-life. Without limitation, a cryoprotectant selected from the group consisting of sucrose, maltose, maltodextrin, trehalose, mannitol, sorbitol, inulin, glycerol, DMSO, ethylene glycol, propylene glycol, 2- methyl-2,4-pentanediol, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyglycerol, skim milk powder, milk protein, whey protein, UHT milk, betaine, adonitol, sucrose, glucose, lactose or any combination thereof, may be employed.

[0080] The species / bacterium or composition(s) comprising said as according to the present disclosure may be administered by enteral, preferably by oral, nasal or rectal administration, and / or by nasoduodenal tube administration. The species / bacterium according to the present disclosure may be administered to the gastrointestinal tract of the subject. It is also possible to administer the species to the colon (or cecum) of the subject.

[0081] The species / bacterium may be applied in an effective amount, i.e. a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g. an amount which results in the treatment and / or prevention of the respective condition. In the context of therapeutic or prophylactic applications, the amount to be administered to the subject may depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It may also depend on the degree, severity and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

[0082] The composition according to the present disclosure may be used as medicament and / or accompanied by a physiologically acceptable carrier which may be any inert carrier. For instance, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any well-known physiological or pharmaceutical carriers, buffers, diluents, and excipients. It will be appreciated that the choice for a suitable physiological carrier will depend upon the intended mode of administration of the composition as taught herein (e.g., oral). The skilled person knows how to select a physiologically acceptable carrier, which is suitable for or compatible with the compositions for use as taught herein.

[0083] It is envisaged that the species / bacterium or composition(s) according to the present disclosure is / are comprised in and / or encapsulated by an (enteric) coating, preferable wherein said coating does not dissolute and / or disintegrate in the gastric environment of the recipient. Such coating may help the composition to reach the intended site for delivery, e.g. the duodenum or colon, without suffering breakdown due to the acidic environment of the stomach. Preferred (enteric) coatings work by presenting a surface that is stable at the highly acidic pH found in the stomach, but breaking down more rapidly at a lower pH. For example, it will not dissolve in the gastric acids of the stomach (pH ~3), but it will dissolve in the alkaline (pH 7-9) environment present in the small intestine, or duodenum.

[0084] In an embodiment, the present disclosure is concerned with the composition for use as a probiotic. Accordingly, ‘probiotics’ as used herein refers to microorganisms such as intestinal bacteria, which - when administered or ingested in effective amounts - confer health benefits to the host (e.g. humans or mammals). Preferably, probiotics should be alive or viable when administered to a subject so as to allow the probiotics to colonize the large intestine of the host. However, under certain conditions, probiotics may also be dead when administered provided that substances produced by the probiotics still exert probiotic, beneficial effects on the host.

[0085] In an embodiment, the present combination as taught herein may be for use as a symbiotic. The term ‘symbiotic’ or ‘symbiotic products’ as used herein generally refers to compositions and / or nutritional supplements combining probiotics and one or more compounds that promote the growth and / or activity of Gl microorganisms, such as prebiotics, into one product. The symbiotic beneficially affects the host by improving the survival and colonization of the probiotic in the Gl tract, by selectively stimulating the growth and / or by activating the metabolism of the probiotic, thus improving host welfare. The skilled person is well- acquainted with symbiotics and knows how to select ingredients that may be combined into a symbiotic.

[0086] The present inventors furthermore surprisingly found that micro-encapsulation of the Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (or the (gut) bacterium) as according to the present disclosure, may provide a further synergistic therapeutic effect in the prevention or treatment of hepatic steatosis, MASLD and / or MASH.

[0087] The term ‘micro-encapsulation’ is used to describe the encapsulation of bacteria in a matrix, coating, or membrane, generally a protective matrix or protective membrane. The (average) diameter of the microcapsules may be between 50 nm and 2 mm, preferably between 100 nm and 1 mm. The matrix, coating or membrane is typically comprised of milk, milk protein, and / or a polymer. The purpose of micro-encapsulation, among other possible purposes, may be to protect bacteria and their components against destruction by the surrounding environment, such as the gastrointestinal environment. The micro-encapsulation of bacteria may also support improved incorporation of bacteria into dairy products, food products, pharmaceutical formulations, and / or pharmaceutical compositions. The micro-encapsulation of bacteria may also support the therapeutic effect.

[0088] Various materials may be used for the micro-encapsulation of bacteria, such as pea protein, milk, milk protein, whey protein, casein, xanthan gum, alginate, gelatin, chitosan, carboxymethyl cellulose, starch, and / or carrageenan, and combinations thereof. In a preferred embodiment, the species / bacterium as according to the present disclosure is micro- encapsulated in one or more polymers.

