Colucci G, Stella S, Castagnetti A, Di Rito L, Lucà M, Tosetti G, D’Ambrosio R, Lampertico P. Gut microbiome in patients with cirrhosis and clinically significant portal hypertension: A pilot study. World J Hepatol 2026; 18(10): 124666 [DOI: 10.4254/wjh.124666]
Corresponding Author of This Article
Giuseppe Colucci, MD, Associate Research Scientist, Division of Gastroenterology and Hepatology, IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, via F. Sforza 28, Milan 20122, Italy. giuseppe.colucci@guest.unimi.it
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Colucci G, Stella S, Castagnetti A, Di Rito L, Lucà M, Tosetti G, D’Ambrosio R, Lampertico P. Gut microbiome in patients with cirrhosis and clinically significant portal hypertension: A pilot study. World J Hepatol 2026; 18(10): 124666 [DOI: 10.4254/wjh.124666]
Giuseppe Colucci, Sonia Stella, Martina Lucà, Giulia Tosetti, Roberta D’Ambrosio, Division of Gastroenterology and Hepatology, IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Milan 20122, Italy
Andrea Castagnetti, Laura Di Rito, Wellmicro Laboratories, Bologna 40100, Italy
Pietro Lampertico, CRC “A. M. and A. Migliavacca”, Center for Liver Disease, Department of Pathophysiology and Transplantation, University of Milan, Milan 20122, Italy
Co-first authors: Giuseppe Colucci and Sonia Stella.
Author contributions: Colucci G designed and coordinated the research study, analyzed the data and wrote the manuscript; Stella S collected and managed fecal samples; Colucci G and Stella S contributed equally to this article, they are the co-first authors of this manuscript; Castagnetti A and Di Rito L performed the research and provided experimental results; Lucà M and Tosetti G selected patients and collected clinical data; D’Ambrosio R and Lampertico P reviewed the data and the manuscript; and all authors thoroughly reviewed and endorsed the final manuscript.
AI contribution statement: No AI has ever been used in this study design, implementation and analysis.
Institutional review board statement: This study was approved by the Medical Ethics Committee of Comitato Etico Milano Area 2, approval No. 1037_2022.
Clinical trial registration statement: Not applicable.
Informed consent statement: The subjects have signed the informed consent form.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
CONSORT 2010 statement: The authors have read the CONSORT 2010 Statement, and the manuscript was prepared and revised according to the CONSORT 2010 Statement.
Data sharing statement: All data can be shared as for the Journal SOP.
Corresponding author: Giuseppe Colucci, MD, Associate Research Scientist, Division of Gastroenterology and Hepatology, IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, via F. Sforza 28, Milan 20122, Italy. giuseppe.colucci@guest.unimi.it
Received: June 26, 2026 Revised: August 1, 2026 Accepted: September 20, 2026 Published online: October 27, 2026 Processing time: 117 Days and 17.9 Hours
Abstract
BACKGROUND
An altered intestinal microbiota may affect the gut-liver axis and influence the outcome of patients with cirrhosis and clinically significant portal hypertension (CSPH).
AIM
To analyze microbiota and mycobiota in patients with cirrhosis and CSPH to identify pathological profiles amenable to treatment.
METHODS
Twenty-five patients with cirrhosis and CSPH were recruited and compared to 25 healthy controls (HC). The 16S rRNA hypervariable regions V3 and V4 (bacterial component) and the ITS2 region (fungal component) isolated from fecal DNA, sequenced on the MiSeq - Illumina®, were analyzed using the QIIME2 (v.2021.11) and DADA2 (v.1.16) software.
RESULTS
Patients’ microbiota included 5 phyla, Actinobacteria (2.4%), Bacteroidetes (35%), Firmicutes (57%), Protobacteria (2.5%), and Elusimicrobia (3%) with significant differences compared to HC in the proportion of Elusimicrobia (3% vs 1%, P < 0.001), Protebacteria (2.5% vs 1.4%, P < 0.003) and Verrucomicrobia (0.1% vs 0.34%, P < 0.004). Pathobionts were detected in the microbiota of 6 (26%) patients, mainly Escherichia Coli and Klebsiella Pneumoniae, and in the mycobiota of 8 (32%) patients, mostly Malassezia Restricta and Candida Albicans. Six patients showed mixed infections with both micro and mycobiota pathobionts. Overall, 20 patients (86%) were affected. The Dysbiosis and Diversity Indexes were also significantly different in patients vs HC (median 2.2 vs 1.3, P < 0.01, and 74 vs 130, P < 0.01).
