Supplementary Material for: Long-Term Effect of Rifaximin with and without Lactulose on the Active Bacterial Assemblages in the Proximal Small Bowel and Faeces in Patients with Minimal Hepatic Encephalopathy

<b><i>Background:</i></b> Gut microbiota play an essential role in the pathogenesis of hepatic encephalopathy (HE). Treatment strategies are directed to modulate intestinal microbiota profiles and their function by the administration of the non-absorbable disaccharide lactulose and the non-absorbable antibiotic rifaximin, which are required for long terms, but little is known on their long-term effect on gut microbiota composition and function. <b><i>Aim:</i></b> To characterize the active bacterial assemblages in duodenum and faeces in patients with minimal HE (MHE) before, during and after long-term therapy with rifaximin. <b><i>Methods:</i></b> We analysed the microbiota composition in 5 patients with liver cirrhosis and MHE treated either with rifaximin 550 mg bid alone continuously for a period of 3 months or combined with lactulose 30–60 mL daily for 3 months. In addition to clinical assessments of HE, biopsies from duodenum and stool samples were analysed for their specific bacterial community applying NGS after RNA isolation before treatment, after 3 months of treatment and 3 months after the end of treatment. <b><i>Results:</i></b> All 5 patients had a significant improvement of their MHE. Bacterial communities were different and distinct in duodenal samples and faeces. No statistically significant changes were found in the bacterial community profile at the different time points. <b><i>Conclusion:</i></b> Rifaximin therapy with and without lactulose over a period of 3 months does not affect the bacterial community composition. The improvement of HE with rifaximin is lasting also after the end of treatment and therefore a prolonged effect on microbiota metabolic function is suggested.