[0089] The subject receiving the species, combination or composition(s) as taught herein may be selected from the group consisting of human being, non-human primate, mouse, rat, dog, cow, and pig. In a preferred embodiment, the subject is a human. The subject may have Diabetes mellitus, e.g. type 1 or type 2. In addition or alternatively, the subject may be obese and / or having a Body Mass Index (BMI) of at least 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35.

[0090] Preferably, the subject is characterized by having (1, 2, 3, 4, 5% or more) elevated (endogenously produced) ethanol level in the gut, e.g. as compared to healthy reference or average in humans. In addition or alternatively, the subject preferably is or has been using proton pump inhibitors (PPIs). As will be clear, the present treatment is preferably not performed as control or placebo treatment and / or within a clinical trial, i.e. a study in which participants are assigned to groups that either receive one or more intervention / treatment, one or more control or placebo intervention / treatment, or no intervention, so that researchers can evaluate the effects of the interventions on biomedical or health-related outcomes.

[0091] The present disclosure may also allow for the prevention and / or treatment of the following diseases, but optionally these diseases are excluded from the scope of the present disclosure: autoimmune disease, autoimmune hepatitis, hepatic inflammation, gastrointestinal disorders, Clostridium difficile infection, Morbus Crohn (Crohn’s disease), ulcerative colitis or Inflammatory Bowel Disease (IBD), and / or Irritable bowel syndrome (IBS). Alternatively and / or additionally, the present disclosure may allow for the prevention and / or treatment of the following diseases, but optionally these diseases are excluded from the present disclosure: intestinal (colon) cancer or liver I pancreatic cancer (e.g. due to senescence of cells induced by endogenous ethanol), (exogenous alcohol induced) hepatitis and cirrhosis, autobrewery syndrome, systemic and localized (organ specific) autoimmune diseases, endocrine autoimmune disease, Type 1 Diabetes mellitus, Type 2 Diabetes mellitus, Hashimoto’s disease, Graves’s disease, or Addison’s disease, skin autoimmune disease, Psoriasis or Vitiligo, rheumatoid autoimmune diseases, rheumatoid arthritis, Bechterew’s disease, and gastrointestinal autoimmune disease, Celiac disease, vasculitis, COPD, CIDP, MS, SLE, Guillain-Barre.

[0092] The Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (and / or (gut) bacterium) as according to the present disclosure may be comprised in the combination or composition in an amount ranging from 104to 1015colony forming units (CFU). For instance, the Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (and / or (gut) bacterium) may be comprised in the combination in an amount of 106CFU to 1013CFU, preferably 107CFU to 1012CFU, preferably 108CFU to 1011CFU, more preferably 109CFU to 1011CFU, e.g. per dose or per ml or per g of formulation or composition comprising said.

[0093] In one of the embodiments, the Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (and / or (gut) bacterium) in the combination or composition taught herein may be incorporated in lyophilized form and / or, micro-encapsulated form (reviewed by, for example, Solanki et al. BioMed Res. Int. 2013, Article ID 620719), or any other form preserving the activity and / or viability of the bacterial strain. In an embodiment, the species / bacterium, combination or composition as taught herein may comprise one or more ingredients which are suitable for promoting survival and / or viability of the bacterium or strain derived therefrom as taught herein during storage and / or during exposure to bile and / or during passage through the Gl tract of a mammal (e.g. a human being). Non-limiting examples of such ingredients include an enteric coating, and controlled release agents allowing passage through the stomach. The skilled person knows how to select suitable ingredients for maintaining a bacterium as taught herein viable and functional i.e. able to carry out intended function(s).

[0094] It may be advantageous to add one or more prebiotic ingredients to the combination as taught herein, for example, to supplement the effects (e.g. production of propionic acid / propionate and / or butyric acid / butyrate or a derivative thereof) of the bacterium as taught herein. The prebiotic ingredients may also enhance the activity and / or stimulate the growth of the bacterium, or a strain derived therefrom, as taught herein. A ‘prebiotic’ as used herein generally refers to a non-digestible food ingredient that promotes the growth of beneficial microorganisms in the intestines. Prebiotics or prebiotic products consist mainly of fermentable fibres or non-digestible carbohydrates. The fermentation of these fibres by probiotics promotes the production of beneficial end products, such as SCFAs, particularly butyrate. Non-limiting examples of suitable prebiotics include fibres such as inulin, pectin, and resistant starch, as well as cellobiose, maltose, mannose, salicine, trehalose, amygdalin, arabinose, melibiose, sorbitol, rhamnose and / or xylose. The skilled person is well-acquainted with the field of prebiotics and knows how to select ingredients endowed with prebiotic activity.

[0095] In addition or alternative to preventing and / or treating hepatic steatosis, MASLD and / or MASH, the present disclosure may be used for (enhancing) acetate, butyric acid and / or butyrate production, preferably in situ, i.e. in the small intestine.

