Potential Therapeutic Options for Ethanol-Producing Ethanol-Resistant Gut Microbes associated with Liver Diseases

Potential Therapeutic Options for Ethanol-Producing Ethanol-Resistant Gut Microbes associated with Liver Diseases | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Potential Therapeutic Options for Ethanol-Producing Ethanol-Resistant Gut Microbes associated with Liver Diseases View ORCID Profile Anissa Idrissa Abdoulaye , Babacar Mbaye , View ORCID Profile Reham Magdy Wasfy , View ORCID Profile Louis Carmarans , Mamadou Beye , Claudia Andrieu , View ORCID Profile Sofiane Bakour , View ORCID Profile Aïcha Hamieh , View ORCID Profile Nicholas Armstrong , View ORCID Profile Patrick Borentain , View ORCID Profile Stéphane Ranque , View ORCID Profile Jean-Marc Rolain , Gregory Dubourg , View ORCID Profile Jean-Christophe Lagier , View ORCID Profile Maryam Tidjani Alou , René Gerolami , View ORCID Profile Matthieu Million doi: https://doi.org/10.1101/2025.11.13.25340061 Anissa Idrissa Abdoulaye 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anissa Idrissa Abdoulaye Babacar Mbaye 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Reham Magdy Wasfy 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Reham Magdy Wasfy Louis Carmarans 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France 5 Unité Hépatologie, Hôpital de la Timone , APHM, 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Louis Carmarans Mamadou Beye 3 Aix Marseille Univ, MEPHI, Marseille, France; APHM (Assistance Publique Hôpitaux de Marseille) , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Claudia Andrieu 3 Aix Marseille Univ, MEPHI, Marseille, France; APHM (Assistance Publique Hôpitaux de Marseille) , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sofiane Bakour 3 Aix Marseille Univ, MEPHI, Marseille, France; APHM (Assistance Publique Hôpitaux de Marseille) , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sofiane Bakour Aïcha Hamieh 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aïcha Hamieh Nicholas Armstrong 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicholas Armstrong Patrick Borentain 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France 5 Unité Hépatologie, Hôpital de la Timone , APHM, 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Borentain Stéphane Ranque 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stéphane Ranque Jean-Marc Rolain 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jean-Marc Rolain Gregory Dubourg 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jean-Christophe Lagier 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jean-Christophe Lagier Maryam Tidjani Alou 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maryam Tidjani Alou René Gerolami 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France 5 Unité Hépatologie, Hôpital de la Timone , APHM, 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthieu Million 1 Aix Marseille Univ, MEPHI , Marseille, France 2 IHU-Méditerranée Infection , Marseille, France 4 Assistance Publique-Hôpitaux de Marseille (APHM) , 13005 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthieu Million For correspondence: matthieumillion{at}gmail.com Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Background Endogenous ethanol (EtOH) production is an emerging pathophysiological mechanism involved in metabolic dysfunction-associated steatohepatitis (MASH). Therefore, characterizing EtOH-producing species enriched in patients with liver disease could help identify putative pathobionts and potential therapeutic treatments. Methods We investigated EtOH production and tolerance, antimicrobial susceptibility and antimicrobial resistance gene(s) in 33 strains enriched in MASH, alcoholic hepatitis (AH) and HBV patients. An antimicrobial was considered a potential therapeutic option when the ratio of the fecal concentration/minimum inhibitory concentration (FC/MIC) was > 10 (1 log 10 ). Results 92% of species enriched in patients with liver diseases produced detectable amounts of ethanol with a strong association between EtOH tolerance and production (p < 0.05). Candida albicans , Nakaseomyces glabratus and Pichia kudriavzevii produced the highest concentrations of EtOH (1.8 to 3.3 g/L). Enterocloster , a strictly anaerobic bacterial genus, was the bacterial genus with the highest EtOH production (0.8 to 1.6 g/L) in 5 g/L of glucose. Poorly absorbed drugs, amphotericin B, rifaximin and vancomycin, together constituted potential therapeutic options for all tested strains. Conclusions In addition to yeasts, Lactobacillaceae and Klebsiella , strains of Enterocloster genus isolated from patients with liver diseases produced significant amount of EtOH. Most gut microbial species associated with liver diseases produce and tolerate ethanol, suggesting a possible vicious microbial-ethanol cycle in such context. Finally, non-absorbed antimicrobials (rifaximin, vancomycin and amphotericin B) already used for gut microbiota-targeted therapies may open new avenues in the design of precision medicine. Introduction Liver diseases are a major public health problem. A study estimated that in 2023, over 2 million deaths or 4% of the global mortality rate, are caused by cirrhosis, viral hepatitis and liver cancer annually ( 1 ). The main causes of cirrhosis are chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic hepatitis (AH), and metabolic dysfunction-associated steatohepatitis (MASH) ( 2 ). The emergence of metabolic hepatic diseases, including metabolic dysfunction-associated fatty liver disease (MAFLD) and its severe form, MASH, is growing worldwide. Indeed, in 2019, the estimated prevalence of MAFLD was 1.6 billion worldwide ( 3 ). It increased from 25% between 1990 and 2006 to 38% between 2016 and 2019 ( 4 ). The global prevalence of MASH is estimated to be 5%, with the highest prevalence in Latin America (44%) ( 4 ). Alterations in the gut microbiota have been observed in viral hepatitis ( 5 , 6 ), AH ( 7 ), MAFLD and MASH ( 8 ). Moreover, one feature of MASH is endogenous ethanol production ( 9 ), which was initially demonstrated by Yuan et al. with Klebsiella pneumoniae strains isolated from patients