Increased fecal ethanol and enriched ethanol-producing gut bacteria Limosilactobacillus fermentum, Enterocloster bolteae, Mediterraneibacter gnavus and Streptococcus mutans in nonalcoholic steatohepatitis

BackgroundNon-alcoholic steatohepatitis (NASH) has become a major public health issue as one of the leading causes of liver disease and transplantation worldwide. The instrumental role of the gut microbiota is emerging but still under investigation. Endogenous ethanol (EtOH) production by gut bacteria and yeasts is an emerging putative mechanism. Microbial metagenomics and culture studies targeting enterobacteria or yeasts have been reported, but no culturomics studies have been conducted so far.AimTo assess fecal EtOH and other biochemical parameters, characterize NASH-associated dysbiosis and identify EtOH-producing gut microbes associated with the disease, fecal samples from 41 NASH patients and 24 controls were analyzed. High-performance liquid chromatography (HPLC) was used for EtOH, glucose, total proteins, triglyceride and total cholesterol. Viable bacteria were assessed with microbial culturomics. Microbial genetic material was assessed using 16S metagenomics targeting the hypervariable V3V4 region.ResultsFecal EtOH and glucose was elevated in the stools of NASH patients (p < 0.05) but not triglyceride, total cholesterol or proteins. In culturomics, EtOH-producing Enterocloster bolteae and Limosilactobacillus fermentum were enriched in NASH. V3V4 16S rRNA amplicon sequencing confirmed the enrichment in EtOH-producing bacteria including L. fermentum, Mediterraneibacter gnavus and Streptococcus mutans, species previously associated with NASH and other dysbiosis-associated diseases. Strikingly, E. bolteae was identified only by culturomics. The well-known Lacticaseibacillus casei was identified in controls but never isolated in patients with NASH (p < 0.05).ConclusionElevated fecal EtOH and glucose is a feature of NASH. Several different EtOH-producing gut bacteria may play an instrumental role in the disease. Culturomics and metagenomics, two complementary methods, will be critical to identify EtOH-producing bacteria for future diagnostic markers and therapeutic targets for NASH. Suppression of EtOH-producing gut microbes and L. casei administration are options to be tested in NASH treatment

Caractérisation du microbiote intestinal chez les patients hépatiques : Infection par le virus de l'hépatite B et hépatite alcoolique