[0096] The term ‘butyrate’ or ‘butyric acid’ (also known under the systematic name butanoic acid) as used herein refers to a carboxylic acid with the structural formula CH3CH2CH2COOH. The term may include derivatives thereof, i.e. compounds derived from butyric acid and includes salts and esters of butyric acid, which are known as butyrate or butanoate. Non-limiting examples of butyrate salts include sodium butyrate, calcium butyrate, magnesium butyrate, manganese butyrate, cobalt butyrate, barium butyrate, lithium butyrate, zinc butyrate, potassium butyrate, ferrous butyrate and the like. Non-limiting examples of butyrate esters (i.e. esters of butyric acid) include cellulose acetate butyrate, methyl butyrate, ethyl butyrate, butyl butyrate, pentyl butyrate, and the like. Without wishing to be bound by any theories, it is believed that the bacterial strain(s) according to the present disclosure, when administered to a human being or when ingested by a human being in an adequate amount, is / are able to survive and at least transiently colonize the gastrointestinal tract of said human being. This colonization may typically enable greater in situ production of butyric acid / butyrate, although other mechanisms cannot be excluded. Increased in situ production may underlie, at least in part, the beneficial effects in the combination as taught herein, e.g. preventing and / or treatment of hepatic steatosis, MASLD and / or MASH.

[0097] In an embodiment, the Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (and / or (gut) bacterium) may be comprised in a food formulation, feed formulation, feed supplement formulation, food supplement formulation or pharmaceutical formulation. At the same time or alternatively, the Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (and / or (gut) bacterium) may be comprised in a liquid, liquid beverage (including dairy beverage and fermented beverage), yogurt, cheese, gel, gelatine, gelatine capsule, powder, paste, tablet, or a capsule.

[0098] The food or food supplement formulation is preferably a dairy product, more preferably a fermented dairy product, most preferably a yogurt or a yogurt drink.

[0099] The pharmaceutical formulation may be for example a liquid or solid form, more preferably a solid form solid dosage form, e.g., may be a capsule, a tablet, or a powder. Preferably, a pharmaceutical formulation does not relate to pure water or aqueous medium comprising more than 99 wt.% water.

[0100] The formulations as taught herein comprising the combination for use according to the present disclosure may further comprise any acceptable carrier that is suitable for keeping the Desulfovibrio species and / or the Anaerobutyricum species or Eubacterium species (and / or (gut) bacterium) as according to the present herein viable until consumption by a subject (e.g. human or animal). For instance, non-limiting examples of acceptable carriers that are suitable for this purpose include any of well-known physiological or pharmaceutical carriers, buffers, and excipients. It will be appreciated that the choice for a suitable physiological or pharmaceutical carrier will depend upon the intended mode of administration of the formulations as taught herein (e.g. oral) and the intended form of the formulations (e.g. beverage, yogurt, powder, capsules, and the like). The skilled person knows how to select a physiological or pharmaceutical carrier, which is suitable for the formulations as taught herein. Preferably, the (gut) bacterium is Escherichia coli strain Niss / e. (1917), more preferably a colibactin knockout Escherichia coli strain Niss / e. (1917).

[0101] It was found that a combination of species / bacterium as disclosed herein is synergistic, leading to an unexpected reduction in hepatic necroinflammatory activity score.

[0102] The (gut) bacterium may be administered separately, sequentially or simultaneously with the Desulfovibrio species, or with the Anaerobutyricum species or Eubacterium species.

[0103] Accordingly, said (gut) bacterium) may be comprised in the same or in a separate composition with respect to said Desulfovibrio species or Anaerobutyricum species or Eubacterium species.

[0104] Preferably, an amount of at least 104, 105, 106, 107, 108(gut) bacterium) cells may be used, e.g. in a composition wherein the bacterium is comprised, for example per ml or per g of said composition. Alternatively or additionally, a total of between 104and 1016, 104and 1015, 104and 1014, 104and 1012, 106and 1012, preferably between 108and 1010, (gut) bacterium cells may preferably be used, e.g. per ml or per g composition.

[0105] In a preferred embodiment, the prevention and / or treatment according to the present disclosure involves at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or at most 10, 20, 30, 40, 50 separate administrations of the species / bacterium, preferably with intervals of at least 1 , 2, 3, 4, 5, 6, 7, 8, 10, and / or at most 10, 20, 30, 40, 50 weeks between said separate administrations. The prevention and / or treatment may also involve daily, weekly, monthly administrations, such as once or twice within every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 days / weeks / months and / or may be during a period of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 weeks (or months or even years).

[0106] The terms ‘comprising’ or ‘to comprise’ and their conjugations, as used herein, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb ‘to consist essentially of’ and ‘to consist of’.

