Background & Aims The gut microbiome is altered in cirrhosis; however its evolution with disease progression is partly understood. We aimed to study changes in microbiome over cirrhosis severity, its stability over time and its longitudinal alterations with decompensation. Methods Controls and age-matched cirrhotics (compensated/decompensated/hospitalized) were included. Their stool microbiota was quantified using multi-tagged pyrosequencing. Ratio of autochthonous to non-autochthonous taxa was calculated as the cirrhosis dysbiosis ratio(CDR); a low number indicating dysbiosis. Firstly, microbiome was compared between controls and cirrhotic sub-groups. Second, for stability assessment, stool collected twice within 6 months in compensated outpatients was analyzed. Thirdly, changes after decompensation were assessed using (a) longitudinal comparison in patients before/after hepatic encephalopathy development (HE), (b) longitudinal cohort of hospitalized infected cirrhotics MELD-matched to uninfected cirrhotics followed for 30 days. Results 244 subjects [219 cirrhotics (121 compensated outpatients,54 decompensated outpatients,44 inpatients) and 25 age-matched controls)] were included. CDR was highest in controls(2.05) than compensated(0.89), decompensated(0.66) and inpatients(0.32,p<0.0001) and negatively correlated with endotoxin. Microbiota and CDR remained unchanged in stable outpatient cirrhotics (0.91 vs. 0.86, p=0.45). In patients studied before/after HE development, dysbiosis occurred post-HE(CDR:1.2 to 0.42, p=0.03). In the longitudinal matched-cohort, microbiota were significantly different between infected/uninfected cirrhotics at baseline and a low CDR was associated with death and organ failures within 30 days. Conclusions Progressive changes in the gut microbiome accompany cirrhosis and become more severe in the setting of decompensation. The cirrhosis dysbiosis ratio may be a useful quantitative index to describe microbiome alterations accompanying cirrhosis progression.

Background and Aims The 7α-dehydroxylation of primary bile acids (BAs), chenodeoxycholic (CDCA) and cholic acid (CA) into the secondary BAs, lithocholic (LCA) and deoxycholic acid (DCA) is a key function of the gut microbiota. We aimed to study the linkage between fecal BAs and gut microbiota in cirrhosis since this could help understand cirrhosis progression. Methods Fecal microbiota were analyzed by culture-independent multitagged-pyrosequencing, fecal BAs using HPLC and serum BAs using LC-MS in controls, early (Child A), and advanced cirrhotics(Child B/C). A subgroup of early cirrhotics underwent BA and microbiota analysis before/after eight weeks of rifaximin. Results Cross-sectional: 47 cirrhotics(24 advanced) and 14 controls were included. In feces, advanced cirrhotics had the lowest total, secondary, secondary/primary BA ratios, and highest primary BAs compared to early cirrhotics and controls. Secondary fecal BAs were detectable in all controls but in a significantly lower proportion of cirrhotics (p<0.002). Serum primary BAs were higher in advanced cirrhotics compared to the rest. Cirrhotics, compared to controls, had a higher Enterobacteriaceae (potentially pathogenic) but lower Lachonospiraceae, Ruminococcaceae and Blautia (7α-dehydroxylating bacteria) abundance. CDCA was positively correlated with Enterobacteriaceae(r=0.57, p<0.008) while Ruminococcaceae were positively correlated with DCA(r=0.4, p<0.05). A positive correlation between Ruminococcaceae and DCA/CA (r=0.82, p<0.012) and Blautia with LCA/CDCA (r=0.61, p<0.03) was also seen. Prospective study: Post-rifaximin, six early cirrhotics had reduction in Veillonellaceae and in the secondary/primary BA ratios. Conclusions Cirrhosis, especially advanced disease, is associated with a decreased conversion of primary to secondary fecal BAs which is linked with abundance of key gut microbiome taxa.

Hepatic encephalopathy (HE) has been related to gut bacteria and inflammation in the setting of intestinal barrier dysfunction. We aimed to link the gut microbiome with cognition and inflammation in HE using a systems biology approach. Multitag pyrosequencing (MTPS) was performed on stool of cirrhotics and age-matched controls. Cirrhotics with/without HE underwent cognitive testing, inflammatory cytokines, and endotoxin analysis. Patients with HE were compared with those without HE using a correlation-network analysis. A select group of patients with HE (n = 7) on lactulose underwent stool MTPS before and after lactulose withdrawal over 14 days. Twenty-five patients [17 HE (all on lactulose, 6 also on rifaximin) and 8 without HE, age 56 ± 6 yr, model for end-stage liver disease score 16 ± 6] and ten controls were included. Fecal microbiota in cirrhotics were significantly different (higher Enterobacteriaceae, Alcaligeneceae, and Fusobacteriaceae and lower Ruminococcaceae and Lachnospiraceae) compared with controls. We found altered flora (higher Veillonellaceae), poor cognition, endotoxemia, and inflammation (IL-6, TNF-α, IL-2, and IL-13) in HE compared with cirrhotics without HE. In the cirrhosis group, Alcaligeneceae and Porphyromonadaceae were positively correlated with cognitive impairment. Fusobacteriaceae, Veillonellaceae, and Enterobacteriaceae were positively and Ruminococcaceae negatively related to inflammation. Network-analysis comparison showed robust correlations (all P < 1E-5) only in the HE group between the microbiome, cognition, and IL-23, IL-2, and IL-13. Lactulose withdrawal did not change the microbiome significantly beyond Fecalibacterium reduction. We concluded that cirrhosis, especially when complicated with HE, is associated with significant alterations in the stool microbiome compared with healthy individuals. Specific bacterial families (Alcaligeneceae, Porphyromonadaceae, Enterobacteriaceae) are strongly associated with cognition and inflammation in HE.