Liver diseases are responsible for increasing global health burden. In recent years, extensive research has demonstrated that gut dysbiosis is an instrumental factor contributing to the pathophysiology of liver diseases. Different patterns of gut microbiota have been observed in alcohol-related liver disease (ALD), hepatitis B virus (HBV) infection, or even their associated complications such as cirrhosis, and hepatocellular carcinoma (HCC). Indeed, metagenomics revealed significant alterations in gut microbiota composition in HBV-infected patients or ALD, with a decrease in butyrate-producing Clostridiales and an increase in inflammation-inducing Enterobacteriaceae. Few research, however, have studied gut dysbiosis at the species level. Furthermore, no study has investigated the gut microbiota in such patients using culturomics approach.Therefore, in the present work, we first performed case-control studies to investigate gut microbiota in HBV and ALD patients, using culturomics and metagenomics. Moreover, we first performed a systematic literature review using artificial intelligence (AI)-powered research tools to decipher gut microbiota signatures associated with HBV that could be used as non-invasive diagnostic biomarkers in different disease stages.Our qualitative analysis in the review allowed us to identify reproducible results for a signature microbiota profile in HBV-associated diseases characterized by a consistent increase in most members of p_Pseudomonadota, c_Bacilli, g_Prevotella, g_Streptococcus, g_Veillonella and a consistent decrease in c_Clostridia, f_Lachnospiraceae, and g_Roseburria. In addition, Saccharomyces cerevisae and Candida tropicalis yeast species were highlyabundant. Both S. cerevisiae and C. tropicalis produce high amounts of ethanol. These results are consistent with the emerging role of endogenous ethanol production in liver diseases.The characteristics of the HBV-associated dysbiosis in our investigation revealed an enrichment in the recently emerging genus Enterocloster (formerly Clostridium), whose significance in HBV infection has not been identified. However, metagenomics could not detect this enrichment due to low taxonomic resolution. At the species level, Enterocloster bolteae species were highly enriched with two strains producing a considerable amount of ethanol (27 and 200 mM), which is known to be harmful to the liver. Interestingly, members of the uncultivated Candidate Phyla Radiation (CPR) phylum, such as Candidatus Saccharibacteria and Atribacter phylum, were discovered to be adversely connected with prothrombin activity in HBV-associated dysbiosis for the first time.Furthermore, Thomasclavelia ramosa (formerly Clostridium ramosum) was found for the first time to be significantly prevalent in patients with ALD who developed HCC. Both culturomics and metagenomic approaches validated this result. Interestingly, T. ramosa has already been found in colorectal and nasopharyngeal cancer patients, indicating its possible oncogenic role.In this context, microbial culturomics helped us isolate viable bacterial species that could be used for further manipulations. Our results suggest that endogenous ethanol production by E. bolteae might participate in HBV-related liver disease. Additionally, the role of CPR in HBV-associated complications deserves further investigation. Future studies should also focus on elucidating the mechanisms by which T. ramosa might contribute to liver carcinoma.Finally, our results provide insight into the potential role of those species in the pathophysiology of ALD and HBV-related disease. This helps pave the way for further development of microbiome-targeted therapeutic options restoring healthy gut microbiota in hepatic patients, such as probiotics and fecal microbiota transplantation.Les maladies du foie sont responsables d'un fardeau sanitaire mondial de plus en pluslourd. Ces dernie res anne es, des recherches approfondies ont de montre que la dysbioseintestinale est un facteur de terminant qui contribue a la physiopathologie des maladiesdu foie. Des profils diffe rents du microbiote intestinal ont e te observe s dans les maladieshe patiques lie es a l'alcool (ALD), l'infection par le virus de l'he patite B (VHB), ou me meces complications associe es telles que la cirrhose et le carcinome he patocellulaire (CHC).En effet, la me tage nomique a re ve le des alte rations significatives de la composition dumicrobiote intestinal chez les patients infecte s par le VHB ou en ALD, avec une diminutiondes Clostridiales produisant du butyrate et une augmentation des Enterobacteriaceaeinduisant l'inflammation. Cependant, peu de recherches ont e tudie la dysbiose intestinaleau niveau des espe ces microbiennes. En outre, aucune e tude n'a examine le microbioteintestinal de ces patients a l'aide de l’approche de culturomique.Par conse quent, dans le pre sent travail, nous avons d'abord re alise des e tudes cas-te moinspour e tudier le microbiote intestinal chez les patients atteints du VHB et de l'ALD, enutilisant la culturomique et la me tage nomique. En outre, nous avons d'abord effectue uneanalyse syste matique de la litte rature a l'aide d'outils de recherche assiste s par ordinateurafin de de crypter les signatures du microbiote intestinal associe es au VHB qui pourraiente tre utilise es comme biomarqueurs diagnostiques non invasifs a diffe rents stades de lamaladie.Notre analyse qualitative dans la revue nous a permis d'identifier des re sultatsreproductibles pour un profil de microbiote signature dans les maladies associe es au VHB,caracte rise par une augmentation constante de la plupart des membres de -Pseudomonadota, Bacilli, Prevotella, Streptococcus, Veillonella et une diminution constantede Clostridia, Lachnospiraceae, et Roseburria. En outre, les espe ces de levureSaccharomyces cerevisae et Candida tropicalis ont e te tre s abondantes. S. cerevisiae et C.tropicalis produisent toutes les deux de grandes quantite s d'e thanol. Ces re sultats sontcohe rents avec le ro le e mergent de la production endoge ne d'e thanol dans les maladiesdu foie.Les caracte ristiques de la dysbiose associe e au VHB dans notre e tude ont re ve le unenrichissement dans le genre Enterocloster (anciennement Clostridium), re cemmentapparu, dont l'importance dans l'infection par le VHB n'a pas e te identifie e. Cependant, lame tage nomique n'a pas pu de tecter cet enrichissement en raison d'une faible re solutiontaxonomique. Au niveau des espe ces bacte riennes, les espe ces Enterocloster bolteaee taient fortement enrichies avec deux souches produisant une quantite conside rabled'e thanol (27 et 200 mM), qui est connue pour e tre nocive pour le foie. Il est inte ressantde noter que les membres du phylum Candidate Phyla Radiation (CPR) non cultive s, telsque les Candidatus Saccharibacteria et le phylum Atribacter, ont e te de couverts pour lapremie re fois en relation ne gative avec l'activite de la prothrombine dans la dysbioseassocie e au VHB.En outre, on a constate pour la premie re fois que Thomasclavelia ramosa (anciennementClostridium ramosum) e tait tre s re pandu chez les patients atteints d'ALD qui ontde veloppe un CHC. Les approches culturomics et me tage nomiques ont valide ce re sultat.Il est inte ressant de noter que T. ramosa a de ja e te trouve chez des patients atteints decancer colorectal et nasopharynge , ce qui indique son ro le oncoge ne possible.Dans ce contexte, la culturomique microbienne nous a permis d'isoler des espe cesbacte riennes viables qui ont pu e tre utilise es pour d'autres manipulations. Nos re sultats sugge rent que la production endoge ne d'e thanol par E. bolteae pourrait participer a lamaladie he patique lie e au VHB. En outre, le ro le de la CPR dans les complications associe esau VHB me rite d'e tre approfondi. Les e tudes futures devraient e galement se concentrersur l'e lucidation des me canismes par lesquels T. ramosa pourrait contribuer au carcinomehe patique.Enfin, nos re sultats ouvrent de nouvelles perspectives sur le ro le potentiel du microbioteintestinal dans la physiopathologie de l'ALD et des maladies lie es au VHB. Ils ouvrent lavoie au de veloppement d'options the rapeutiques cible es sur le microbiome et visant arestaurer un microbiote intestinal sain chez les patients he patiques, telles que lesprobiotiques et la transplantation du microbiote fe cal