[0107] Reference to an element by the indefinite article ’a’ or ‘an’ does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article ‘a’ or ‘an’ thus usually means ‘at least one’. The terms ‘to increase’ and ‘increased level’ and the terms ‘to decrease’ and ‘decreased level’ refer to the ability to significantly increase or significantly decrease or to a significantly increased level or significantly decreased level. Generally, a level is increased or decreased when it is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher or lower, respectively, than the corresponding level in a control or reference. Alternatively, a level in a sample may be increased or decreased when it is statistically significantly increased or decreased compared to a level in a control or reference.

[0108] As used herein, the term “identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" perse has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. For example NCBI Nucletide Blast with standard settings (blastn, https: / / blast.ncbi.nlm.nih.gov / ). Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

[0109] As an illustration, by a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence is identical to the reference sequence except that there may be up to five point mutations per each 100 nucleotides of the reference polypeptide sequence. In other words, to obtain a nucleotide sequence being at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or substituted with another nucleotide, and / or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. In a sequence listing, a “n” may denote a, t, g, or c.

[0110] Should there be an inconsistency between the sequences disclosed in the description and the sequences disclosed in the sequence listing, the sequences disclosed in the description are preferred. Alternatively, the sequences of the sequence listing may be used.

[0111] Sequence listing

[0112]

[0113]

[0114] Figure descriptions

[0115] Figure 1 - Butyrate and acetate production from ethanol was observed, see Figure 1 , wherein acetate is produced from ethanol by d piger and also butyrate was produced when co- cultured with L2-7 A soehngenii.

[0116] Figure 2 - Increasing pH in fecal sample of Caucasian MASLD-MASH subject alters endogenous ethanol production from added fructose, with more ethanol production at pH 6-7 than pH 5 during 48 hours anaerobic culture b) coculture of D piger and A soehngenii again converts ethanol (light green) produced from fructose (purple) into butyrate (pink) and acetate (orange )optimally at pH levels 5.6-6.7. Fig. 3 . Unsupervised clustering of the genomes of D.piger isolates (top) and those of other D.piger, Desulfovibrio and other related bacterial species. Two clusters of genomes of D.piger isolates are highlighted, represented by the type strain D.piger ATCC29089 and D.piger G0R1 (indicated by the open and closed stars, respectively). Also the genomes of other Desulfovibrio species including that of D. vulgaris (open circle) are shown together with several species of related genera (all obetained from NCBI). The genome of the used strain CH- 104 is indicated in the picture and it shares with the genomes of the other strains of the GOR group an Average Nucleotide Identity (AN I) of more than approximately 98 % while it has an ANI level of less than approximatley 95 % with that of the genomes belonging to Group ATCC29098.

[0117] Fig. 4. Reduction of the expression of inflammatory liver genes after D.piger CH-104 administration compared to placebo. The star indicates p< 0.05.

[0118] Fig. 5. Reduction of the expression of liver metabolic genes involved in fat storage or MASH after D.piger CH-104 administration compared to placebo. A single star indicates p< 0.05 and two starts p<0.01.

[0119] Fig. 6. Increased expression of small intestinal genes coding for tight junction proteins involved in increased barrier function after D.piger CH-104 administration as compared to placebo. A single star indicates p< 0.05.

[0120] Fig. 7. Reduced cytokine production after LPS induction of bone marrow cells isolated from mice after administration of D.piger CH-104 as compared to placebo. Two stars indicate p< 0.01 and 3 stars p< 0.001.

[0121] EXPERIMENTAL EXAMPLE 1

[0122] Culture conditions

[0123] Desulfovibrio piger was routinely maintained in a modified YCFA medium supplemented with 20mM lactate and 20mM sodium sulfate. Anaerobutyricum soehgenii was routinely maintained in a modified YCFA medium supplemented with 20mM glucose. YCFA medium composed of (g / l): 10 yeast extract, 10 soy peptone, 4 sodium bicarbonate, 2,7 sodium acetate, 4,5 monopotassium phosphate, 0.9 dipotassium phosphate, 0.9 ammonium chloride, 0.9 sodium chloride, 0.09 magnesium sulfate, 0.0005 resazurin, 0.5 cysteine and 1 ml of vitamin mixture.

[0124] Ethanol conversion and relation to pH To test the capability to convert ethanol, D. piger was inoculated in YCFA medium containing 5mM ethanol and 20mM sodium sulfate. The bacterial cultures were collected at initial time point and during the growth up to 96 hours for metabolite measurement by HPLC.

[0125] To test the capability of D. piger to convert ethanol, YCFA medium was used. The substrates (ethanol and sodium sulfate) were added to these preadjusted pH media, then subsequently inoculated with D. piger. The ethanol consumption was monitored during 48h growth.