with auto-brewing syndrome (ABS) and MASH ( 10 ). Similarly, Meijnikman et al. reported a positive correlation between the abundance of Lactobacillaceae and endogenous ethanol production in MAFLD patients ( 11 ). Interestingly, the causal role of these microbes in endogenous ethanol production was confirmed through in vitro and in vivo experimental studies ( 10 , 12 ). Moreover, Casanas et al. recently treated a patient with metabolic dysfunction-associated steatosis liver disease (MASLD) and ABS using fecal microbiota transplantation (FMT) ( 13 ). While prior research has established aerobic species-mediated endogenous ethanol production in MASH and ABS ( 10 , 12 ), our culturomics approach applied to the gut microbiota from MASH and viral hepatitis patients also highlighted enriched anaerobic bacterial species. These studies revealed enrichment of yeasts in the intestinal microbiota of MASH patients, specifically Candida albicans , Nakaseomyces glabratus and Pichia kudriavzevii ( 14 ). Similarly, enriched bacterial species included Limosilactobacillus fermentum , Mediterraneibacter gnavus , Streptococcus mutans and Enterocloster bolteae in the intestinal microbiota of MASH and HBV patients ( 15 , 16 ), as well as enrichment of Thomasclavelia ramosa in the intestinal microbiota of patients with alcohol-related hepatocellular carcinoma ( 17 ). As the etiology of MASH and ABS is related to endogenous ethanol production by gut microbial taxa, microbiota-targeted therapies might be relevant for these pathologies ( 9 , 18 ). The identification and characterization of potential microbial biomarkers represent crucial steps for that purpose ( 19 ). In this study, we investigated ethanol production, ethanol tolerance, antibiotic susceptibility and antibiotic resistance gene expression in thirty-three ethanol-producing strains that were enriched and isolated from patients afflicted with MASH, AH and HBV. Materials and methods Ethical approval All the strains used in this study were isolated in the HEPATGUT project, which was approved by the ethics and personal protection committees (CPP: 21.04391.000046 - 21075). In accordance with the Declaration of Helsinki (World Medical Association, 2013), written informed consent was obtained from each participant. Studied strains Thirty-three strains, including bacteria and yeasts, obtained from culturomics case–control studies on MASH, HVB and AH patients conducted in our laboratory and significantly enriched in these pathologies were included in this study ( 14 – 16 ). Strains of species enriched in patients and those enriched in controls were studied regardless of the sample of origin ( Table 1 ). Hence, we included the following species enriched in the intestinal microbiota of MASH patients: Bacteroides thetaiotaomicron , C. albicans, E. bolteae , Enterocloster clostridioformis , K. pneumoniae , Klebsiella michiganensis , L. fermentum , M. gnavus , N. glabratus , Peptoniphilus grossensis, P. kudriavzevii and T. ramosa ( 14 , 15 , 20 ). Strains of T. ramosa enriched in the gut microbiota of patients with AH and chronic HBV infection were also studied ( 17 ). In addition, species significantly depleted in the intestinal microbiota of MASH patients compared to healthy individuals were included, specifically Alistipes shahii and Bacteroides uniformis ( 15 ). In fact, A. shahii is known to have beneficial effects on various pathologies, including liver fibrosis ( 21 ). Moreover, B. uniformis is a known gut commensal that has been shown to alleviate MASH ( 22 ). View this table: View inline View popup Download powerpoint Table 1. Strains of interest In vitro ethanol production assay For each species, ethanol production was quantified in triplicate in the initial isolation medium. Hence, bacteria were grown in Columbia broth base enriched with 5% defibrinated sheep blood (COS) at 37°C for 24 hours for aerobic bacteria and 48 hours for anaerobic bacteria. Yeasts were grown in Sabouraud broth (Oxoid, Basingstoke, UK) at 30°C for 24 hours. One milliliter of each culture and of a negative control (sterile culture medium) were subsequently transferred to 20 mL glass Headspace vials (Supplementary materials and methods). Considering the difference in glucose concentration between the COS (5 g/L) used for bacteria and Sabouraud broth (20 g/L) used for yeast, the highest concentration of ethanol-producing bacterial strains was also tested using modified COS broth (COS with 20 g/L glucose). The ethanol concentration was determined using a headspace gas chromatography–mass spectrometry (HS–GC–MS) system (Perkin Elmer, Villebon sur Yvette, France) in the Swafer D7 setup, which included an HS110 headspace injector, a Clarus 690 gas chromatograph and an SQ8T mass spectrometer, as described by Sissoko et al. ( 23 ). Ethanol tolerance test The ethanol tolerance of bacteria was assessed in COS broth containing 0%, 5% and 10% ethanol, whereas that of yeast was assessed in Sabouraud broth (Oxoid) supplemented with 0%, 5% and 10% ethanol (Supplementary materials and methods). Statistics To compare quantitative variables, Mann–Whitney or Student’s t tests were applied on the basis of their distribution. All tests were two-tailed. A p value < 0.05 was used to determine significance. All statistical analyses were performed using GraphPad Prism version 10.4.1 (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com ). Antifungal and antibiotic susceptibility tests Antifungal susceptibility testing to amphotericin B, caspofungin, flucytosine, micafungin and voriconazole was conducted using the VITEK 2 automated system (bioMérieux, Marcy l’Etoile, France) and VITEK 2 AST-YS08 antifungal cards (bioMérieux, Durham, USA). Susceptibility to fluconazole was assessed using E-test strips (bioMerieux, Marcy l’Etoile, France) (Supplementary materials and methods). The susceptibility to 25 antibiotics was assessed with the disc diffusion method using E-test strips (bioMerieux) (Supplementary materials and methods). Considering the widespread use of rifaximin in the treatment of patients with liver disease, we also assessed their susceptibility to rifaximin. The minimum inhibitory concentration (MIC) of rifaximin