Caractérisation du microbiote intestinal chez les patients hépatiques : Infection par le virus de l'hépatite B et hépatite alcoolique

Liver diseases are responsible for increasing global health burden. In recent years, extensive research has demonstrated that gut dysbiosis is an instrumental factor contributing to the pathophysiology of liver diseases. Different patterns of gut microbiota have been observed in alcohol-related liver disease (ALD), hepatitis B virus (HBV) infection, or even their associated complications such as cirrhosis, and hepatocellular carcinoma (HCC). Indeed, metagenomics revealed significant alterations in gut microbiota composition in HBV-infected patients or ALD, with a decrease in butyrate-producing Clostridiales and an increase in inflammation-inducing Enterobacteriaceae. Few research, however, have studied gut dysbiosis at the species level. Furthermore, no study has investigated the gut microbiota in such patients using culturomics approach.Therefore, in the present work, we first performed case-control studies to investigate gut microbiota in HBV and ALD patients, using culturomics and metagenomics. Moreover, we first performed a systematic literature review using artificial intelligence (AI)-powered research tools to decipher gut microbiota signatures associated with HBV that could be used as non-invasive diagnostic biomarkers in different disease stages.Our qualitative analysis in the review allowed us to identify reproducible results for a signature microbiota profile in HBV-associated diseases characterized by a consistent increase in most members of p_Pseudomonadota, c_Bacilli, g_Prevotella, g_Streptococcus, g_Veillonella and a consistent decrease in c_Clostridia, f_Lachnospiraceae, and g_Roseburria. In addition, Saccharomyces cerevisae and Candida tropicalis yeast species were highlyabundant. Both S. cerevisiae and C. tropicalis produce high amounts of ethanol. These results are consistent with the emerging role of endogenous ethanol production in liver diseases.The characteristics of the HBV-associated dysbiosis in our investigation revealed an enrichment in the recently emerging genus Enterocloster (formerly Clostridium), whose significance in HBV infection has not been identified. However, metagenomics could not detect this enrichment due to low taxonomic resolution. At the species level, Enterocloster bolteae species were highly enriched with two strains producing a considerable amount of ethanol (27 and 200 mM), which is known to be harmful to the liver. Interestingly, members of the uncultivated Candidate Phyla Radiation (CPR) phylum, such as Candidatus Saccharibacteria and Atribacter phylum, were discovered to be adversely connected with prothrombin activity in HBV-associated dysbiosis for the first time.Furthermore, Thomasclavelia ramosa (formerly Clostridium ramosum) was found for the first time to be significantly prevalent in patients with ALD who developed HCC. Both culturomics and metagenomic approaches validated this result. Interestingly, T. ramosa has already been found in colorectal and nasopharyngeal cancer patients, indicating its possible oncogenic role.In this context, microbial culturomics helped us isolate viable bacterial species that could be used for further manipulations. Our results suggest that endogenous ethanol production by E. bolteae might participate in HBV-related liver disease. Additionally, the role of CPR in HBV-associated complications deserves further investigation. Future studies should also focus on elucidating the mechanisms by which T. ramosa might contribute to liver carcinoma.Finally, our results provide insight into the potential role of those species in the pathophysiology of ALD and HBV-related disease. This helps pave the way for further development of microbiome-targeted therapeutic options restoring healthy gut microbiota in hepatic patients, such as probiotics and fecal microbiota transplantation.Les maladies du foie sont responsables d'un fardeau sanitaire mondial de plus en pluslourd. Ces dernie res anne es, des recherches approfondies ont de montre que la dysbioseintestinale est un facteur de terminant qui contribue a la physiopathologie des maladiesdu foie. Des profils diffe rents du microbiote