CONCLUSION
Differences in the microbiome composition, dysbiosis and biodiversity indexes between patients and HC, the presence of bacterial and fungal pathobionts suggest an active role of the microbiome in cirrhosis with CSPH.
Core Tip: The gut-liver axis is affected in cirrhosis with clinically significant portal hypertension and may contribute to disease progression and liver decompensation. In a group of patients recruited in a pilot study, we detected significant differences in the intestinal microbiota and mycobiota compared to healthy controls, including the presence of specific pathobionts. Dietary modifications and the supplementation of specific pre and probiotics may help preventing disease progression in patients with cirrhosis and clinically significant portal hypertension.
Citation: Colucci G, Stella S, Castagnetti A, Di Rito L, Lucà M, Tosetti G, D’Ambrosio R, Lampertico P. Gut microbiome in patients with cirrhosis and clinically significant portal hypertension: A pilot study. World J Hepatol 2026; 18(10): 124666
Recent studies have confirmed the important role of the microbiome in maintaining body homeostasis[1]. The intestinal microbiota supports the integrity of the intestinal epithelium by ensuring the persistence of the mucous barrier and by competing with aggressive pathobionts. In addition, they act as primers of the immune system and providers of short chain fatty acids, key elements in several metabolic pathways[2,3].
Cirrhosis is the endpoint of chronic hepatitis of any etiology, characterized by the formation of fibrotic septa surrounding nodules of regenerative liver parenchyma[4,5]. These changes may lead to the obstruction of portal blood flow with the development of portal hypertension (PH), up to the formation of collateral vessel that by-pass the liver, being eventually responsible for toxic hepatic encephalopathy (HE). The intestinal epithelium is also affected by a reduction in the tight junction integrity and by the alterations in the composition of the intestinal microbiota with the proliferation of pathobionts and bacterial translocation[6-9]. As a consequence, the impaired gut-liver axis may influence the status of cirrhosis with clinically significant PH (CSPH), a condition at risk of decompensation and HE[10]. The involvement of the microbiota in the pathogenesis of liver diseases has been investigated in the past, mainly confirming its role in causing the hyperammonemia that characterize decompensated cirrhosis and trigger HE[11,12]. Several bacterial strains, including Enterobacteriaceae, Proteobacteria and Streptococcaceae are known to produce urease that increase the production of ammonia. In addition, dysbiotic microbiota may lack the bacteria that produce glutamate dehydrogenase and utilize ammonium in their metabolic pathways. Normally, these bacteria incorporate ammonium to generate glutamate, acting as a crucial detoxification pathway[13].
Several studies have investigated the role of the microbiota in the pathogenesis of liver diseases, including decompensated cirrhosis. A recent study, focused on hepatitis B surface antigen positive Asiatic patients with cirrhosis and PH vs non-cirrhotic patient with chronic hepatitis, showed a dysbiosis microbiota with related metabolomics alterations. Pathobionts were detected that were responsive to antibiotics treatment in a mouse animal model[14].
To analyze both the microbiota and mycobiota composition in patients with cirrhosis and CSPH caused by different etiologies, we investigated, in the present pilot study, the microbiome composition of a representative group of clinically well characterized patients with cirrhosis and CSPH to identify pathological profiles potentially amenable to treatment with specific diets, antibiotics and appropriate probiotic supplementation. A group of matched normal individuals served as controls.
MATERIALS AND METHODS
Study population
In this prospective, single-center, pilot study performed at the Gastroenterology and Hepatology Division, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 25 cirrhotic patients with a diagnosis of CSPH were studied. Cirrhosis was diagnosed based on clinical grounds, hematological, imaging techniques, transient elastography and liver histology. CSPH was defined according to the Baveno VI-VII criteria, including the presence of splenomegaly (> 12 cm) or low platelets count (< 150) or esophageal varices, or hepatic venous pressure gradient (> 10 mmHg) or liver elastography (> 15 kPa)[15]. Twenty-five healthy individuals, matched for age, sex and ethnicity, rigorously screened to exclude any health issue and selected according to the human microbiome project, served as controls[16].
