Thomasclavelia ramosa and Alcohol-Related Hepatocellular Carcinoma: A Microbial Culturomics Study

Thomasclavelia ramosa and Alcohol-Related Hepatocellular Carcinoma: A Microbial Culturomics Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thomasclavelia ramosa and Alcohol-Related Hepatocellular Carcinoma: A Microbial Culturomics Study Reham MAGDY WASFY, Anissa ABDOULAYE, Patrick BORENTAIN, Babacar MBAYE, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5053117/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2025 Read the published version in Gut Pathogens → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Gut microbiota alteration is implicated in the pathogenesis of alcoholic liver disease (ALD) and associated hepatocellular carcinoma (HCC). No study has characterized the dysbiosis associated with ALD by microbial culturomics, which certifies viability and allows pathobiont strain candidates to be characterized. Methods: A case-control study (n = 59) was conducted on patients with ALD without HCC (ALD-NoHCC, n=16), ALD with HCC (ALD-HCC, n=19) and controls (n=24) groups. 16S rRNA amplicon sequencing and microbial culturomics were used as complementary methods for gut microbiome profiling. Results: Compared to the control group, Thomasclavelia ramosa and Gemmiger formicilis were significantly increased in the ALD-HCC group and Mediterraneibacter gnavus was significantly increased in the ALD-NoHCC group using 16S rRNA sequencing. By microbial culturomics, T. ramosa was detected in all ALD samples (100%), and the most enriched since cultivated in only a small proportion of controls (20%, p < 0.001). Conclusions: T. ramosa , identified by culturomics and 16 rRNA sequencing, may be associated with ALD and ALD-HCC. These results highlight the potential role of T. ramosa in liver cancer, in line with its genotoxic properties and its tumor growth-promoting effect in gnotobiotic mice recently reported. Thomasclavelia ramosa Enterocloster bolteae Mediterraneibacter gnavus Alcoholic-related liver disease Hepatocellular carcinoma Gut microbiome Microbial culturomics 16S rRNA sequencing Cancer Figures Figure 1 Figure 2 HIGHLIGHTS The gut microbiota signature of ALD and ALD-HCC was explored by microbial culturomics and 16S amplicon sequencing. By culturomics, T. ramosa was the most enriched and cultured from all included ALD patients, but in only 20% of controls (p < 0.05). T. ramosa has recently been reported to have genotoxic properties and to promote the development of colon tumors in gnotobiotic mice. T. ramosa is identified as a putative oncobiont associated with ALD-HCC, thus opening new avenues for diagnosis and treatment. INTRODUCTION Hepatocellular carcinoma (HCC) is the most common form of liver cancer (90% of cases), primarily associated with chronic liver disease. Its major risk factors include chronic viral hepatitis infection (HBV, HCV), Metabolic dysfunction-associated Steato-Hepatitis (MASH) and Alcoholic Liver Disease (ALD) ( 1 ). Liver cancer ranks sixth for incidence and third for mortality across diverse cancers, which has caused a great cancer burden globally ( 2 ). The link between alcohol, liver disease, and cancer is well-established. Its mechanism would include direct toxicity of alcohol on the liver, but persistent instrumental factors are suspected, as the evolution of the disease is not reversible upon withdrawal ( 3 ). The gut-liver axis and gut microbiota are potential candidates to explain the persistence of a vicious circle that would explain the persistent excess risk up to 10 years after weaning ( 4 ). Recent studies suggest that alcohol dependence syndrome and ALD are both associated with gut microbiota alteration with distinct features ( 5 , 6 ). This indicates that a specific and persistent gut dysbiosis may enhance cirrhosis and hepatocarcinogenesis by the gut-liver axis. Indeed, some data from human studies ( 7 – 12 ) and animal experimental models ( 13 – 18 ) indicate that HCC occurrence is related to gut microbiota, and treatment with broad-spectrum antibiotics decreases HCC tumor growth in mice ( 19 ). Several metagenomic studies have investigated gut dysbiosis in patients with ALD based on 16S rRNA amplicon sequencing ( 20 – 22 ) or shotgun (whole-genome) sequencing ( 6 ). However, no study described gut dysbiosis in patients with ALD or ALD-associated HCC (ALD-HCC) based on the culturomics approach. It has been shown that the reliability of metagenome assembled genomes (MAGs) obtained through deep sequencing is limited and biased by the production of erroneous MAGs compared to isolation especially for the handful of MAGs that are critical for the study of pathogens ( 23 ). This evidences that culture remains essential and complementary to sequencing when investigating gut pathogens associated with liver cancer. Culture-based studies focusing on intestinal dysbiosis and liver cancer are scarce. We found a study reporting cultured microbial counts but with minimal microbial taxonomic accuracy and evidencing an Escherichia coli enrichment ( 7 ). Microbial culturomics is a new -omics strategy developed in our center as a high-throughput culture method based on Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) and diversified physicochemical culture conditions mimicking the natural microenvironment ( 24 ). Our team applied fungal and bacterial culturomics in the context of liver diseases, demonstrating the importance of ethanol-producing yeast and bacteria in MASH and HBV-associated liver disease ( 25 , 26 ). However, to our knowledge, no study has yet characterized microbiota associated with ALD using microbial culturomics. We, therefore, decided to carry out this microbial culturomics study to complete the spectrum of dysbiosis of the gut microbiota, whose instrumental role had been demonstrated