[0126] The coculture between D. piger and Anaerobutyricum soehgenii in fructose plus ethanol was performed in YCFA medium containing 20mM fructose and 10mM ethanol. These two bacteria were simultaneously inoculated at the start and the bacterial supernatants were collected during the growth up to 60 hours. Fructose, ethanol and short chain fatty acids were quantified by HPLC analyses.

[0127] Results

[0128] Butyrate and acetate production from ethanol was observed, see Figure 1 , wherein acetate is produced from ethanol by d piger and also butyrate was produced when co-cultured with L2-7 A soehngenii.

[0129] EXPERIMENTAL EXAMPLE 2

[0130] The present inventors consider that increased pH in the gut, e.g. due to use of proton pump inhibitors (PPIs), is linked with MASLD-MASH. The inventors hypothesize that gut microbial ethanol production from dietary sugars such as fructose is intestinal pH dependent.

[0131] Method

[0132] Fresh stool sample of a human subject was suspended in anaerobic PBS solution in an anaerobic bottle to make fecal suspension for the fecal fructose study. This fecal suspension was stored at 4oC up to a few weeks. Fructose fermentation at different pH values was performed in a minimal medium (per liter the medium contains 0.53 g Na2HPO4.2H2O, 0.41 g KH2PO4, 0.3 g NH4CI, 0.11 g CaCI2.2H2O, 0.10 g MgCI2.6H2O, 0.3 g NaCI, 4.0 g NaHCO3 and 0.48 g Na2S.9H2O as well as alkaline and acid trace elements (each 1 ml 1-1) and vitamins (0.2 ml 1-1). The alkaline trace element solution contained the following (mM): 0.1 Na2SeO3, 0.1 Na2WO4, 0.1 Na2MoO4 and 10 NaOH. The acid trace element solution was composed of the following (mM): 7.5 FeCI2, 1 H3BO4, 0.5 ZnCI2, 0.1 CuCI2, 0.5 MnCI2, 0.5 CoCI2, 0.1 NiCI2 and 50 HCI. The vitamin solution had the following composition (g / l): 0.02 biotin, 0.2 niacin, 0.5 pyridoxine, 0.1 riboflavin, 0.2 thiamine, 0.1 cyanocobalamin, 0.1 p- aminobenzoic acid and 0.1 pantothenic acid. The pH values were pre-adjusted by using 3M / 1M of NaOH or 3M HCI / 1M HCI. 20mM fructose was supplemented in the medium as substrate.

[0133] To test how fecal microbiome metabolize fructose, the fecal suspension was added to the pH pre-adjusted media containing fructose as substrate. The bacterial supernatants were collected during the growth up to 48 h for substrate and metabolite measurements.

[0134] To investigate the influence of D.piger and A. soehgenii on fecal fructose metabolism, these two bacteria were co-inoculated with the fecal suspension to the pH pre-adjusted media containing fructose as substrate. The bacterial supernatants were collected during 48 hours for substrate and metabolite measurements by HPLC.

[0135] Results

[0136] See Figure 2. Increasing pH in fecal sample of Caucasian MASLD-MASH subject alters endogenous ethanol production from added fructose, with more ethanol production at pH 6-7 than pH 5 during 48 hours anaerobic culture b) coculture of D pigerand A soehngenii again converts ethanol (light green) produced from fructose (purple) into butyrate (pink) and acetate (orange )optimally at pH levels 5.6-6.7.

[0137] EXPERIMENTAL EXAMPLE 3

[0138] The present inventors found that (co-)administration of Desulfovibrio species with a Anaerobutyricum species and / or Eubacterium species (or an NADH overexpressing (gut) bacterium) has a beneficial and synergistic effect in patients having or at risk of acquiring hepatic steatosis.

[0139] METHODS

[0140] Participants

[0141] Caucasian, treatment-naive, omnivorous individuals with hepatic steatosis on ultrasound are included. The main inclusion criteria are age 21-69 years, male or postmenopausal female, body mass index (BMI) > 25 kg / m2 with hepatic steatosis on previous ultrasound with suspicion of MASLD (based on elevated liver enzymes, impaired glucose tolerance, and severity of steatosis on ultrasound). Exclusion criteria are any history of cardiovascular disease, T2DM, renal disease, cholecystectomy, or compromised immunity; use of protonpump inhibitors, antibiotics, or anticoagulants in the past 3 months; any current use of medication; a history of moderate to heavy alcohol use (>12 g per day); or other causes of liver disease besides MASLD (e.g. hemochromatosis, auto-immune hepatitis, cirrhosis, hepatitis B or C, hemochromatosis, alpha-1 antitrypsin deficiency, alcoholic liver disease).

[0142] Intervention

[0143] Subjects are treated for at least 24 weeks according to the single or combinatorial treatment arms shown in Table 1. The hepatic necroinflammatory activity score (MASLD activity score) is measured at baseline and after treatment. Microbiota treatment is given in capsule form, at 1010living units per capsule, once daily.