Microbiome sequence analysis
Fecal samples were collected at a single time point and shipped at room temperature to the Wellmicro laboratory. The hypervariable regions V3 and V4 of the 16S rRNA gene (bacterial component) and the ITS2 region (fungal component) were analyzed by massively parallel sequencing on the MiSeq - Illumina® platform. Sequences were selected and grouped into amplicon sequence variants using the QIIME2 (v.2021.11) and DADA2 (v.1.16) software, while the taxonomic assignment was performed using a proprietary bacterial and fungal database. The dysbiosis index measures the overall deviation of the relative abundances of the sample with respect to the reference intervals of healthy subjects at the taxonomic level of genus. The biodiversity index is based on number of species, phylogenetic distance (P.D. Whole Tree) and distribution of species (Pielou Index)[17].
Statistical analysis
Statistical analysis was performed using the MedCalcTM program. Continuous variables are reported as medians and range, and categorical variables as counts and proportions. χ2 test, Fisher’s exact and Wilcoxon test were applied as appropriate. A two-sided P value less than 0.05 was considered statistically significant.
The study received the approval of the Milano Area 2 ethical review committee on November 24, 2022.
RESULTS
Twenty-five Caucasian patients with compensated cirrhosis and CSPH were included in the study (Table 1). Most of the patients were males (80%), median age of 65 (40-72) years, 96% with esophageal varices and 84% with splenomegaly (12-18 cm), liver elastography ranging from 18 kPa to 64 kPa, model for end-stage liver diseases (MELD) score between 6 and 16. Among the etiological factors, metabolic and alcohol related liver disease (MetALD) was significantly more prevalent than viral hepatitis, hepatitis C virus and hepatitis B virus combined (P < 0.01). No previous episodes of decompensation were reported. Patients were under treatment with carvedilol (88%), lactulose (44%) and rifaximin (22%), with no differences among groups.
Table 1 Patients’ main clinical features, n (%)/median (range).
The microbiota composition of our patients included mainly Firmicutes (57%) and Bacteroidetes (35%) with no significant differences compared to healthy controls (HC) (Figure 1A). Likewise, the Firmicutes/Bacteroidetes ratio were similar in patients and in HC (1.7, 0.27-4.9 vs 2.3, 0.8-4.3).
Figure 1 Intestinal microbiota phyla in patients with cirrhosis and clinically significant portal hypertension.
A: Distribution of the predominant microbiota, Firmicutes and Bacteroidetes phyla in cirrhotic patients with clinically significant portal hypertension and in healthy controls; B: Significant differences have been observed between patients and controls in the proportion of Proteobacteria (aP < 0.03). Elusimicrobia (bP < 0.01) and Verrucomicrobia (cP < 0.04). HD: Healthy donors; CSPH: Clinically significant portal hypertension.
Four additional phyla were detected both in patients and HC: While actinobacteria were similar (2.4% vs 3.2%, not significant the prevalence of Proteobacteria and Elusimicrobia was higher in patients than in controls (2.5% vs 1.4%, P < 0.003) and (0.3% vs 0.1%, P < 0.001), respectively. In contrast, patients had lower prevalence of Verrucomicrobia (0.1% vs 0.3%, P < 0.004) than in HC Figure 1B). The Dysbiosis index was significantly higher in patients than in HC (median 2.2 vs 1.3, P < 0.01) while the biodiversity index was significant lower (74 vs 130, P < 0.01) (Figures 2 and 3).
Figure 2 Dysbiosis index in cirrhotic patients with clinically significant portal hypertension and healthy controls.aP < 0.001, significant differences have been observed between patients and controls. Data are expressed as median and range. CSPH: Clinically significant portal hypertension; HC: Healthy controls.
Figure 3 Biodiversity index in cirrhotic patients with C clinically significant portal hypertension clinically significant portal hypertension and healthy controls.aP < 0.001, significant differences have been observed between patients and controls. Data are expressed as median and range. HD: Healthy donors; CSPH: Clinically significant portal hypertension.