intestinal ont e te observe s dans les maladieshe patiques lie es a l'alcool (ALD), l'infection par le virus de l'he patite B (VHB), ou me meces complications associe es telles que la cirrhose et le carcinome he patocellulaire (CHC).En effet, la me tage nomique a re ve le des alte rations significatives de la composition dumicrobiote intestinal chez les patients infecte s par le VHB ou en ALD, avec une diminutiondes Clostridiales produisant du butyrate et une augmentation des Enterobacteriaceaeinduisant l'inflammation. Cependant, peu de recherches ont e tudie la dysbiose intestinaleau niveau des espe ces microbiennes. En outre, aucune e tude n'a examine le microbioteintestinal de ces patients a l'aide de l’approche de culturomique.Par conse quent, dans le pre sent travail, nous avons d'abord re alise des e tudes cas-te moinspour e tudier le microbiote intestinal chez les patients atteints du VHB et de l'ALD, enutilisant la culturomique et la me tage nomique. En outre, nous avons d'abord effectue uneanalyse syste matique de la litte rature a l'aide d'outils de recherche assiste s par ordinateurafin de de crypter les signatures du microbiote intestinal associe es au VHB qui pourraiente tre utilise es comme biomarqueurs diagnostiques non invasifs a diffe rents stades de lamaladie.Notre analyse qualitative dans la revue nous a permis d'identifier des re sultatsreproductibles pour un profil de microbiote signature dans les maladies associe es au VHB,caracte rise par une augmentation constante de la plupart des membres de -Pseudomonadota, Bacilli, Prevotella, Streptococcus, Veillonella et une diminution constantede Clostridia, Lachnospiraceae, et Roseburria. En outre, les espe ces de levureSaccharomyces cerevisae et Candida tropicalis ont e te tre s abondantes. S. cerevisiae et C.tropicalis produisent toutes les deux de grandes quantite s d'e thanol. Ces re sultats sontcohe rents avec le ro le e mergent de la production endoge ne d'e thanol dans les maladiesdu foie.Les caracte ristiques de la dysbiose associe e au VHB dans notre e tude ont re ve le unenrichissement dans le genre Enterocloster (anciennement Clostridium), re cemmentapparu, dont l'importance dans l'infection par le VHB n'a pas e te identifie e. Cependant, lame tage nomique n'a pas pu de tecter cet enrichissement en raison d'une faible re solutiontaxonomique. Au niveau des espe ces bacte riennes, les espe ces Enterocloster bolteaee taient fortement enrichies avec deux souches produisant une quantite conside rabled'e thanol (27 et 200 mM), qui est connue pour e tre nocive pour le foie. Il est inte ressantde noter que les membres du phylum Candidate Phyla Radiation (CPR) non cultive s, telsque les Candidatus Saccharibacteria et le phylum Atribacter, ont e te de couverts pour lapremie re fois en relation ne gative avec l'activite de la prothrombine dans la dysbioseassocie e au VHB.En outre, on a constate pour la premie re fois que Thomasclavelia ramosa (anciennementClostridium ramosum) e tait tre s re pandu chez les patients atteints d'ALD qui ontde veloppe un CHC. Les approches culturomics et me tage nomiques ont valide ce re sultat.Il est inte ressant de noter que T. ramosa a de ja e te trouve chez des patients atteints decancer colorectal et nasopharynge , ce qui indique son ro le oncoge ne possible.Dans ce contexte, la culturomique microbienne nous a permis d'isoler des espe cesbacte riennes viables qui ont pu e tre utilise es pour d'autres manipulations. Nos re sultats sugge rent que la production endoge ne d'e thanol par E. bolteae pourrait participer a lamaladie he patique lie e au VHB. En outre, le ro le de la CPR dans les complications associe esau VHB me rite d'e tre approfondi. Les e tudes futures devraient e galement se concentrersur l'e lucidation des me canismes par lesquels T. ramosa pourrait contribuer au carcinomehe patique.Enfin, nos re sultats ouvrent de nouvelles perspectives sur le ro le potentiel du microbioteintestinal dans la physiopathologie de l'ALD et des maladies lie es au VHB. Ils ouvrent lavoie au de veloppement d'options the rapeutiques cible es sur le microbiome et visant arestaurer un microbiote intestinal sain chez les patients he patiques, telles que lesprobiotiques et la transplantation du microbiote fe cal