[0144] Liver biopsy

[0145] Percutaneous liver biopsies are performed on the basis of clinical indications according to local standard procedure. All histologic specimens are scored by a liver pathologist who was blinded to any other results. The MASH Clinical Research Network (MASH-CRN) classification (Kleiner et al Volume41 , Issue 6 June 2005) is assessed with use of hematoxylin and eosin-stained slides for steatosis, inflammation and ballooning, and with a sirius red-stained slide for evaluation of fibrosis. The necroinflammatory activity score (NAS) is determined as described herein.

[0146] Plasma measurement

[0147] Bile acid plasma level is determined by liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0148] RESULTS

[0149] As shown, the present inventors determine the therapeutic effect of Desulfovibrio species with an Anaerobutyricum species and / or Eubacterium species (or an NADH overexpressing (gut) bacterium) when administered alone, or when administered in combination.

[0150] Desulfovibrio species alone has ability to improve necroinflammatory activity score, but a combination with Anaerobutyricum species and / or Eubacterium species increases the effect dramatically and well beyond additive effect (synergistic combination). Also a (gut) bacterium overexpressing NADH dehydrogenase has the ability to improve necroinflammatory activity score.

[0151] The efficacy in reduction of the necroinflammatory activity score following treatment is shown in Table 1 accordingly to the following ranking system, wherein the first rank describes the lowest effect and the last rank describes the highest effect: ‘non-measurable’, ‘very low, ‘low’, ‘low / medium’, ‘medium’, ‘high’, ‘very high’. In healthy subjects, a lower necroinflammatory activity score can prevent onset of hepatic steatosis, MASLD and / or MASH. It is expected that results similar to the putative effects as shown in Table 1 can be obtained with larger patient cohorts.

[0152] Table 1 : treatment scheme and effect on lowered necroinflammatory activity score (NAS)

[0153] EXPERIMENTAL EXAMPLE 4

[0154] To study the diversity of human D.piger strains we isolated 11 new sulfate reducing bacteria from fresh fecal samples of healthy subjects using lactate as electron donor and sulfate as electron acceptor using standard methods (Faith et al 2011). Based on 16S rRNA analysis all isolates were found belonged to the species D. piger. To further characterize the strains their genomes were sequenced using deep Illumina sequencing resulting 90-130 contigs. Detailed comparative analysis of the genomes of 11 isolates with available genomes of D.piger and other Desulfovibrio species revealed an unusual characteristic: two clearly separated clusters were observed, one contained the type strain D.piger ATCC 29098 (termed Group 29098) and one contained the earlier described strain D.piger GOR1 (Rey et al 2013) (termed Group GOR) (Fig. 3).

[0155] It is important to note that (except for D. alkaliphilus) all other Desulfovibrio species, including D. vulgaris, clearly separated from the D. piger strains based on their genomes, indicative of different physiological properties (Fig.3). Similarly, it is to be expected that strains belonging to either of the two observed D.piger clusters would show different properties as their genomes differ considerably.

[0156] Earlier studies with D.piger GOR1 showed it to be maintained stably without negative host effects in a gnotobiotic mouse model, consisting of a synthetic community colonizing germ- free mice. No effect of the presence or absence of D.piger GOR1 was observed on total body weight or epididymal fat pad weights of these gnotobiotic mice (Rey et al 2013). Because of the absence of negative health effects of strain GOR1 , we selected an isolate from the GOR1 cluster and termed this CH-104 and used it for further studies (Fig.3).

[0157] Fig. 3 shows unsupervised clustering of the genomes of D.piger isolates (top) and those of other D.piger, Desulfovibrio and other related bacterial species. Two clusters of genomes of D.piger isolates are highlighted, represented by the type strain D.piger ATCC29089 and D.piger GOR1 (indicated by the open and closed stars, respectively). Also the genomes of other Desulfovibrio species including that of D. vulgaris (open circle) are shown together with several species of related genera (all obtained from NCBI). The genome of the used strain CH- 104 is indicated in the picture and it shares with the genomes of the other strains of the GOR group an Average Nucleotide Identity (ANI) of more than approximately 98 % while it has an ANI level of less than approximatley 95 % with that of the genomes belonging to Group ATCC29098.

[0158] Detailed genome analysis showed strain CP- 104 it to be devoid of antibiotic resistance genes that are linked to mobile elements. Like the D. piger type strain ATCC 29098 and D.piger GOR, strain CH-104 has intrinsic resistance at a moderate level to vancomycin, gentamycin, and kanamycin as well as ampicillin. However, strain CH-104 was found to be highly sensitive to tetracycline (MIC of 0.25 pg / mL), chloramphenicol (MIC of 4 pg / mL), erythromycin (MIC of 1 pg / mL), and rifampicin (MIC of 2 pg / mL), as determined according to EFSA reported protocols guidance (EFSA 2018). This is highly relevant as several other D.piger isolates show resistance to tetracyclin of 0.5- 64 ug / mL, including strain GOR1.