in ALD with experimental evidence ( 27 ). Accordingly, this study aims to characterize the microbial signature in patients with ALD and ALD-associated HCC using both culturomics and large-scale sequencing (v3v4 region 16S rRNA amplicon sequencing). METHODS Full methods are reported in the supplementary data. Briefly, 59 participants were investigated, including 35 patients with ALD (19 with HCC) and 24 controls (CTL). Liver stiffness measurements in cirrhotic patients were conducted using a FibroScan® instrument (Echosens, Paris, France). Routine biochemistry, including prothrombin index (PT), platelets count (PLT), total bilirubin (TBIL), serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and serum creatinine, were measured. These analyses were performed only among cases. The fecal samples from all participants were collected and analyzed by v3-v4 16S rRNA amplicon sequencing (see Supplementary methods). We selected 11 samples from patients with ALD and 10 samples from controls for microbial culturomics (see Supplementary methods). For culturomics, 21 samples were selected as this approach is of unequaled value since it is the only one that ascertains viable strains, but it is associated with a huge workload (6 weeks by sample). Statistical analyses were performed using GraphPad Prism Software for Windows (GraphPad Software, San Diego, CA, USA) (version 9.0). P values less than 0.05 were considered significant differences. MAIN RESULTS The bacterial genera Thomasclavelia, Enterocloster, Clostridium, and Peptoniphilus are associated with ALD by culturomics The characteristics of the study participants and the general results of culturomics are detailed in the supplementary results. Overall, twenty-one samples were analyzed using microbial culturomics (5 ALD-HCC, 6 ALD-NoHCC, and 10 CTL), allowing the isolation and identification of 32,088 colonies (Table S1). Diversity at the species level was found to be increased in the ALD group for only four bacterial genera, all belonging to one phylum ( Bacillota ), including Thomasclavelia (p = 0.0016), Enterocloster (p = 0.0058), Clostridium (p = 0.0021) and Peptoniphilus (p = 0.0044, Fig. 1a). Thomasclavelia ramosa is the most enriched species in ALD by culturomics. At the cultured species level, surprisingly, T. ramosa was detected in all ALD patients (11/11 (100%), including those with ALD-HCC (n = 5) and ALD-NoHCC (n = 6), but in only 20% of the controls (2/10, two-tailed Fisher exact test p = 0.00044, Fig. 1b). Moreover, among all the cultured species, T. ramosa also had the most significant difference in frequency between cases and controls (100% vs. 20%: +80%). This was consistent with the increased diversity of the Thomasclavelia genus, as reported above (Fig. 1a). Enrichment of Enterocloster species in ALD by culturomics We recently reported an enrichment in Enterocloster bolteae in liver diseases associated with metabolic-associated steatohepatitis (26) and hepatitis B virus (28). Here, we found again an enrichment in Enterocloster bolteae in liver disease associated with alcoholism (Fig. 1b). This was consistent with the increased diversity of this genus in ALD (Fig. 1a), particularly the significant increase of 3 species of this genus, including also E. clostridioformis and E. aldenensis (Fig. 1b & Fig. 1c). Strikingly, this association was found only by culturomics but not by 16S rRNA sequencing, as observed in our previous studies (26, 28). The species Thomasclavelia ramosa and Mediterraneibacter gnavus are enriched in ALD both by culturomics and 16S rRNA amplicon sequencing Linear discriminant analysis (LDA) effect size modeling was applied on the v3v4 sequencing data of the 59 samples to identify specific bacterial taxa associated with ALD. Compared to the control group, the gut microbiota of all ALD patients showed a significantly increased abundance of Streptococcus_salivarius/Atribacter_sp223 (OTU39314), Escherichia_albertii / Escherichia_coli (OTU2689), Mediterraneibacter_gnavus (formerly, Ruminococcus_gnavus (29)), and Thomasclavelia_ramosa (formerly Clostridium ramosum (30)). Accordingly, the only two species enriched in ALD both by culturomics and 16S rRNA sequencing were T. ramosa and Mediterraneibacter gnavus . Thomasclavelia ramosa was the only species identified by culturomics and sequencing and associated with hepatocellular carcinoma Including all three groups (ALD-NoHCC, ALD-HCC, CTL) in a Linear Discriminant Analysis, the pathobiont Mediterraneibacter_gnavus (31, 32) was significantly increased in ALD-NoHCC, while T_ramosa and Gemmiger_formicilis were the only 2 operational taxonomic units (OTUs) significantly associated with the ALD-HCC group (Fig. 2). Accordingly, T. ramosa was the only species identified by culturomics and sequencing and associated with ALD-HCC, identifying it as one of the best oncobiont candidates for further exploration of an instrumental role of a gut microbe for liver cancer associated with alcoholism. Genomic analysis did not identify a clonal specificity (Supplementary Table 2 & Supplementary Fig. 2). DISCUSSION This brief preliminary report, which uses a combined approach of microbial culturomics and 16S rRNA sequencing, identifies T. ramosa as one of the best candidates for the intestinal pathogenic oncobiont associated with ALD and HCC associated with ALD. T. ramosa is a human gut pathogen associated with several cases of severe infection (bacteremia, infection of aortic aneurysm, osteomyelitis, arthritis, gas gangrene, Fournier's gangrene, fatal infections) recently associated with human cancer, notably colorectal cancer ( 33 , 34 ), but also HCC in a 2023 Chinese study ( 35 ). One of the first discovered genotoxin produced by a gut commensal was colibactin from Escherichia coli strains which alkylates DNA, and associated with