for each strain was assessed using the agar dilution method, according to the recommendations of the Clinical Laboratory Standards Committee (CLSI), as there were no E-test strips commercially available for this molecule. Potential efficacy of rifaximin, vancomycin and amphotericin B in vivo To determine the potential efficacy of antimicrobials on the species mentioned above, we selected the least absorbable molecules (amphotericin B, rifaximin and vancomycin) and compared their fecal concentrations (FC) reported in the literature ( 24 – 26 ) to the MICs obtained in this study (Supplementary materials and methods). This ratio (FC/MIC) aim to determine the potential efficacy of these drugs in liver diseases associated to endogenous ethanol production, as our target is the gut microbiota. We established an FC/MIC threshold equal to 10, which indicates an efficacy of antimicrobials on species targeted in gut microbiota. Antimicrobial(s) resistance gene(s) After bacterial genome sequencing and assembly (Supplementary materials and methods), the presence of antimicrobial resistance gene(s) was assessed by the command abritAMR with AMRFinder Plus via Galaxy Australia ( https://usegalaxy.org.au/ ). Additionally, for strains presenting a high resistance to rifampin and rifaximin (> 256 µg/mL), the presence of mutation(s) in the rifampin resistance-determining region (RRDR) in the β subunit of bacterial RNA polymerase (rpoB) which is the main reported cause of resistance to rifampicin ( 27 ) was assessed after genomes annotation with PROKKA via Galaxy Australia ( https://usegalaxy.org.au/ , last accessed on October 16 th , 2025) and rpoB alignment in MEGA 12 (version 12.0.14). Results High ethanol production by microbial species associated with liver diseases The production of ethanol by strains of species enriched in the intestinal microbiota of the controls ( A. shahii and B. uniformis ) was low, always under 0.03 g/L (Table S1), compared with that of species enriched in the intestinal microbiota of liver disease patients (n= number of strains, median= median of ethanol dosed in g/L [interquartile range], n= 33, 0.58 [0.21–1.86] vs. n=3, 0.026 [0.025–0.028], 22.3-fold, two-tailed Mann–Whitney test, p <0.0001, Figure S1). Indeed, ethanol production was 22-fold greater in species enriched in the intestinal microbiota of patients with liver diseases than in those enriched in the intestinal microbiota of the controls. A total of 92% (11/12) of the species associated with liver diseases produced detectable levels of ethanol, whereas none of the strains enriched in the controls (0/3) produced detectable levels of ethanol. B. thetaiotaomicron was the only species enriched in the microbiota of patients with liver diseases that did not produce any detectable amount of ethanol. The analysis of the amount of ethanol produced by each strain revealed that compared to the bacterial strains, the yeast strains produced 11 times more ethanol (n=9, 2.70 [2.11–2.98] vs. n=27, 0.24 [0.03–0.61], 11.25-fold, two-tailed Mann–Whitney test; p <0.0001) (Figure S2). The two strains of N. glabratus produced the greatest amount of ethanol (3.29 g/L and 3.06 g/L), followed by the P. kudriavzevii strains (2.89 g/L–2.48 g/L) and C. albicans strains (2.34 g/L–1.84 g/L) ( Figure 1.A ). Download figure Open in new tab Figure 1. Ethanol production by yeast and bacteria dosed by GC–MS and their ethanol tolerance. (A) NG: N. glabratus , CA: C. albicans , PK: P. kudriavzevii , KM: K. michiganensis , KP: K. pneumoniae , BU: B. uniformis , AS: A. shahii , BT: B. thetaiotaimicron , MG: M. gnavus , LF: L. fermentum , Pgro: P. grossensis , EB: E. bolteae , EC: E. clostridioformis , TR: T. ramosa , EtOH: Ethanol quantity dosed, *: Strains isolated from controls. (B) Ethanol production by species (EtOH: Ethanol produced dosed). Level of significance: *: p = 0.0286 (two-tailed Mann–Whitney test). (C) Comparison of strains that grew in 10% ethanol medium with those that did not grow in terms of ethanol production (EtOH: Ethanol quantity dosed). Level of significance: ****: p<0.0001 (two-tailed Mann–Whitney test). The two E. bolteae strains produced ethanol concentrations greater than 1 g/L (1.26 g/L and 1.05 g/L), as did the two E. clostridioformis strains (1.59 g/L and 0.85 g/L). L. fermentum produced 1.02 g/L ethanol. All the strains of Klebsiella spp. produced ethanol at concentrations above 0.5 g/L (0.54–0.62 g/L). T. ramosa strains produced ethanol concentrations ranging from 0.18 g/L to 0.29 g/L. M. gnavus (MG14) and P. grossensis (Pgro) produced 0.10 g/L and 0.03 g/L ethanol, respectively. B. thetaiotaomicron strains produced the lowest concentration (0.01 g/L) ( Figure 1.A ). Notably, strains from the same species produced similar amounts of ethanol, suggesting an overall species-dependent effect ( Figure 1A ). Enterocloster spp. produced twice as much ethanol as Klebsiella spp. did ( Figure 1.B ) (n=4, 1.16 [0.90–1.51]; n=4, 0.59 [0.55–0.62]; 1.97-fold, two-tailed Mann–Whitney test; p=0.0286). The ethanol concentration measured in sterile media was consistently under 0.006 g/L (Table S2). As Sabouraud broth, which is used for yeast culture, contains 20 g/L glucose, we also assessed the ethanol production of the highest bacterial ethanol producers of each species in modified COS broth with 20 g/L of glucose, namely, B. thetaiotaomicron (BTN3), E. clostridioformis (EC38), K. pneumoniae (KPS6), L. fermentum (LF46) and T. ramosa (TRS5, Figure S3.A). The increased glucose concentration resulted in an increased ethanol production in some species (( L. fermentum (LF46) and K. pneumoniae (KPS6)), whereas it resulted in a decreased ethanol production in others (( E. clostridioformis (EC38) and T. ramosa (TRS5)) (Figure S3.A). A comparison of ethanol production between bacterial and yeast strains in the presence of the same amount of glucose in the medium (20 g/L) (Figure S3.B) revealed that compared with bacteria, yeast still produced significantly more ethanol (n=3, 2.8 [2.40–3.25] vs. n=5, 0.58 [0.12–0.96], 4.8-fold, two-tailed Mann–Whitney test; p <0.0001). At 20 g/L, the same gradient of ethanol production per species was maintained, except for