Taxonogenomics of Culturomica massiliensis gen. nov., sp. nov., and Emergencia timonensis gen. nov., sp. nov. new bacteria isolated from human stool microbiota

Abstract Two new bacterial strains, Marseille-P2698T (CSUR P2698 = DSM 103,121) and Marseille-P2260T (CSUR P2260 = DSM 101,844 = SN18), were isolated from human stools by the culturomic method. We used the taxonogenomic approach to fully describe these two new bacterial strains. The Marseille-P2698T strain was a Gram-negative, motile, non-spore-forming, rod-shaped bacterium. The Marseille-P2260T strain was a Gram-positive, motile, spore-forming rod-shaped bacterium. Major fatty acids found in Marseille-P2698T were C15:0 iso (63%), C15:0 anteiso (11%), and C17:0 3-OH iso (8%). Those found in Marseille-P2260T strain were C16:00 (39%), C18:1n9 (16%) and C18:1n7 (14%). Strains Marseille-P2698T and Marseille-P2260T had 16S rRNA gene sequence similarities of 91.50% with Odoribacter laneus T, and of 90.98% and 95.07% with Odoribacter splanchnicus T and Eubacterium sulci T, respectively. The exhibited digital DNA-DNA Hybridization values lower than 20.7%, and Orthologous Average Nucleotide Identity values lower than 73% compared to their closest related bacterial species O. splanchnicus T and E. sulci T respectively. Phenotypic, biochemical, phylogenetic, and genomic results obtained by comparative analyses provided sufficient evidence that both of the two studied strains Marseille-P2698T and Marseille-P2260T are two new bacterial species and new bacterial genera for which the names Culturomica massiliensis gen. nov., sp. nov., and Emergencia timonensis gen. nov., sp. nov. were proposed, respectively

Limosilactobacillus fermentum, Lactococcus lactis and Thomasclavelia ramosa are enriched and Methanobrevibacter smithii is depleted in patients with non-alcoholic steatohepatitis

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Ethanol-Producing Enterocloster bolteae Is Enriched in Chronic Hepatitis B-Associated Gut Dysbiosis: A Case–Control Culturomics Study