[0159] Importantly, D.piger CH-104 showed relevant physiological properties including the use of ethanol, fructose, lactate, formate and pyruvate electron donor with sulphate as acceptor - also other electron acceptors could be used such sulfite and the natural occurring isothionate, a degradation product of taurine. Here the use of ethanol as electron donor is important to note as it may explain the use of D. piger CH- 104 to reduce the ethanol levels in the intestinal tract of subjects with the so called autobrewery syndrome and subsequent liver damage. In addition, efficient growth on pyruvate alone was observed and this property was used in large scale fermentations where no sulphate was added and hence no large amounts of hydrogen sulphide were produced that could damage the fermentation equipment or lead to toxic sulphide concentrations in the final product. To grow D.piger CH-104 with high growth rate on a plant-based medium, we used a basal salt medium with added yeast extract (10 g / L), guar gum (10 g / L) and sodium pyruvate (55 g / L). We grew cells to optical density at 600 nm of over 2.5 under anaerobic conditions in this medium, harvested and washed the cells by centrifugation, and prepared in an anaerobic cabinet a series of vials containing a D. piger suspension in phosphate-buffered saline (PBS) containing 10% glycerol and 10% maltodextrin, with a concentration of 10e8 CFU / ml. When frozen at -80 these vials showed a high stability over time of the viability of D.piger CH-104 as determined as active fluorescent units by flow cytometry as described in ISO 19344:2015 | IDF232:2015.

[0160] Subsequently, we tested the effectiveness of D.piger CH-104 in protecting against liver damage and development of NAFLD and NASH (presently termed MAFLD and MASH for metabolic dysfunction-associated fatty liver disease and steatohepatitis, respectively). For this we performed an experiment in a diabetic mouse model where we gavaged 3 times per week 13 mice with a dose of 10e8 CFU of D.piger CH-104 prepared as describe above and 11 mice with a placebo consisting of PBS with 10% glycerol and 10% maltodextrin. This treatment was performed for a period of 4 weeks and following that period all mice were sacrificed and analyzed for the expression of genes involved in liver function, barrier function, and inflammation.

[0161] Remarkably, the expression of liver genes including those for CD3 and CD11c and F480 were to various degrees decreased after the D.piger intervention as compared to placebo (Fig. 2). All of these point to a reduction of liver inflammation: the CD3 gene codes for a crucial T-cell-marker that responds external and endogenous inflammatory stimulants, the CD11c gene encoding the CD11c protein, which is a marker for dendritic cells and inflammation, and the F480 gene coding for the F4 / 80 antigen that is typically expressed in livers after a high fat diet inducing inflammation.

[0162] Fig. 4 shows reduction of the expression of inflammatory liver genes after D.piger CH-104 administration compared to placebo. The star indicates p< 0.05.

[0163] Further analysis of the expression of liver genes involved in metabolic functions related to fat storage or markers for MASH confirmed the strong reduction in inflammation in the liver of mice treated with D.piger CH-104 over placebo. These included the Crat, Car3, Cyp39a1, and SLc23a1 genes implicated in fat transport and storage, lipid synthesis or anti-oxydant transport. Several of these are considered markers for MASH while at least Crat, Car and SLc23a1 are implicated carcinogenesis (Smith et al 2020) (Fig.5).

[0164] Fig. 5 shows reduction of the expression of liver metabolic genes involved in fat storage or MASH after D.piger CH-104 administration compared to placebo. A single star indicates p< 0.05 and two starts p<0.01.

[0165] Of note the Lbp gene codes for the LPS binding protein and its decreased expression is indicative of less inflammatory LPS that is circulating. In general LBP levels are decreased when barrier function is increased and hence we also addressed the impact of the D.piger CH-104 administration on intestinal function (Fig 6). The small intestinal expression of genes for Cln4 for claudin 4 and ZO1 for zona occludes 1 (ZO1) were found to be increased to various levels after the D.piger intervention. Claudin 4 is a crucial tight junction protein involved in maintaining cell polarity and controlling paracellular permeability similar to ZO1 that maintains the tight junctions. This is also the case for the Ocln gene coding for a similar tight junction protein. In conclusion, the increased expression of these tight junction protein genes in the small intestine testifies for an increased barrier function due to the administration of D.piger CH-104. It is likely that a similar increase barrier function occurs in the large intestine and explains the reduced levels of Lbp gene expression in the liver (Fig. 5).

[0166] Fig. 6 shows increased expression of small intestinal genes coding for tight junction proteins involved in increased barrier function after D.piger CH-104 administration as compared to placebo. A single star indicates p< 0.05.