E. clostridioformis (EC38), which produced less ethanol than K. pneumoniae (KPS6) did (Figure S3.B). High ethanol production is associated with high ethanol tolerance All yeast strains grew in 10% ethanol, as did two bacterial strains, L. fermentum (LF46) and E. clostridioformis (EC39) (Table S3). All bacterial strains grew at a concentration of 5% ethanol, except E. bolteae (EB42) and A. shahii (ASS4) (Table S3). None of the species enriched in the intestinal microbiota of the controls grew in the presence of 10% ethanol (Table S3). The strains that grew in 10% ethanol produced significantly more ethanol (p < 0.0001) than those that did not ( Figure 1.C ). Those that were tolerant to 10% ethanol produced ten times more ethanol than those that were not (n = strains, m= median of ethanol dose in g/L [inter quartile range], n=11, 2.49 [1.85–2.90] vs. n=25, 0.24 [0.03–0.56], 10.4-fold, two-tailed Mann–Whitney test, p <0.0001) ( Figure 1.C ). High potential activity of amphotericin B, rifaximin and vancomycin in vivo The detailed antibiotic and antifungal susceptibility results are reported in Table S4 and Table S5. Antibiotic susceptibility is usually determined according to standards recommended by the EUCAST in Europe, and the MICs are determined according to concentrations of the molecule of interest in the blood, which is not relevant for our specific target, which is the gut microbiota. Therefore, it is important to assess the potential efficacy of molecules of interest in the gut as we target gut commensals. Hence, based on four characteristics for each antibiotic (no absorbability by the intestinal barrier, a low MIC, a fecal concentration available in the literature dosed in humans and availability of an oral galenic form), we selected antimicrobials that could have high activity in vivo (Table S6). In fact, EUCAST recommendations are generally available only for renown clinical pathogens. This limits the recommendations for most anaerobic bacteria (Table S6). The FC/MIC ratio is more convenient for the estimation of the potential efficacy of poorly absorbed drugs. This ratio is particularly relevant in liver diseases associated with endogenous ethanol production, where gut microbiota plays a crucial role. According to our results, amphotericin B was the most suitable for yeast. Furthermore, it has been shown to be highly effective in in vitro MAFLD models ( 28 , 29 ). Finally, its fecal concentration for the treatment of digestive candidiasis (2 g/day) was estimated at 60 μg/g of stool ( 24 ), which gives a ratio of the fecal concentration to the MIC of at least greater than 1 log 10 ( Figure 2 ) (15 to 30-fold (Table S6)). Download figure Open in new tab Figure 2. Potential in vivo efficacy of rifaximin, vancomycin and amphotericin B. Ratio of the fecal concentration after the recommended dose of amphotericin B, rifaximin and vancomycin to the MIC obtained after antifungal and antibiotic susceptibility tests. FC: Fecal concentration, MIC: Minimal inhibitory concentration, T: Threshold. For most bacteria, rifaximin appeared as the better choice. Rifaximin is a water-soluble antibiotic that is poorly absorbed through the gut barrier and has broad-spectrum bactericidal activity ( 30 ). The fecal concentration of rifaximin needed to prevent relapses in encephalopathy (1100 mg/day) is estimated to be 11000 μg/g of stool ( 25 ). Therefore, the fecal concentration/MIC (FC/MIC) ratio ranged from 2 to 5 log 10 (347 to 355,000-fold greater than the rifaximin FC/MIC (Table S6)). This ratio was highly variable depending on each genus and species: 2–3 log 10 for K. pneumoniae ; 4–5 log 10 for B. thetaiotaomicron and L. fermentum; and > 5 log 10 for all Enterocloster strains, M. gnavus and P. grossensis ( Figure 2 ). However, this ratio could not be estimated for T. ramosa, as its MIC was >256 µg/mL. Therefore, vancomycin seemed to be the most suitable antibiotic for T. ramosa . Furthermore, vancomycin could be used with the recommended posology for Clostridium difficile infection (CDI), as T. ramosa was previously known as Clostridium ramosum prior to reclassification. The fecal concentration of vancomycin used to treat CDI (500 mg/day) has been estimated at 1000 μg/g of stool ( 26 ), which resulted in a FC/MIC ratio > 2 log 10 (333–500-fold higher than T. ramosa MICs (Table S6)) for all 9 T. ramosa strains ( Figure 2 ). Notably, all these ratios were at least 15-fold higher than the MICs obtained (Table S6). Vancomycin resistant gene in E. clostridioformis align with phenotypic profile The assessment of resistance genes towards rifaximin and vancomycin is crucial before the implementation of antibiotic therapy. Using AMRFinder Plus, which detects all resistance genes present in the NCBI database with at least 90% identity and contain gene arr alleles, we did not identify any arr genes in the bacterial genomes examined (Tables S7). T. ramosa strains were the only ones with high resistance to rifampicin and rifaximin (> 256 µg/mL, Table S5). Due to the lack of susceptible T. ramosa strain and the important phylogenetical distance between T. ramosa and main species studied in this context ( M. tuberculosis or E. coli or S. aureus ) ( 31 ), the mapping of rpoB regions was performed with T. ramosa genome of reference (ASM1672878v1). This mapping showed high similarities between our genomes and the reference one (only two mutations, Table S8) with no mutations found in the RRDR region (Table S8). However, vancomycin resistance genes were detected in the genomes of E. clostridioformis . Specifically, strains EC38 and EC39 possessed four and ten vancomycin resistance genes, respectively (Table S7). These results are consistent with the in vitro vancomycin susceptibility profile observed for E. clostridioformis EC39 (Table S5). Resistance genes against macrolides, streptogramin, lincosamide and tetracycline were the most important genes within the studied genomes (Table S7). Discussion The increasing incidence of liver diseases and the instrumental role of gut dysbiosis in their pathogenesis highlight the need for improved therapeutic approaches. Therefore, in this study, we characterized species enriched in these pathologies