International audienceBackground: Hepatitis B virus (HBV) infection is a global health epidemic that causes fatal complications, leading to liver cirrhosis and hepatocellular carcinoma. The link between HBV-related dysbiosis and specific bacterial taxa is still under investigation. Enterocloster is emerging as a new genus (formerly Clostridium), including Enterocloster bolteae, a gut pathogen previously associated with dysbiosis and human diseases such as autism, multiple sclerosis, and inflammatory bowel diseases. Its role in liver diseases, especially HBV infection, is not reported. Methods: The fecal samples of eight patients with chronic HBV infection and ten healthy individuals were analyzed using the high-throughput culturomics approach and compared to 16S rRNA sequencing. Quantification of ethanol, known for its damaging effect on the liver, produced from bacterial strains enriched in chronic HBV was carried out by gas chromatography-mass spectrometry. Results: Using culturomics, 29,120 isolated colonies were analyzed by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-TOF); 340 species were identified (240 species in chronic HBV samples, 254 species in control samples) belonging to 169 genera and 6 phyla. In the chronic HBV group, 65 species were already known in the literature; 48 were associated with humans but had not been previously found in the gut, and 17 had never been associated with humans previously. Six species were newly isolated in our study. By comparing bacterial species frequency, three bacterial genera were serendipitously found with significantly enriched bacterial diversity in patients with chronic HBV: Enterocloster, Clostridium, and Streptococcus (p = 0.0016, p = 0.041, p = 0.053, respectively). However, metagenomics could not identify this enrichment, possibly concerning its insufficient taxonomical resolution (equivocal assignment of operational taxonomic units). At the species level, the significantly enriched species in the chronic HBV group almost all belonged to class Clostridia, such as Clostridium perfringens, Clostridium sporogenes, Enterocloster aldenensis, Enterocloster bolteae, Enterocloster clostridioformis, and Clostridium innocuum. Two E. bolteae strains, isolated from two patients with chronic HBV infection, showed high ethanol production (27 and 200 mM). Conclusions: Culturomics allowed us to identify Enterocloster species, specifically, E. bolteae, enriched in the gut microbiota of patients with chronic HBV. These species had never been isolated in chronic HBV infection before. Moreover, ethanol production by E. bolteae strains isolated from the chronic HBV group could contribute to liver disease progression. Additionally, culturomics might be critical for better elucidating the relationship between dysbiosis and chronic HBV infection in the future

Endogenous Ethanol and Triglyceride Production by Gut Pichia kudriavzevii, Candida albicans and Candida glabrata Yeasts in Non-Alcoholic Steatohepatitis

International audienceNonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably by high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn). Candida yeasts are the main causes of auto-brewery syndromes but have been neglected in NASH. Here, the fecal ethanol and microbial content of 10 cases and 10 controls were compared. Ethanol was measured by gas chromatography-mass spectrometry. Species identification was performed by MALDI-TOF MS, and triglyceride production was assessed by a colorimetric enzymatic assay. The fecal ethanol concentration was four times higher in patients with NASH (median [interquartile range]: 0.13 [0.05–1.43] vs. 0.034 [0.008–0.57], p = 0.037). Yeasts were isolated from almost all cases but not from controls (9/10 vs. 0/10, p = 0.0001). Pichia kudriavzevii was the most frequent (four patients), while Candida glabrata, Candida albicans, and Galactomyces geotrichum were identified in two cases each. The concentration of ethanol produced by yeasts was 10 times higher than that produced by bacteria (median, 3.36 [0.49–5.60] vs. 0.32 [0.009–0.43], p = 0.0029). Using a 10% D-fructose restricted medium, we showed that NASH-associated yeasts transformed fructose in ethanol. Unexpectedly, yeasts isolated from NASH patients produced a substantial amount of triglycerides. Pichia kudriavzevii strains produced the maximal ethanol and triglyceride levels in vitro. Our preliminary human descriptive and in vitro experimental results suggest that yeasts have been neglected. In addition to K. pneumoniae, gut Pichia and Candida yeasts could be linked with NASH pathophysiology in a species- and strain-specific manner through fructose-dependent endogenous alcohol and triglyceride production

Description of Agathobaculum massiliense sp. nov., a new bacterial species prevalent in the human gut and predicted to produce indole and tryptophan based on genomic analysis

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