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.

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1 ) lyophilization of the stool sample; 2 ) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3 ) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterifi ed bile acids; 4 ) extraction of bile acids from particulate material using 0.1 N NaOH; 5 ) isolation of deconjugated bile acids by solid phase extraction; 6 ) formation of phenacyl esters by derivatization using phenacyl bromide; and 7 ) HPLC separation measuring eluted peaks at 254 nm.

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