and assessed their ethanol production and tolerance as well as antimicrobial susceptibility. We found that gut microbial species enriched in the intestinal microbiota of patients with liver diseases produce significant quantities of ethanol in vitro . Notably, most of them are capable of both producing ethanol and demonstrating ethanol tolerance, as reported by Yuan et al. ( 10 ). Previous studies have reported ethanol production by yeasts ( 14 ), K. pneumoniae ( 10 ), E. bolteae ( 16 ), L. fermentum and M. gnavus ( 15 ), as well as ethanol tolerance in yeast and L. fermentum at concentrations exceeding 10% ( 32 , 33 ). This finding indicates a potentially vicious cycle in diseases such as ABS and MASH when a high-carbohydrate diet is sustained ( Figure 3 ). To our knowledge, this study is the first to report in vitro ethanol production by E. clostridioformis , K. michiganensis, P. grossensis , and T. ramosa . Additionally, this is the first report of E. clostridioformis growth in the presence of 10% ethanol. Download figure Open in new tab Figure 3. A vicious cycle caused by ethanol-producing ethanol-tolerant species due to a maintained high-glucose diet and the potential utilization of low-carbohydrate diet combined with antimicrobials as gut microbiota-targeted and precision medicine to disrupt the cycle. Although anaerobic bacteria constitute the predominant microbial community within the gut microbiota ( 34 ), previous studies have used culture conditions that are not suitable for the isolation of strictly anaerobic bacteria such as Enterocloster that are enriched in the microbiota of patients with liver diseases. For instance, Yuan et al. ( 10 ) used yeast peptone dextrose (YPD) medium with a 24-hour incubation under both aerobic and anaerobic conditions, which did not allow the isolation of strictly anaerobic bacteria. This underscores the importance of using broader culture methods, such as culturomics, to better identify potential pathobionts in diseases such as MASH. According to our findings, ethanol production varies with the species and glucose concentration in the growth medium. For instance, while K. pneumoniae increased ethanol production as the glucose concentration increased, Enterocloster decreased ethanol production under the same conditions. These results suggest the influence of glucose intake on endogenous ethanol production and suggest that carbohydrate intake may play a key role in the management of these diseases ( 35 ). Enriched species in the intestinal microbiota of MASH patients, including yeast, Enterocloster , Klebsiella and T. ramosa, produce ethanol and hence may contribute to MASH pathogenesis. Xue et al. previously reported that reducing the abundance of ethanol-producing bacteria significantly using antibiotics could be a novel therapeutic approach ( 12 ). In our study, these pathobionts demonstrated high susceptibility to nonabsorbable antibiotics such as amphotericin B, rifaximin, and vancomycin (FC/MIC = 15–355,000), all of which have established efficacy in the treatment of various conditions ( 29 , 36 , 37 ). Nonetheless, it is noteworthy that fecal antimicrobial concentrations may vary because of individual patient characteristics and comorbidities ( 26 ). Most importantly, in in vivo models for the treatment of MASH, amphotericin B, rifaximin and vancomycin have been demonstrated to reduce steatohepatitis and fibrosis ( 29 , 36 , 37 ). In clinical practice, patients diagnosed with both ABS and MASH have been successfully managed with antimicrobials in combination with low-carbohydrate diets and probiotics ( Table 2 ). In particular, fluconazole is the most prescribed antifungal agent in these scenarios in this context ( Table 2 ). View this table: View inline View popup Download powerpoint Table 2. Cases of ABS cured by antimicrobials in literature Antibiotic resistance represents a significant concern that must be addressed during antibiotic therapy. This underscores the importance of analyzing the resistome of isolated species of interest to minimize the emergence of resistance. Here, genomic resistome analysis further supported the potential application of rifaximin and vancomycin for managing MASH associated with bacterial ethanol producers based on the principal bacteria isolated. The rpoB alignment of T. ramosa did not elucidate the nature of the resistance. Additionally, the lack of T. ramosa strain susceptible to rifampicin and or rifaximin evoked a natural resistance of these species. For a better understanding of resistance mechanisms identified here, further investigations should investigate and try to decipher vancomycin resistant genes in E. clostridioformis and the phenotypic resistance of T. ramosa to rifampicin and rifaximin. Furthermore, this study highlights the importance of developing a microbiota-targeted and precision medicine approach for managing the gut microbiota dysbiosis associated with liver diseases ( 13 ). As previously discussed, compared with other species, certain species may produce more ethanol, exhibit greater tolerance to ethanol, or display increased resistance to antibiotics. This variation highlights the necessity for personalized, microbiota-targeted treatments. Ideally, one or two antimicrobials of interest can be used based on the patient’s microbial profile. Given the potential for antibiotic resistance in the identified species and the possibility of significant changes in the composition of the microbiota due to antibiotic therapy ( 37 ), further research involving animal models and eventually human models is essential to validate the beneficial effects of combined antimicrobial agents in this context. A limitation of this study is the relatively small number of strains analyzed for each species. A greater number of strains would strengthen the evidence for ethanol production and tolerance at the species level. Furthermore, an animal model would substantially enhance our understanding of how these species influence the development and progression of liver diseases, whether through individual or synergistic effects. However, besides all these limits, our work enables a full characterization of 33 strains associated