An integrated analysis of fecal microbiome and metabolomic features distinguish non-cirrhotic NASH from healthy control populations

BACKGROUND AND AIMS: There is great interest in identifying microbiome features as reliable noninvasive diagnostic and/or prognostic biomarkers for non-cirrhotic NASH fibrosis. Several cross-sectional studies have reported gut microbiome features associated with advanced NASH fibrosis and cirrhosis, where the most prominent features are associated with cirrhosis. However, no large, prospectively collected data exist establishing microbiome features that discern non-cirrhotic NASH fibrosis, integrate the fecal metabolome as disease biomarkers, and are unconfounded by BMI and age. APPROACH AND RESULTS: Results from shotgun metagenomic sequencing performed on fecal samples prospectively collected from 279 US patients with biopsy-proven NASH (F1-F3 fibrosis) enrolled in the REGENERATE I303 study were compared to those from 3 healthy control cohorts and integrated with the absolute quantification of fecal bile acids. Microbiota beta-diversity was different, and BMI- and age-adjusted logistic regression identified 12 NASH-associated species. Random forest prediction models resulted in an AUC of 0.75-0.81 in a receiver operator characteristic analysis. In addition, specific fecal bile acids were significantly lower in NASH and correlated with plasma C4 levels. Microbial gene abundance analysis revealed 127 genes increased in controls, many involving protein synthesis, whereas 362 genes were increased in NASH many involving bacterial environmental responses (false discovery rate < 0.01). Finally, we provide evidence that fecal bile acid levels may be a better discriminator of non-cirrhotic NASH versus health than either plasma bile acids or gut microbiome features. CONCLUSIONS: These results may have value as a set of baseline characteristics of non-cirrhotic NASH against which therapeutic interventions to prevent cirrhosis can be compared and microbiome-based diagnostic biomarkers identified.