colon cancer ( 36 ). A another recent study reported that two other gut commensals, Morganella morganii and T. ramosa , exhibited genotoxicity and promoted tumor growth in an experimental model ( 37 ). While indolimines have been characterized as the genotoxic molecules for M. morganii , the small molecules (< 3 kDa) that lead to the genotoxic and pro-cancer role are not known for T. ramose ( 37 ). Notably, T. ramosa lacks known biosynthetic gene clusters (BGCs) and induces DNA damage via colibactin-independent and indolimines-independent mechanisms ( 37 ). Limitations of our study included a small sample size and lack of experimental evidence. However, the clear T. ramosa signature (LDA score > 5, p < 0.05) identified here with a limited sample size means that the difference (effect size) is huge, supporting the strength of the association as the first Bradford-Hill criteria for causality. Consistency and reproducibility are fulfilled by two previous studies confirming the association with CRC ( 33 , 34 ) and the confirmation of the T. ramosa -liver cancer association by another team ( 35 ). The linear discriminant analysis fulfills the biological gradient (the higher the T. ramosa 16S rRNA number of reads - the higher the risk of HCC). The plausibility and experimental evidence are supported by the recently reported in vitro genotoxicity and in vivo tumor growth-promoting properties of T. ramosa ( 37 ). The robustness of our results is not due to the number of samples or deep sequencing, but to microbial culturomics, the only approach to certify the presence of viable and biologically active microbes with high fidelity taxonomic characterization at the species level thanks to MALDI-TOF MS. Studies with higher statistical power could identify other microbial candidates, and T. ramosa is very likely part of a group of liver cancer oncobionts. Future studies could use deep sequencing and automated microbial culturomics (45) to focus on a larger sample to obtain a better match or at least larger groups, allowing for the stratification of all comorbidities and the stage of liver disease. We found that T. ramosa was the most different in culturomics between ALD and controls after analysis of 21 samples and 32088 colonies, and the most significantly associated with liver cancer by DNA sequencing after analysis of 59 samples and 2,840,773 reads. Even if future studies with larger sample size and better representation of participant groups according to the comorbidities, cause, and stage of liver disease are necessary to confirm the association and the potential role of T. ramosa in hepatocellular carcinoma, we believe that our results and recent literature ( 37 ) evidenced that T. ramosa could be a potential candidate among the gut commensals favored by alcoholism, and which can then contribute to liver carcinogenesis through its recently demonstrated genotoxic and tumor growth-promoting properties. Abbreviations ALD, Alcoholic liver disease; HCC, Hepatocellular carcinoma; MASH, Metabolic dysfunction-associated steatohepatitis; NGS, next-generation sequencing. Declarations Ethics approval and consent to participate The HEPATGUT study was approved by the local ethics committee of the Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France (IHUMI, 2020-004), approved by the Protection of Persons Committee (Approval No. CPP: 21.04391.000046—21075), and carried out according to the 2013 Declaration of Helsinki (World Medical Association, 2013). Patient consent (non-opposition) was obtained according to French regulations. Consent for publication Not applicable Availability of data and materials The authors confirm that the data supporting the findings of this study within the article and/or supplementary materials are available upon request. The raw sequencing data of fecal samples are available in the NCBI Sequence Read Archive with accession number PRJEB62828. The genome of six strains of Thomasclavelia ramosa (strains CSUR Q9705, Q9779, Q9849, QA0117, QA0118, and QA0666, available on request) have been sequenced, and genome sequencing data are publicly available under the Bioproject NCBI PRJEB76822. Competing interests The authors declare that they have no competing interests. Funding This work was funded by the Agence Nationale de la Recherche under two programs: ANR-15-CE36-0004-01 and ANR "Investissements d'avenir", Méditerranée Infection 10-IAHU-03. The Région Provence-Alpes-Côte d'Azur also supported this study, which received financial support from the Fondation Méditerranée Infection. Authors' contributions Conceptualization, funding acquisition, and Project administration: MM, RG. Methodology and visualization: RMW, MM. Resources: AG, PB. Investigation: RMW, AA, BM, AC, CA, GM. Data curation and formal analysis: RMW, AG, AL. Supervision: MTA, MM, RG. Validation: RWM, MM, RG. 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Supplementary Files Graphicalabstract.png Graphical abstract Supplementarydata.docx Cite Share Download PDF Status: Published Journal Publication published 07 May, 2025 Read the published version in Gut Pathogens → Version 1 posted Editorial decision: Revision requested 02 Apr, 2025 Reviews received at journal 02 Apr, 2025 Reviews received at journal 01 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 29 Mar, 2025 Submission checks completed at journal 29 Mar, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5053117","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436921665,"identity":"83b5b7b5-4a0c-4c80-8898-c3832070c3a0","order_by":0,"name":"Reham MAGDY WASFY","email":"","orcid":"","institution":"MEPHI, Aix-Marseille Université","correspondingAuthor":false,"prefix":"","firstName":"Reham","middleName":"MAGDY","lastName":"WASFY","suffix":""},{"id":436921666,"identity":"aa8c7c1e-2a49-4dbe-8ea0-c55cf3877903","order_by":1,"name":"Anissa