with liver diseases as well as their disposition for the scientific community. These findings may offer valuable insights for managing hepatic disorders and could potentially have implications for liver cancer treatment. Conclusions Most gut microbes associated with liver diseases in this study exhibited both tolerance to ethanol and the ability to produce it. The enrichment and characterization of endogenous ethanol-producing microbes, including C. albicans , N. glabratus , P. kudriavzevii , E. clostridioformis , E. bolteae , L. fermentum , K. pneumoniae , K. michiganensis , T. ramosa and M. gnavus, provide evidence that these species may play a significant role in liver diseases. Consequently, gut microbiota may serve as a target for precision medicine strategies tailored to individual patients. Potential antimicrobial therapies could include amphotericin B, rifaximin, and vancomycin. These findings may offer valuable insights for managing hepatic disorders and potentially have implications for cancer treatment. Data Availability All the 33 genomes of the 33 bacterial strains of species enriched in the intestinal microbiota of patients with liver diseases are available in NBCI under BIOPROJECT: PRJNA1254750, except Q9705 and QA0666 which were previously deposited under the BIOPROJECT: PRJEB76822. The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. All the 36 strains (33 associated with liver diseases and 3 associated with controls) reported in this study are publicly available and can be ordered in our microbial collection, the CSUR https://csur.eu/ and by mail contact{at}csur.eu . Data deposition All the 33 genomes of the 33 bacterial strains of species enriched in the intestinal microbiota of patients with liver diseases are available in NBCI under BIOPROJECT: PRJNA1254750, except Q9705 and QA0666 which were previously deposited under the BIOPROJECT: PRJEB76822. Data availability The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Strains availability All the 36 strains (33 associated with liver diseases and 3 associated with controls) reported in this study are publicly available and can be ordered in our microbial collection, the CSUR https://csur.eu/ and by mail contact{at}csur.eu . The number of each strain is reported in Table 1 . Disclosure Statement The authors report there are no competing interests to declare. Funding This work was supported by a grant from the French Government managed by the National Research Agency under the “Investissements d’avenir (Investments for the Future)” program with the reference ANR-10-IAHU-03 (Méditerranée Infection) by the Contrat Plan Etat-Région and the European funding FEDER IHUPERF. Author contributions Writing – original draft: AIA, MB, CA, SB; Writing – review & editing: MM, MTA, JCL, GD, RG; Conceptualization: RG, MM; Investigation: AIA, BM,NA RMW, AH, JMR, SR, GD; Data curation: BM, RMW, AIA, MB, CA, SB; Methodology: MM, RG, MTA; Supervision: MM, RG, MTA; Formal analysis: NA, AIA; Project administration: RG, MM; Validation: RG, MM, MTA; Funding acquisition: RG, MM; Resources: PB, RG, LC; Visualization: AIA, MM, MTA. Acknowledgments We thank Nicolas ORAIN for technical help as well as Hanh NGUYEN THI MY from the hepatology unit. We also thank Dr Fadi BITTAR for his help in antimicrobials resistance genes interpretations. Figure 3 (VN28ZL02DT) were Created in https://BioRender.com . Claude ai (version Haiku 4.7) and Copilot ai (version: bizchat.20251030.51.2) were used only for rephrasing. The final meaning was checked and ensured that the message that we went to vehiculate was intact. Abbreviations ABS Auto-brewery syndrome COS Columbia plus 5% sheep blood EtOH Ethanol FC Fecal concentration HS-GC-MS Headspace gas-gas chromatography coupled with mass spectrometry MIC Minimal inhibitory concentration YPG Yeast peptone glucose References 1. ↵ Devarbhavi H , Asrani SK , Arab JP , Nartey YA , Pose E , Kamath PS . Global burden of liver disease: 2023 update . J Hepatol . 2023 ; 79 ( 2 ): 516 – 37 . doi: 10.1016/j.jhep.2023.03.017 OpenUrl CrossRef PubMed 2. ↵ Ge PS , Runyon BA . Treatment of Patients with Cirrhosis. Campion EW, editor . N Engl J Med . 2016 ; 375 ( 8 ): 767 – 77 . doi: 10.1056/NEJMra1504367 OpenUrl CrossRef PubMed 3. ↵ Younossi Z , Tacke F , Arrese M , Chander Sharma B , Mostafa I , Bugianesi E , et al. Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis . Hepatology . 2019 ; 69 ( 6 ): 2672 – 82 . doi: 10.1002/hep.30251 . OpenUrl CrossRef PubMed 4. ↵ Younossi ZM , Golabi P , Paik JM , Henry A , Van Dongen C , Henry L . The global epidemiology of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH): a systematic review . Hepatology . 2023 ; 77 ( 4 ): 1335 – 47 . doi: 10.1097/HEP.0000000000000004 . OpenUrl CrossRef PubMed 5. ↵ Milosevic I , Russo E , Vujovic A , Barac A , Stevanovic O , Gitto S , et al. Microbiota and viral hepatitis: State of the art of a complex matter . World J Gastroenterol . 2021 ; 27 ( 33 ): 5488 – 501 . doi: 10.3748/wjg.v27.i33.5488 . OpenUrl CrossRef PubMed 6. ↵ Neag MA , Mitre AO , Catinean A , Buzoianu AD . Overview of the microbiota in the gut-liver axis in viral B and C hepatitis . World J Gastroenterol . 2021 ; 27 ( 43 ): 7446 – 61 . doi: 10.3748/wjg.v27.i43.7446 . OpenUrl CrossRef PubMed 7. ↵ Wang SC , Chen YC , Chen SJ , Lee CH , Cheng CM . Alcohol addiction, gut microbiota, and alcoholism treatment: a review . Int J Mol Sci . 2020 ; 21 ( 17 ): 6413 . doi: 10.3390/ijms21176413 . OpenUrl CrossRef 8. ↵ Lang S , Schnabl B Microbiota and fatty liver disease—the known, the unknown, and the future . Cell Host Microbe . 2020 ; 28 ( 2 ): 233 – 44 . doi: 10.1016/j.chom.2020.07.007 . OpenUrl CrossRef PubMed 9. ↵ Zhu L , Baker SS , Gill C , Liu W , Alkhouri R , Baker RD , et al. Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: A connection between endogenous alcohol and NASH . Hepatology . 2013 ; 57 ( 2 ): 601 – 9 . doi: 10.1002/hep.26093 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Yuan J , Chen C , Cui J , Lu J , Yan C , Wei X , et al. Fatty liver disease caused by high-alcohol-producing klebsiella pneumoniae . Cell Metab . 