[0167] Finally, ex vivo experiments were performed to demonstrate the long lasting effect of D.piger CH-104 administration by isolating bone marrow cells from the mice of the study. These were treated by LPS (100 ng / ml) as to induce an inflammatory response and subsequently the level of induced cytokines were determined. Remarkably, the levels of the pro-inflammatory cytokine IL-6 and TN Fa were reduced upon LPS treatment when the bone marrow cells were isolated from mice treated with D.piger CH-104 over the placebo treated mice (Fig. 7).

[0168] Fig. 7 shows reduced cytokine production after LPS induction of bone marrow cells isolated from mice after administration of D.piger CH-104 as compared to placebo. Two stars indicate p< 0.01 and 3 stars p< 0.001.

[0169] This again points to a lasting anti-inflammatory effect of the D.piger CH-104 treatment that is in line with its other effects in reducing liver inflammation, damage and markers of MASH as well as increasing barrier function. It is known that long term liver inflammation as in MASH will lead to the development of MAFLD that is irreversible and leads to liver steatosis. Hence, administration of D.piger CH-104 and strains that share high genomic identity and hence physiological similarity will have the capacity to reduce MAFLD or MASH development.

[0170] References

[0171] EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Rychen G, Aquilina G, et al. Guidance on the characterisation of microorganisms used as feed additives or as production organisms. EFSA J. 2018;16(3):e05206. Published 2018 Mar 28. doi : 10.2903 / j.efsa.2018.5206

[0172] Faith JJ, McNulty NP, Rey FE, Gordon JI (2011) Predicting a human gut microbiota’s response to diet in gnotobiotic mice. Science 333(6038):101-104.

[0173] Smith, B.P., Auvil, L.S., Welge, M. et al. Identification of early liver toxicity gene biomarkers using comparative supervised machine learning. Sci Rep 10, 19128 (2020). https : / / doi.org / 10.1038 / s41598- 020-76129-8

[0174] Rey FE, Gonzalez MD, Cheng J, Wu M, Ahern PP, Gordon JI. Metabolic niche of a prominent sulfatereducing human gut bacterium. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33): 13582-7. doi: 10.1073 / pnas.1312524110. Epub 2013 Jul 2

[0175] Review Nat Rev Gastroenterol Hepatol. 2024 Aug;21 (8):556-571 . doi: 10.1038 / s41575-024-00937-w. Epub 2024 Jun 3.

[0176] Endogenous ethanol production in health and disease

[0177] Meijnikman AS, Nieuwdorp & B Schnabl

Claims

CLAIMS1. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO:16, for use in preventing and / or treating hepatic steatosis.

2. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to claim 1 , wherein said Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, is combined with Anaerobutyricum soehngenii, or Anaerobutyricum species having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO:2 or SEQ ID NO:

33. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the previous claims, wherein the use is further in preventing and / or treating autobrewery syndrome by decreasing in situ ethanol level, preferably by in situ converting ethanol into short-chain fatty acid(s), particularly acetate and / or butyrate.

4. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the previous claims, wherein the hepatic steatosis is metabolic dysfunction-associated steatotic liver disease (MASLD) and / or metabolic dysfunction-associated steatohepatitis (MASH).

5. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the previous claims, which is administered by enteral, preferably oral, or nasal or by rectal administration, and / or by nasoduodenal tube administration.

6. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the previous claims, which is in lyophilized form and / or encapsulated by an enteric coating, preferable wherein said enteric coating does not dissolute and / or disintegrate in a gastric environment.

7. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of theprevious claims, wherein the Desulfovibrio GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, is comprised in a composition, preferably- a pharmaceutical composition, preferably in a liquid or solid dosage form, such as a capsule, a tablet, or a powder; and / or- a food composition, preferably a dairy product, more preferably a fermented dairy product, most preferably a yogurt or a yogurt drink.

8. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to claim 7, wherein said Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16 is present in the composition in an amount ranging from 104to 1015colony forming units (CFU).

9. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the previous claims, wherein the use involves at least 2, 3, 4, 5, 6, 7, 8, 9, 10 separate administrations of said Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, preferably with intervals of at least 1 , 2, 3, 4, 5, 6, 7, 8 weeks between said separate administrations.

10. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the previous claims, wherein the use is in a mammal, preferably a human.

11. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the claims 1-10, wherein the Desulfovibrio piger GOR is a Desulfovibrio piger having a genome comprising a sequence with at least 98% sequence identity with SEQ ID NO: 17.

12. Desulfovibrio piger CH-104, deposited as CBS153622.

13. Desulfovibrio piger GOR, or Desulfovibrio piger strain having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO: 16, for use according to any one of the claims 1-11 , wherein the Desulfovibrio piger GOR is Desulfovibrio piger CH-104 deposited as CBS153622.