ABDOULAYE","email":"","orcid":"","institution":"IHU Méditerranée 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MILLION","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYJACZgYDEMnceCCBgUEOJHLgAX4NjM0QLYwNIC3GYC0JBLWAAVALkExsALHxadFtP3/8cUGBTeLadqCWBxV30ueHHX4ItMVOTrcBuxazM8mMzTMM0hK3HQY57Myz3I230wyAWpKNzQ7g0HIAqIXH4DBES2Lb4dyNsxNAWg4kbsOl5fxjkJb/cC3phrPTP+DXcgNsywG4lgR56RwCttx4bDibxyDZGOqXw4YbpHMKDiQY4PHL+cQHn3n+2MluO3/44MMfFYfl5Wenb/7wocJODpcWTGAAVmlArHIQkG8gRfUoGAWjYBSMBAAAKuZuPUIN/IMAAAAASUVORK5CYII=","orcid":"","institution":"IHU Méditerranée Infection","correspondingAuthor":true,"prefix":"","firstName":"Matthieu","middleName":"","lastName":"MILLION","suffix":""},{"id":436921678,"identity":"8b5c0b7b-ad34-4d90-9da5-cdb41ef5785a","order_by":10,"name":"Rene GEROLAMI","email":"","orcid":"","institution":"IHU Méditerranée Infection","correspondingAuthor":false,"prefix":"","firstName":"Rene","middleName":"","lastName":"GEROLAMI","suffix":""}],"badges":[],"createdAt":"2024-09-08 14:30:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5053117/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5053117/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13099-025-00703-6","type":"published","date":"2025-05-07T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79810721,"identity":"095cbd23-465e-41af-9ddf-c630da4eab5b","added_by":"auto","created_at":"2025-04-03 06:37:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4176679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCulturomics results evidenced \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThomasclavelia ramosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eas the gut bacteria the most enriched in ALD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Species diversity by genus (only four genera with a significant difference are shown). b. Species with a significantly different frequency of detection between ALD and controls. Barnard’s bilateral exact test *p \u0026lt; 0.05, **p\u0026lt; 0.005, ***p\u0026lt;0.0005. At least two species were enriched in ALD for each of the four genera identified in Figure 1a (\u003cem\u003eThomasclavelia, Enterocloster, Clostridium, Peptoniphilus\u003c/em\u003e). c. Number of species for each of the four genera associated with ALD for each participant.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5053117/v1/42427ce7fed0df2eff4d2d4f.png"},{"id":79810726,"identity":"1a5946e5-1b7f-4e46-a2cb-11b9db6761eb","added_by":"auto","created_at":"2025-04-03 06:37:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3394099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinear discriminant analysis (LDA) on 16S rRNA sequencing results identified association between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThomasclavelia ramosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand liver cancer in alcohol liver disease patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALD: Alcoholic liver disease; CTL: Controls; HCC: Hepatocellular carcinoma. A logarithmic LDA score \u0026gt; 2 indicated a higher relative abundance in the corresponding group than in other groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5053117/v1/6211e1ea28525a4b78cd2f4f.png"},{"id":82537493,"identity":"e84f3475-0ebe-4d02-a47d-3c06eac79a1f","added_by":"auto","created_at":"2025-05-12 16:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6914026,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5053117/v1/b23d1565-47b3-4b15-953d-fd36dc9e1030.pdf"},{"id":79810723,"identity":"3b8d937e-1c40-4b1b-94c8-07fe9b6a94f3","added_by":"auto","created_at":"2025-04-03 06:37:55","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":199393,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5053117/v1/5607815802933063a89e1d82.png"},{"id":79811566,"identity":"ca60021b-df06-4472-b87f-0a410d0c974b","added_by":"auto","created_at":"2025-04-03 06:45:56","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":323355,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5053117/v1/f61fb5cdc4aecb5ba1e6bb6f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thomasclavelia ramosa and Alcohol-Related Hepatocellular Carcinoma: A Microbial Culturomics Study","fulltext":[{"header":"HIGHLIGHTS","content":"\u003cul\u003e\n \u003cli\u003eThe gut microbiota signature of ALD and ALD-HCC was explored by microbial culturomics and 16S amplicon sequencing.\u003c/li\u003e\n \u003cli\u003eBy culturomics,\u003cem\u003e\u0026nbsp;T. ramosa\u003c/em\u003e was the most enriched and cultured from all included ALD patients, but in only 20% of controls (p \u0026lt; 0.05).\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eT. ramosa\u0026nbsp;\u003c/em\u003ehas recently been reported to have genotoxic properties and to promote the development of colon tumors in gnotobiotic mice.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eT. ramosa\u003c/em\u003e is identified as a putative oncobiont associated with ALD-HCC, thus opening new avenues for diagnosis and treatment.