2019 ; 30 ( 4 ): 675 – 688.e7 . doi: 10.1016/j.cmet.2019.08.018 . OpenUrl CrossRef PubMed 11. ↵ Meijnikman AS , Davids M , Herrema H , Aydin O , Tremaroli V , Rios-Morales M , et al. Microbiome-derived ethanol in nonalcoholic fatty liver disease . Nat Med . 2022 ; 28 ( 10 ): 2100 – 6 . doi: 10.1038/s41591-022-02016-6 . OpenUrl CrossRef PubMed 12. ↵ Xue G , Feng J , Zhang R , Du B , Sun Y , Liu S , et al. Three Klebsiella species as potential pathobionts generating endogenous ethanol in a clinical cohort of patients with auto-brewery syndrome: a case control study . eBioMedicine . 2023 ; 91 : 104560 . doi: 10.1016/j.ebiom.2023.104560 . OpenUrl CrossRef PubMed 13. ↵ Casañas-Martínez M , Barbero-Herranz R , Alegre-González D , Mosquera-Lozano JD , Del Campo R , Llorente-Artero M , et al. Fecal microbiota transplantation in a long-standing auto-brewery syndrome with complex symptomatology . J Hepatol . 2025 ; 82 ( 4 ): e186 – 8 . doi: 10.1016/j.jhep.2024.12.005 . OpenUrl CrossRef PubMed 14. ↵ Mbaye B , Borentain P , Magdy Wasfy R , Alou MT , Armstrong N , Mottola G , et al. Endogenous ethanol and triglyceride production by gut pichia kudriavzevii , candida albicans and candida glabrata yeasts in non-alcoholic steatohepatitis . Cells . 2022 ; 11 ( 21 ): 3390 . doi: 10.3390/cells11213390 . OpenUrl CrossRef 15. ↵ Mbaye B , Magdy Wasfy R , Borentain P , Tidjani Alou M , Mottola G , Bossi V , et al. Increased fecal ethanol and enriched ethanol-producing gut bacteria Limosilactobacillus fermentum , Enterocloster bolteae , Mediterraneibacter gnavus and Streptococcus mutans in nonalcoholic steatohepatitis . Front Cell Infect Microbiol . 2023 ; 13 : 1279354 . doi: 10.3389/fcimb.2023.1279354 . OpenUrl CrossRef PubMed 16. ↵ Magdy Wasfy R , Mbaye B , Borentain P , Tidjani Alou M , Murillo Ruiz ML , Caputo A , et al. Ethanol-producing Enterocloster bolteae is enriched in chronic hepatitis B-associated gut dysbiosis: a case–control culturomics study . Microorganisms . 2023 ; 11 ( 10 ): 2437 . doi: 10.3390/microorganisms11102437 . OpenUrl CrossRef PubMed 17. ↵ Magdy Wasfy R , Abdoulaye A , Borentain P , Mbaye B , Tidjani Alou M , Caputo A , et al. Thomasclavelia ramosa and alcohol-related hepatocellular carcinoma: a microbial culturomics study . Gut Pathog . 2025 ; 17 ( 1 ): 27 . doi: 10.1186/s13099-025-00703-6 . OpenUrl CrossRef PubMed 18. ↵ Trebicka J , Macnaughtan J , Schnabl B , Shawcross DL , Bajaj JS . The microbiota in cirrhosis and its role in hepatic decompensation . J Hepatol . 2021 Jul ; 75 : S67 – 81 . doi: 10.1016/j.jhep.2020.11.013 . OpenUrl CrossRef PubMed 19. ↵ Zhao Z , Chen J , Zhao D , Chen B , Wang Q , Li Y , et al. Microbial biomarker discovery in Parkinson’s disease through a network-based approach . NPJ Parkinsons Dis . 2024 ; 10 ( 1 ): 203 . doi: 10.1038/s41531-024-00802-2 . OpenUrl CrossRef PubMed 20. ↵ Mbaye B , Wasfy RM , Alou MT , Borentain P , Andrieu C , Caputo A , et al. Limosilactobacillus fermentum , Lactococcus lactis and Thomasclavelia ramosa are enriched and Methanobrevibacter smithii is depleted in patients with non-alcoholic steatohepatitis . Microb Pathog . 2023 ; 180 : 106160 . doi: 10.1016/j.micpath.2023.106160 . OpenUrl CrossRef PubMed 21. ↵ Parker BJ , Wearsch PA , Veloo ACM , Rodriguez-Palacios A . The genus Alistipes : gut bacteria with emerging implications to inflammation, cancer, and mental health . Front Immunol . 2020 ; 11 : 906 . doi: 10.3389/fimmu.2020.00906 . OpenUrl CrossRef PubMed 22. ↵ Nie Q , Luo X , Wang K , Ding Y , Jia S , Zhao Q , et al. Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway . Cell . 2024 ; 187 ( 11 ): 2717 – 2734.e33 . doi: 10.1016/j.cell.2024.03.034 . OpenUrl CrossRef 23. ↵ Sissoko S , Konate S , Armstrong N , Traore I , Kone AK , Djimde A , et al. Candida tropicalis , Clavispora lusitaniae , Limosilactobacillus fermentum , Liquorilactobacillus mali , and Leuconostoc pseudomesenteroides are associated with ethanol in Malian traditional fermented milk products . Microb Pathog . 2025 ; 200 : 107298 . doi: 10.1016/j.micpath.2025.107298 . OpenUrl CrossRef PubMed 24. ↵ Hofstra W , De Vries-Hospers HG , Van Der Waaij D . Concentrations of amphotericin B in faeces and blood of healthy volunteers after the oral administration of various doses . Infection . 1982 ; 10 ( 4 ): 223 – 7 . doi: 10.1007/BF01666915 . OpenUrl CrossRef PubMed 25. ↵ Jiang ZD , Ke S , Palazzini E , Riopel L , Dupont H . In vitro activity and fecal concentration of rifaximin after oral administration . Antimicrob Agents Chemother . 2000 ; 44 ( 8 ): 2205 – 6 . doi: 10.1128/AAC.44.8.2205-2206.2000 . OpenUrl Abstract / FREE Full Text 26. ↵ Gonzales M , Pepin J , Frost EH , Carrier JC , Sirard S , Fortier LC , et al. Faecal pharmacokinetics of orally administered vancomycin in patients with suspected Clostridium difficile infection . BMC Infect Dis . 2010 ; 30 : 10 : 363 . doi: 10.1186/1471-2334-10-363 . OpenUrl CrossRef 27. ↵ Boll L , Kern WV , Schuster S , Schultheiß M , Schneider C , Vavra M , et al. Frequent high-level rifaximin resistance in Escherichia coli associated with long-term treatment of patients with liver cirrhosis: a prospective, controlled study . Microbiol Spectr . 2025 13 ( 11 ): e0334724 . doi: 10.1128/spectrum.03347-24 . OpenUrl CrossRef 28. ↵ Fotis D , Liu J , Dalamaga M . Could gut mycobiome play a role in NAFLD pathogenesis? Insights and therapeutic perspectives. Metab Open . 2022 ; 14 : 100178 . doi: 10.1016/j.metop.2022.100178 . OpenUrl CrossRef 29. ↵ Demir M , Lang S , Hartmann P , Duan Y , Martin A , Miyamoto Y , et al. The fecal mycobiome in non-alcoholic fatty liver disease . J Hepatol . 2022 ; 76 ( 4 ): 788 – 99 . doi: 10.1016/j.jhep.2021.11.029 . OpenUrl CrossRef PubMed 30. ↵ Ouyang-Latimer J , Jafri S , VanTassel A , Jiang ZD , Gurleen K , Rodriguez S , et al. In vitro antimicrobial susceptibility of bacterial enteropathogens isolated from international travelers to Mexico, Guatemala, and India from 2006 to 2008 . Antimicrob Agents Chemother . 2011 55 ( 2 ): 874 – 8 . doi: 10.1128/AAC.00739-10 . Epub 2010 Nov 29. OpenUrl Abstract / FREE Full Text 31. ↵ Goldstein BP . Resistance to rifampicin: a review . J Antibiot (Tokyo ). 