Pathobionts distribution
Overall, 20 patients (86%) had pathobionts in their intestinal flora: 6 in the microbiota, 8 in the mycobiota and 6 in both populations. In the microbiota of 6 patients (26%), we detected the presence of pathobionts, with the predominance of Escherichia Coli (E. Coli) and Klebsiella Pneumoniae alone or in combination (Figure 4A). In the mycobiota, pathobionts were observed in 8 patients (35%), harboring mainly Malassezia Restricta and Candida Albicans (C. Albicans), alone or in combination, respectively detected in 22%, 4% and 9% of cases (Figure 4B). Six additional patients had a mixed microbiota and mycobiota pathobionts infection, including Malassezia Restricta + Bacteroides fragilis (1), M. Restricta + Shigella flexneri (2), C. Albicans + Shigella flexneri (1), C. Albicans + Bacteroides fragilis (1) and C. Albicans + Klebsiella Pneumoniae (1) (Figure 4C).
Figure 4 Distribution of pathobionts in the microbiota and mycobiota of patients with clinically significant portal hypertension.
A: Distribution of pathobionts in the microbiota of cirrhotic patients with clinically significant portal hypertension (CSPH); B: Distribution of pathobionts in the mycobiota of cirrhotic patients with CSPH; C: Distribution of pathobionts in both the microbiota and mycobiota of cirrhotic patients with CSPH.
As shown in Table 2, in the overall pathogens’ affected population, 17 (85%) patients presented with type 2 diabetes mellitus (T2DM), 12 (70%) with MetALD and overweight [body mass index (BMI)median and range 31, 24-46]. On the other hand, patients with no pathobionts had more viral etiology (hepatitis C virus and hepatitis B virus), no T2DM, less previous alcohol abuse and overweight. Among these factors, the prevalence of T2DM was significantly lower (P < 0.01) in patients with no pathobionts than in the whole population of patients with pathobionts. No differences were observed among patients with bacterial, fungal, mixed pathobionts and patients without, in terms of age, sex, liver enzymes, BMI, MELD, liver elastography (Table 3). Treatment with lactulose or rifaximin also had no effect on the microbiome distribution. Interestingly, 2 patients, “CV and MA”, who had the highest and lowest Firmicutes/Bacteroidetes (F/B) ratio, 5.4% and 0.3%, respectively, showed a significant different phyla proportion (Figure 5).
Figure 5 Different phyla distribution in patients “CV and MA” with the lowest and highest Firmicutes/Bacteroidetes ratio values.
F/B: Firmicutes/Bacteroidetes.
Table 2 Comparison of baseline parameters between patients with and without fecal pathobionts, n (%)/median (range).
In the current pilot study, we evaluated the microbiome composition of a representative group of cirrhotic patients with CSPH, a clinical condition never specifically analyzed so far in details.
Several studies have investigated the fecal and oral microbiome in various liver diseases, reporting alterations in the gut-liver assay with sometimes discordant results among taxonomic profiles, probably due to different ethnicities, dietary and life style habits[18]. A recent study has analyzed the plasma circulating microbiota of patients with cirrhosis with or without PH and detected an increase of Enterobacteriacea, Escherichia/Shigella, and Prevotella with no difference between peripheral or hepatic vein compartments. In this study no fecal samples were available[19]. In our patients, we found relevant differences compared to HC in the proportion of Elusimicrobia and Proteobacteria phyla that were significantly increased while Verrucomicrobia were significantly reduced. Elusimicrobia, a complex phylum with species known to be active in nitrogen fixation and ammonia production, may contribute to hyperammonemia and the onset of HE[20].
Alterations in the composition of the microbiota are known to be associated with an impairment of intestinal integrity and physiology, favoring the proliferation of pathobionts, including Enterobacteriaceae and Proteobacteria[21,22]. The Increased levels of Proteobacteria, detected in our patient’s population, is known to be linked to dysbiosis and chronic inflammation[23]. Facultative Protobacteria anaerobes, such as Escherichia, Klebsiella, Salmonella, and Helicobacter genera, are associated with several diseases, including inflammatory bowel disease, metabolic syndrome and T2DM[24,25]. Indeed, we detected in our patients the presence of E. Coli and K. Pneumoniae as well as S. flexneri that can disrupt the normal balance of commensal bacteria, reducing beneficial taxa such as Firmicutes and Bacteroidetes, which are important for gut health.