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is the most common form of liver cancer (90% of cases), primarily associated with chronic liver disease. Its major risk factors include chronic viral hepatitis infection (HBV, HCV), Metabolic dysfunction-associated Steato-Hepatitis (MASH) and Alcoholic Liver Disease (ALD) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Liver cancer ranks sixth for incidence and third for mortality across diverse cancers, which has caused a great cancer burden globally (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe link between alcohol, liver disease, and cancer is well-established. Its mechanism would include direct toxicity of alcohol on the liver, but persistent instrumental factors are suspected, as the evolution of the disease is not reversible upon withdrawal (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The gut-liver axis and gut microbiota are potential candidates to explain the persistence of a vicious circle that would explain the persistent excess risk up to 10 years after weaning (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Recent studies suggest that alcohol dependence syndrome and ALD are both associated with gut microbiota alteration with distinct features (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This indicates that a specific and persistent gut dysbiosis may enhance cirrhosis and hepatocarcinogenesis by the gut-liver axis. Indeed, some data from human studies (\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and animal experimental models (\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) indicate that HCC occurrence is related to gut microbiota, and treatment with broad-spectrum antibiotics decreases HCC tumor growth in mice (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral metagenomic studies have investigated gut dysbiosis in patients with ALD based on 16S rRNA amplicon sequencing (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) or shotgun (whole-genome) sequencing (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, no study described gut dysbiosis in patients with ALD or ALD-associated HCC (ALD-HCC) based on the culturomics approach. It has been shown that the reliability of metagenome assembled genomes (MAGs) obtained through deep sequencing is limited and biased by the production of erroneous MAGs compared to isolation especially for the handful of MAGs that are critical for the study of pathogens (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This evidences that culture remains essential and complementary to sequencing when investigating gut pathogens associated with liver cancer.\u003c/p\u003e \u003cp\u003eCulture-based studies focusing on intestinal dysbiosis and liver cancer are scarce. We found a study reporting cultured microbial counts but with minimal microbial taxonomic accuracy and evidencing an \u003cem\u003eEscherichia coli\u003c/em\u003e enrichment (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Microbial culturomics is a new -omics strategy developed in our center as a high-throughput culture method based on Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) and diversified physicochemical culture conditions mimicking the natural microenvironment (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Our team applied fungal and bacterial culturomics in the context of liver diseases, demonstrating the importance of ethanol-producing yeast and bacteria in MASH and HBV-associated liver disease (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, to our knowledge, no study has yet characterized microbiota associated with ALD using microbial culturomics. We, therefore, decided to carry out this microbial culturomics study to complete the spectrum of dysbiosis of the gut microbiota, whose instrumental role had been demonstrated in ALD with experimental evidence (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Accordingly, this study aims to characterize the microbial signature in patients with ALD and ALD-associated HCC using both culturomics and large-scale sequencing (v3v4 region 16S rRNA amplicon sequencing).\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eFull methods are reported in the supplementary data. Briefly, 59 participants were investigated, including 35 patients with ALD (19 with HCC) and 24 controls (CTL). Liver stiffness measurements in cirrhotic patients were conducted using a FibroScan® instrument (Echosens, Paris, France). Routine biochemistry, including prothrombin index (PT), platelets count (PLT), total bilirubin (TBIL), serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and serum creatinine, were measured. These analyses were performed only among cases. The fecal samples from all participants were collected and analyzed by v3-v4 16S rRNA amplicon sequencing (see Supplementary methods). We selected 11 samples from patients with ALD and 10 samples from controls for microbial culturomics (see Supplementary methods). For culturomics, 21 samples were selected as this approach is of unequaled value since it is the only one that ascertains viable strains, but it is associated with a huge workload (6 weeks by sample). Statistical analyses were performed using GraphPad Prism Software for Windows (GraphPad Software, San Diego, CA, USA) (version 9.0). P values less than 0.05 were considered significant differences.\u003c/p\u003e"},{"header":"MAIN RESULTS","content":"\u003ch2\u003eThe bacterial genera Thomasclavelia, Enterocloster, Clostridium, and Peptoniphilus are associated with ALD by culturomics\u003c/h2\u003e\u003cp\u003eThe characteristics of the study participants and the general results of culturomics are detailed in the supplementary results. Overall, twenty-one samples were analyzed using microbial culturomics (5 ALD-HCC, 6 ALD-NoHCC, and 10 CTL), allowing the isolation and identification of 32,088 colonies (Table S1). Diversity at the species level was found to be increased in the ALD group for only four bacterial genera, all belonging to one phylum (\u003cem\u003eBacillota\u003c/em\u003e), including \u003cem\u003eThomasclavelia\u003c/em\u003e (p = 0.0016), \u003cem\u003eEnterocloster\u003c/em\u003e (p = 0.0058), \u003cem\u003eClostridium\u003c/em\u003e (p = 0.0021) and \u003cem\u003ePeptoniphilus\u003c/em\u003e (p = 0.0044, Fig. 1a).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eThomasclavelia ramosa is the most enriched species in ALD by culturomics.