2014 ; 67 ( 9 ): 625 – 30 . doi: 10.1038/ja.2014.107 . OpenUrl CrossRef PubMed 32. ↵ Day AW , Kumamoto CA . Selection of ethanol tolerant strains of Candida albicans by repeated ethanol exposure results in strains with reduced susceptibility to fluconazole . PLoS One . 2024 ; 19 ( 2 ): e0298724 . doi: 10.1371/journal.pone.0298724 . OpenUrl CrossRef PubMed 33. ↵ Pereira GV , Miguel MG , Ramos CL , et al. Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture . Appl Environ Microbiol . 2012 ; 78 ( 15 ): 5395 – 405 . doi: 10.1128/AEM.01144-12 . OpenUrl Abstract / FREE Full Text 34. ↵ Shin W , Wu A , Massidda MW , Foster C , Thomas N , Lee DW , et al. A robust longitudinal co-culture of obligate anaerobic gut microbiome with human intestinal epithelium in an anoxic-oxic interface-on-a-chip . Front Bioeng Biotechnol . 2019 ; 7 : 7 : 13 . doi: 10.3389/fbioe.2019.00013 . OpenUrl CrossRef PubMed 35. ↵ Lê KA , Bortolotti M . Role of dietary carbohydrates and macronutrients in the pathogenesis of nonalcoholic fatty liver disease . Curr Opin Clin Nutr Metab Care . 2008 ; 11 ( 4 ): 477 – 82 . doi: 10.1097/MCO.0b013e328302f3ec . OpenUrl CrossRef PubMed 36. ↵ Qiu T , Zhu X , Wu J , Hong W , Hu W , Fang T . Mechanisms of rifaximin inhibition of hepatic fibrosis in mice with metabolic dysfunction associated steatohepatitis through the TLR4/NFκB pathway . Sci Rep . 2025 ; 15 ( 1 ): 9815 . doi: 10.1038/s41598-025-92282-4 . OpenUrl CrossRef PubMed 37. ↵ Çirkin G , Aydemir S , Açıkgöz B , Çelik A , Güler Y , Kiray M , et al. Hepatic histopathological benefit, microbial cost: oral vancomycin mitigates non-alcoholic fatty liver disease while disrupting the cecal microbiota . Int J Mol Sci . 2025 ; 26 ( 17 ): 8616 . doi: 10.3390/ijms26178616 . OpenUrl CrossRef PubMed 38. Saverimuttu J , Malik F , Arulthasan M , Wickremesinghe P . A Case of Auto-brewery Syndrome Treated with Micafungin . Cureus . 2019 ; 14; 11 ( 10 ): e5904 . doi: 10.7759/cureus.5904 . OpenUrl CrossRef PubMed 39. Malik F , Wickremesinghe P , Saverimuttu J . Case report and literature review of auto-brewery syndrome: probably an underdiagnosed medical condition . BMJ Open Gastroenterol . 2019 ; 6 ( 1 ): e000325 . doi: 10.1136/bmjgast-2019-000325 . OpenUrl Abstract / FREE Full Text 40. Cordell B , McCarthy J . A case study of gut fermentation syndrome (auto-brewery) with Saccharomyces cerevisiae as the causative organism . Int J Clin Med . 2013 ; 04 ( 07 ): 309 – 312 . doi: 10.4236/ijcm.2013.47054 . OpenUrl CrossRef 41. Dahshan A , Donovan K . Auto-brewery syndrome in a child with short gut syndrome: case report and review of the literature . J Pediatr Gastroenterol Nutr . 2001 ; 33 ( 2 ): 214 – 5 . doi: 10.1097/00005176-200108000-00024 . OpenUrl CrossRef PubMed Web of Science 42. Kaji H , Asanuma Y , Yahara O , Shibue H , Hisamura M , Saito N , et al. Intragastrointestinal alcohol fermentation syndrome: report of two cases and review of the literature . J Forensic Sci Soc . 1984 ; 24 ( 5 ): 461 – 71 . doi: 10.1016/s0015-7368(84)72325-5 . OpenUrl CrossRef PubMed 43. Jansson-Nettelbladt E , Meurling S , Petrini B , Sjölin J . Endogenous ethanol fermentation in a child with short bowel syndrome . Acta Paediatr . 2006 ; 95 ( 4 ): 502 – 4 . doi: 10.1080/08035250500501625 . OpenUrl CrossRef PubMed 44. Spinucci G , Guidetti M , Lanzoni E , Pironi L . Endogenous ethanol production in a patient with chronic intestinal pseudo-obstruction and small intestinal bacterial overgrowth : Eur J Gastroenterol Hepatol . 2006 ; 18 ( 7 ): 799 – 802 . doi: 10.1097/01.meg.0000223906.55245.61 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 14, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Potential Therapeutic Options for Ethanol-Producing Ethanol-Resistant Gut Microbes associated with Liver Diseases Anissa Idrissa Abdoulaye , Babacar Mbaye , Reham Magdy Wasfy , Louis Carmarans , Mamadou Beye , Claudia Andrieu , Sofiane Bakour , Aïcha Hamieh , Nicholas Armstrong , Patrick Borentain , Stéphane Ranque , Jean-Marc Rolain , Gregory Dubourg , Jean-Christophe Lagier , Maryam Tidjani Alou , René Gerolami , Matthieu Million medRxiv 2025.11.13.25340061; doi: https://doi.org/10.1101/2025.11.13.25340061 Share This Article: Copy Citation Tools Potential Therapeutic Options for Ethanol-Producing Ethanol-Resistant Gut Microbes associated with Liver Diseases Anissa Idrissa Abdoulaye , Babacar Mbaye , Reham Magdy Wasfy , Louis Carmarans , Mamadou Beye , Claudia Andrieu , Sofiane Bakour , Aïcha Hamieh , Nicholas Armstrong , Patrick Borentain , Stéphane Ranque , Jean-Marc Rolain , Gregory Dubourg , Jean-Christophe Lagier , Maryam Tidjani Alou , René Gerolami , Matthieu Million medRxiv 2025.11.13.25340061; doi: https://doi.org/10.1101/2025.11.13.25340061 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Gastroenterology Subject Areas All Articles Addiction Medicine (568) Allergy and Immunology (863) Anesthesia (300) Cardiovascular Medicine (4435) Dentistry and Oral Medicine (444) Dermatology (382) Emergency Medicine (608) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1509) Epidemiology (15228) Forensic Medicine (30) Gastroenterology (1124) Genetic and Genomic Medicine (6598) Geriatric Medicine (668) Health Economics (997) Health Informatics (4536) Health Policy (1368) Health Systems and Quality Improvement (1613) Hematology (540) HIV/AIDS (1264) Infectious Diseases (except HIV/AIDS) (15916) Intensive Care and Critical Care Medicine (1103) Medical Education (623) Medical Ethics (146) Nephrology (667) Neurology (6599) Nursing (346) Nutrition (998) Obstetrics and Gynecology (1144) Occupational and Environmental Health (957) Oncology (3332) Ophthalmology (974) Orthopedics (369) Otolaryngology (420) Pain Medicine (436) Palliative Medicine (130) Pathology (663) Pediatrics (1693) Pharmacology and Therapeutics (691) Primary Care Research (711) Psychiatry and Clinical Psychology (5447) Public and Global Health (9231) Radiology and Imaging (2198) Rehabilitation Medicine and Physical Therapy (1370) Respiratory Medicine (1196) Rheumatology (593) Sexual and Reproductive Health (712) Sports Medicine (530) Surgery (712) Toxicology (99) Transplantation (289) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a00540a60fc6c13d',t:'MTc3OTU1MTgwNg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();