As previously reported, E. Coli and Klebsiella may worsen liver function and cause severe infections, including abscesses and spontaneous bacterial peritonitis[26]. S. flexneri in case of translocation- induced bacteremia may cause systemic diseases severely affecting the liver[27].
Moreover, impaired gut barrier function (leaky gut), somehow also induced by PH, favors translocation of enteric bacteria or their products into the blood of cirrhotic patients often through porto-systemic shunts that bypass the liver and its detoxification function[28,29]. As a consequence, Lipopolysaccharides and bacterial translocation activates immune responses and systemic inflammation, leading to the release of cytokines which contribute to liver damage and exacerbate PH[30,31]. The lack of Verrucomicrobia in our patients’ microbiota, includes Akkermasia, known for its beneficial functions and whose concentration is inversely correlated to pathological conditions[32]. This is consistent with the reduced variety of microbiota species as indicated by the high index of dysbiosis, a parameter that depends on the relative abundance of phyla. The biodiversity index is also altered by a low number of observed bacterial species, indicating a reduced variability and variety of the microbiota necessary to maintain the intestinal homeostasis and function.
The analysis of the mycobiota composition showed the predominance of M. Restricta and C. Albicans, previously shown to exacerbate liver injury by inducing the inflammatory cytokines cascade[33-35]. The microbiome of the majority of our patients, showed the presence of micro and mycobiota pathobionts, mainly E. Coli, K. Pneuiomia, M. Restricta and C. Albicans, associated to a high prevalence of T2DM and MetALD, the former significantly higher than in patients with no pathobionts. This suggest that the persistence of these metabolic and alcohol associated pathogenic factors maintains and progressively worsens the alterations of the microbiome, compared to past, resolved viral hepatitis infection.
These data support the possible role of dysfunctional microbiota in the pathogenesis of cirrhosis with CSPH and highlight the need to explore novel therapeutic strategies[36]. These may include prebiotics supplementation to foster the growth of beneficial bacteria and probiotics, including L. Kefir, B. Breve and L. Rhamnosus[36]. Several studies have shown the beneficial effects of probiotics in patients with cirrhosis, including a randomized placebo-controlled trial based on the VSL#3 probiotic preparation[37]. Recently another probiotics consortium, VE303, proved able to engraft the intestine of patients with HE improving their overall psychometric performance scores[38]. Dietary modifications, such as increasing fiber and reducing refined carbohydrates may also help as a high fat diet low in fiber may create a gut environment that promotes Proteobacteria growth. Interestingly, in our population, patients with no pathobionts had neither overweight nor T2DM. Moreover, antibiotics treatment that affects the gut microbiota, can cause resilient opportunistic bacteria, including some Proteobacteria, to proliferate[39]. Fecal microbiota transplantation may be indicated in severe dysbiosis cases[40]. Addressing underlying conditions like inflammatory bowel disease or metabolic disorders such as obesity, insulin resistance, and type 2 diabetes, which may be linked to gut dysbiosis, might be advisable[40].
Our study has also some limitations. The small sample size of this pilot study may be appropriate for a pilot, proof of principle study but has to be increased to confirm the observed data and support more treatment options. We also acknowledge that we did not include a control group of patients with cirrhosis without CSPH.
CONCLUSION
In conclusion, the observed difference in the microbiome composition including the dysbiosis and biodiversity indexes, between cirrhotic patients with CSPH and HC, as well as the presence of bacterial and fungal pathobionts confirm a dysfunctional microbiota and gut-liver axis. Changes in dietary habits and the supplementation of specific prebiotics and probiotics may help modulate disease progression in patients with CSPH.
ACKNOWLEDGEMENTS
We thank Dr. P. Serri for initial patients’ recruitment and Dr. A. Colucci-Zelger for graphical editing.
Méheust R, Castelle CJ, Matheus Carnevali PB, Farag IF, He C, Chen LX, Amano Y, Hug LA, Banfield JF. Groundwater Elusimicrobia are metabolically diverse compared to gut microbiome Elusimicrobia and some have a novel nitrogenase paralog.ISME J. 2020;14:2907-2922.
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