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAt the cultured species level, surprisingly, \u003cem\u003eT. ramosa\u003c/em\u003e was detected in all ALD patients (11/11 (100%), including those with ALD-HCC (n = 5) and ALD-NoHCC (n = 6), but in only 20% of the controls (2/10, two-tailed Fisher exact test p = 0.00044, Fig. 1b). Moreover, among all the cultured species, \u003cem\u003eT. ramosa\u003c/em\u003e also had the most significant difference in frequency between cases and controls (100% vs. 20%: +80%). This was consistent with the increased diversity of the \u003cem\u003eThomasclavelia\u003c/em\u003e genus, as reported above (Fig. 1a).\u003c/p\u003e\u003ch3\u003eEnrichment of Enterocloster species in ALD by culturomics\u003c/h3\u003e\u003cp\u003eWe recently reported an enrichment in \u003cem\u003eEnterocloster bolteae\u003c/em\u003e in liver diseases associated with metabolic-associated steatohepatitis (26) and hepatitis B virus (28). Here, we found again an enrichment in \u003cem\u003eEnterocloster bolteae\u003c/em\u003e in liver disease associated with alcoholism (Fig. 1b). This was consistent with the increased diversity of this genus in ALD (Fig. 1a), particularly the significant increase of 3 species of this genus, including also \u003cem\u003eE. clostridioformis\u003c/em\u003e and \u003cem\u003eE. aldenensis\u003c/em\u003e (Fig. 1b \u0026amp; Fig. 1c). Strikingly, this association was found only by culturomics but not by 16S rRNA sequencing, as observed in our previous studies (26, 28).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eThe species Thomasclavelia ramosa and Mediterraneibacter gnavus are enriched in ALD both by culturomics and 16S rRNA amplicon sequencing\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eLinear discriminant analysis (LDA) effect size modeling was applied on the v3v4 sequencing data of the 59 samples to identify specific bacterial taxa associated with ALD. Compared to the control group, the gut microbiota of all ALD patients showed a significantly increased abundance of \u003cem\u003eStreptococcus_salivarius/Atribacter_sp223\u003c/em\u003e (OTU39314), \u003cem\u003eEscherichia_albertii / Escherichia_coli\u003c/em\u003e (OTU2689), \u003cem\u003eMediterraneibacter_gnavus\u003c/em\u003e (formerly, \u003cem\u003eRuminococcus_gnavus\u003c/em\u003e (29)), and \u003cem\u003eThomasclavelia_ramosa\u003c/em\u003e (formerly \u003cem\u003eClostridium ramosum\u003c/em\u003e (30)). Accordingly, the only two species enriched in ALD both by culturomics and 16S rRNA sequencing were \u003cem\u003eT. ramosa\u003c/em\u003e and \u003cem\u003eMediterraneibacter gnavus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eThomasclavelia ramosa was the only species identified by culturomics and sequencing and associated with hepatocellular carcinoma\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIncluding all three groups (ALD-NoHCC, ALD-HCC, CTL) in a Linear Discriminant Analysis, the pathobiont \u003cem\u003eMediterraneibacter_gnavus\u003c/em\u003e (31, 32) was significantly increased in ALD-NoHCC, while \u003cem\u003eT_ramosa\u003c/em\u003e and \u003cem\u003eGemmiger_formicilis\u003c/em\u003e were the only 2 operational taxonomic units (OTUs) significantly associated with the ALD-HCC group (Fig. 2). Accordingly, \u003cem\u003eT. ramosa\u003c/em\u003e was the only species identified by culturomics and sequencing and associated with ALD-HCC, identifying it as one of the best oncobiont candidates for further exploration of an instrumental role of a gut microbe for liver cancer associated with alcoholism. Genomic analysis did not identify a clonal specificity (Supplementary Table 2 \u0026amp; Supplementary Fig. 2).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis brief preliminary report, which uses a combined approach of microbial culturomics and 16S rRNA sequencing, identifies \u003cem\u003eT. ramosa\u003c/em\u003e as one of the best candidates for the intestinal pathogenic oncobiont associated with ALD and HCC associated with ALD. \u003cem\u003eT. ramosa\u003c/em\u003e is a human gut pathogen associated with several cases of severe infection (bacteremia, infection of aortic aneurysm, osteomyelitis, arthritis, gas gangrene, Fournier's gangrene, fatal infections) recently associated with human cancer, notably colorectal cancer (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), but also HCC in a 2023 Chinese study (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the first discovered genotoxin produced by a gut commensal was colibactin from \u003cem\u003eEscherichia coli\u003c/em\u003e strains which alkylates DNA, and associated with colon cancer (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). A another recent study reported that two other gut commensals, \u003cem\u003eMorganella morganii\u003c/em\u003e and \u003cem\u003eT. ramosa\u003c/em\u003e, exhibited genotoxicity and promoted tumor growth in an experimental model (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). While indolimines have been characterized as the genotoxic molecules for \u003cem\u003eM. morganii\u003c/em\u003e, the small molecules (\u0026lt;\u0026thinsp;3 kDa) that lead to the genotoxic and pro-cancer role are not known for \u003cem\u003eT. ramose\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Notably, \u003cem\u003eT. ramosa\u003c/em\u003e lacks known biosynthetic gene clusters (BGCs) and induces DNA damage via colibactin-independent and indolimines-independent mechanisms (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLimitations of our study included a small sample size and lack of experimental evidence. However, the clear \u003cem\u003eT. ramosa\u003c/em\u003e signature (LDA score\u0026thinsp;\u0026gt;\u0026thinsp;5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) identified here with a limited sample size means that the difference (effect size) is huge, supporting the strength of the association as the first Bradford-Hill criteria for causality. Consistency and reproducibility are fulfilled by two previous studies confirming the association with CRC (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and the confirmation of the \u003cem\u003eT. ramosa\u003c/em\u003e-liver cancer association by another team (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The linear discriminant analysis fulfills the biological gradient (the higher \u003cem\u003ethe T. ramosa\u003c/em\u003e 16S rRNA number of reads - the higher the risk of HCC). The plausibility and experimental evidence are supported by the recently reported \u003cem\u003ein vitro\u003c/em\u003e genotoxicity and in vivo tumor growth-promoting properties of \u003cem\u003eT. ramosa\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe robustness of our results is not due to the number of samples or deep sequencing, but to microbial culturomics, the only approach to certify the presence of viable and biologically active microbes with high fidelity taxonomic characterization at the species level thanks to MALDI-TOF MS. Studies with higher statistical power could identify other microbial candidates, and \u003cem\u003eT. ramosa\u003c/em\u003e is very likely part of a group of liver cancer oncobionts. Future studies could use deep sequencing and automated microbial culturomics (45) to focus on a larger sample to obtain a better match or at least larger groups, allowing for the stratification of all comorbidities and the stage of liver disease. We found that \u003cem\u003eT. ramosa\u003c/em\u003e was the most different in culturomics between ALD and controls after analysis of 21 samples and 32088 colonies, and the most significantly associated with liver cancer by DNA sequencing after analysis of 59 samples and 2,840,773 reads.\u003c/p\u003e \u003cp\u003eEven if future studies with larger sample size and better representation of participant groups according to the comorbidities, cause, and stage of liver disease are necessary to confirm the association and the potential role of \u003cem\u003eT. ramosa\u003c/em\u003e in hepatocellular carcinoma, we believe that our results and recent literature (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) evidenced that \u003cem\u003eT. ramosa\u003c/em\u003e could be a potential candidate among the gut commensals favored by alcoholism, and which can then contribute to liver carcinogenesis through its recently demonstrated genotoxic and tumor growth-promoting properties.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALD, Alcoholic liver disease; HCC, Hepatocellular carcinoma; MASH, Metabolic dysfunction-associated steatohepatitis; NGS, next-generation sequencing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HEPATGUT study was approved by the local ethics committee of the Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France (IHUMI, 2020-004), approved by the Protection of Persons Committee (Approval No. CPP: 21.04391.000046—21075), and carried out according to the 2013 Declaration of Helsinki (World Medical Association, 2013). Patient consent (non-opposition) was obtained according to French regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study within the article and/or supplementary materials are available upon request. The raw sequencing data of fecal samples are available in the NCBI Sequence Read Archive with accession number PRJEB62828. The genome of six strains of \u003cem\u003eThomasclavelia ramosa\u003c/em\u003e (strains CSUR Q9705, Q9779, Q9849, QA0117, QA0118, and QA0666, available on request) have been sequenced, and genome sequencing data are publicly available under the Bioproject NCBI PRJEB76822.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Agence Nationale de la Recherche under two programs: ANR-15-CE36-0004-01 and ANR \"Investissements d'avenir\", Méditerranée Infection 10-IAHU-03. The Région Provence-Alpes-Côte d'Azur also supported this study, which received financial support from the Fondation Méditerranée Infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, funding acquisition, and Project administration: MM, RG. Methodology and visualization: RMW, MM. Resources: AG, PB. Investigation: RMW, AA, BM, AC, CA, GM. Data curation and formal analysis: RMW, AG, AL. Supervision: MTA, MM, RG. Validation: RWM, MM, RG. Writing - original draft: RMW – review \u0026amp; editing: AG, MTA, MM, RG. Reviewed the results and approved the final version of the manuscript: all Authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Vincent Bossi for their excellent technical assistance. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest is to be declared.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLlovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al. Hepatocellular carcinoma. Nat Rev Dis Primers. 2021;7(1):6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Description of \u003cem\u003eMediterraneibacter massiliensis\u003c/em\u003e, gen. nov., sp. nov., a new genus isolated from the gut microbiota of an obese patient and reclassification of \u003cem\u003eRuminococcus faecis\u003c/em\u003e, \u003cem\u003eRuminococcus lactaris\u003c/em\u003e, \u003cem\u003eRuminococcus torques\u003c/em\u003e, \u003cem\u003eRuminococcus gnavus\u003c/em\u003e and \u003cem\u003eClostridium glycyrrhizinilyticum\u003c/em\u003e as \u003cem\u003eMediterraneibacter faecis\u003c/em\u003e comb. nov., \u003cem\u003eMediterraneibacter lactaris\u003c/em\u003e comb. nov., \u003cem\u003eMediterraneibacter torques\u003c/em\u003e comb. nov., \u003cem\u003eMediterraneibacter gnavus\u003c/em\u003e comb. nov. and \u003cem\u003eMediterraneibacter glycyrrhizinilyticus\u003c/em\u003e comb. nov. Antonie Van Leeuwenhoek. 2018;111(11):2107-28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawson PA, Saavedra Perez L, Sankaranarayanan K. Reclassification of \u003cem\u003eClostridium cocleatum\u003c/em\u003e, \u003cem\u003eClostridium ramosum\u003c/em\u003e, \u003cem\u003eClostridium spiroforme\u003c/em\u003e and \u003cem\u003eClostridium saccharogumia\u003c/em\u003e as \u003cem\u003eThomasclavelia cocleata\u003c/em\u003e gen. nov., comb. nov., \u003cem\u003eThomasclavelia ramosa\u003c/em\u003e comb. nov., gen. nov., \u003cem\u003eThomasclavelia spiroformis\u003c/em\u003e comb. nov. and \u003cem\u003eThomasclavelia saccharogumia\u003c/em\u003e comb. nov. Int J Syst Evol Microbiol. 2023;73(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrost EH, Coletto E, Bell A, Juge N. \u003cem\u003eRuminococcus gnavus\u003c/em\u003e: friend or foe for human health. FEMS Microbiol Rev. 2023;47(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorowska MT, Drees C, Yarawsky AE, Viswanathan M, Ryan SM, Bunker JJ, et al. 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Oncol Translational Med. 2023;9(1):28\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson MR, Jiang Y, Villalta PW, Stornetta A, Boudreau PD, Carr\u0026aacute; A et al. The human gut bacterial genotoxin colibactin alkylates DNA. Science. 2019;363(6428).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Y, Oh J, Xue M, Huh WJ, Wang J, Gonzalez-Hernandez JA, et al. Commensal microbiota from patients with inflammatory bowel disease produce genotoxic metabolites. Science. 2022;378(6618):eabm3233.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"gut-pathogens","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gutp","sideBox":"Learn more about [Gut Pathogens](http://gutpathogens.biomedcentral.com/)","snPcode":"13099","submissionUrl":"https://submission.nature.com/new-submission/13099/3","title":"Gut Pathogens","twitterHandle":"@GutPathogens","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Thomasclavelia ramosa, Enterocloster bolteae, Mediterraneibacter gnavus, Alcoholic-related liver disease, Hepatocellular carcinoma, Gut microbiome, Microbial culturomics, 16S rRNA sequencing, Cancer","lastPublishedDoi":"10.21203/rs.3.rs-5053117/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5053117/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Gut microbiota alteration is implicated in the pathogenesis of alcoholic liver disease (ALD) and associated hepatocellular carcinoma (HCC). No study has characterized the dysbiosis associated with ALD by microbial culturomics, which certifies viability and allows pathobiont strain candidates to be characterized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A case-control study (n = 59) was conducted on patients with ALD without HCC (ALD-NoHCC, n=16), ALD with HCC (ALD-HCC, n=19) and controls (n=24) groups. 16S rRNA amplicon sequencing and microbial culturomics were used as complementary methods for gut microbiome profiling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCompared to the control group, \u003cem\u003eThomasclavelia ramosa\u003c/em\u003e and \u003cem\u003eGemmiger formicilis\u003c/em\u003e were significantly increased in the ALD-HCC group and \u003cem\u003eMediterraneibacter gnavus\u003c/em\u003e was significantly increased in the ALD-NoHCC group using 16S rRNA sequencing. By microbial culturomics, \u003cem\u003eT. ramosa\u003c/em\u003e was detected in all ALD samples (100%), and the most enriched since cultivated in only a small proportion of controls (20%, p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e \u003cem\u003eT. ramosa\u003c/em\u003e, identified by culturomics and 16 rRNA sequencing, may be associated with ALD and ALD-HCC. These results highlight the potential role of \u003cem\u003eT. ramosa\u003c/em\u003e in liver cancer, in line with its genotoxic properties and its tumor growth-promoting effect in gnotobiotic mice recently reported.\u003c/p\u003e","manuscriptTitle":"Thomasclavelia ramosa and Alcohol-Related Hepatocellular Carcinoma: A Microbial Culturomics Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 06:37:51","doi":"10.21203/rs.3.rs-5053117/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-02T23:54:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-02T15:28:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T07:45:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222038697951228579324213658127595723679","date":"2025-04-01T07:37:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199781370218082317931450673290953105839","date":"2025-04-01T04:16:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T00:01:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-29T14:06:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-29T06:25:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Gut Pathogens","date":"2025-03-28T15:12:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"gut-pathogens","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gutp","sideBox":"Learn more about [Gut Pathogens](http://gutpathogens.biomedcentral.com/)","snPcode":"13099","submissionUrl":"https://submission.nature.com/new-submission/13099/3","title":"Gut Pathogens","twitterHandle":"@GutPathogens","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1f948ac-3807-41c5-b4df-6039d0d53e30","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:01:00+00:00","versionOfRecord":{"articleIdentity":"rs-5053117","link":"https://doi.org/10.1186/s13099-025-00703-6","journal":{"identity":"gut-pathogens","isVorOnly":false,"title":"Gut Pathogens"},"publishedOn":"2025-05-07 15:57:22","publishedOnDateReadable":"May 7th, 2025"},"versionCreatedAt":"2025-04-03 06:37:51","video":"","vorDoi":"10.1186/s13099-025-00703-6","vorDoiUrl":"https://doi.org/10.1186/s13099-025-00703-6","workflowStages":[]},"version":"v1","identity":"rs-5053117","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5053117","identity":"rs-5053117","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}