Studies of the duodenal microbiota have been focused on SIBO, which relies on traditional culture-dependent method or breath testing. Cirrhosis-associated duodenal dysbiosis is not yet clearly defined. In the attempt to shed light on this issue, we used 16S rRNA metagenomics to determine whether the duodenum microbiota differed between cirrhotic patients and healthy controls. Our results suggest that the structure of duodenal mucosa microbiota in cirrhotic patients is dramatically different from normal controls.

As can be observed in this study, at the genus level, Veillonella, Prevotella, Neisseria, and Haemophilus, were the most discriminative taxa between cirrhosis and controls. All these taxa are commonly presented in the oral cavity11, which suggests oral microbiota has a great impact upon duodenal microbiota. The oral cavity is the entry point of bacteria into the body. The human oral microbiota not only play a role in disease of the oral cavity, but also interact with microbiomes from other parts of the human body12. Our earlier study has found that microbes of oral origin could be present in stool of cirrhosis patients13. Recently, a direct evaluation of the salivary microbiome in controls and patients with cirrhosis was performed by Bajaj et al.14. In salivary microbiome of cirrhotic patients with previous hepatic encephalopathy, relative abundance of autochthonous taxa (Lachnospiraceae and Ruminococcaceae) decreased whereas potentially pathogenic taxa (Prevotella and Fusobacteriaceae) increased. Partly in consistent with their results, our results also found the relative abundance of duodenal Prevotella and Fusobacterium in cirrhosis was significantly higher than in controls. It has been reported in a previous study that distinct bacterial populations in the oral microbiota are involved in production of high levels of H 2 S and CH 3 SH in the oral cavity. The H 2 S group showed higher proportions of the genera Neisseria, Porphyromonas and SR1, whereas the CH 3 SH group had higher proportions of the genera Prevotella, Veillonella, Atopobium, Megasphaera, and Selenomonas15. It is interesting that the duodenal bacterial groups enriched in cirrhosis and healthy controls are highly consistent with the oral microbiota in CH 3 SH group and H 2 S group, respectively. Blood levels of CH 3 SH have been suggested as important factors in the pathogenesis of hepatic encephalopathy16. The shift of microbiota toward CH 3 SH generated community might indicate a direct contribution of duodenal microbiota to hepatic encephalopathy in cirrhosis. Our results are in line with recently published data showing that gut dysbiosis links with systemic and neuro-inflammation. When compared with control mice, the small intestinal microbiota in cirrhotic mice showed relative increase in Enterobacteriaceae and Staphylococcaceae along with predominantly oral families such as Streptococcaceae17. Taking together, these results might indicate associations between small intestinal microbiota of oral origins and hepatic encephalopathy.

To investigate if, in addition to compositional impact, there is an impact on bacterial function, PICRUSt was applied to the 16S rRNA amplicon sequencing data to infer bacterial metabolic functions. Interestingly, we found higher abundance of bacterial motility proteins and bacterial secretion system in healthy duodenal microbiota. The motility proteins play an important role in bacterial attachment on epithelial cells and travel to or away from stimulus18. Bacterial secretion system, which can be classified into type I-IV, operates generally on the principal of active transportation of protein from cytoplasm to bacterial surface, which play crucial roles in gut colonization through invasion on mucosal surface19. Both bacterial motility and secretion system are heavily involved in host adhesion and colonization. The enrichment of genes related to bacterial motility and secretion system might indicate a harsher and more competitive environment in healthy duodenum than in cirrhotic duodenum. In normal conditions, intestinal peristalsis, gastric acid, and bile secretion act to control control bacterial colonization, attachment, and infiltration into the host20. Abnormalities in one or more of these host defenses result in bacterial overgrowth of the small intestine2. In cirrhosis, marked decreases in intestinal intraluminal concentrations of bile acids have been ascribed to decreased secretion and increased deconjugation21. Abnormalities in small intestinal motility are related to the degree of chronic liver failure22. Decompensated cirrhotics were found to have slower intestinal transit times as compared with compensated cirrhotics and healthy controls23. Small intestine motility dysfunction is more severe in those with history of spontaneous bacterial peritonitis24. In our research, cirrhotic patients were observed with significantly higher inter-individual variations than healthy controls, which also supports the conclusion that cirrhosis abolish colonization control to certain exogenous bacteria and weaken the normal control of endogenous bacterial community.

The enriched pathways in cirrhosis were related to transporters, amino sugar and nucleotide sugar metabolism, which likely reflect the basic requirements of microbial life in the duodenum of cirrhosis. The bacterial transport systems enable bacteria to accumulate needed nutrients, extrude unwanted by products and maintain cytoplasmic content of protons and salts conducive to growth and development25. Some ABC transporter can be involved in resistance to antimicrobial peptides26. The drug transporters genes are overrepresented in the infant/elderly gut microbiome, perhaps due to the frequent antibiotic treatment of infants and the elderly compared with adults27. Therefore, we speculate that the enrichment of transporter gene might be a selective result of occasionally antibiotics use in cirrhotic patients, who are more susceptible to infections.

Although, at the genus level, Neisseria were found overrepresented in healthy controls. Two OTUs representing Gemella and Neisseria, respectively, were the most discriminative OTUs between two types of cirrhosis. The results here are in line with our recently published data, which showed alterations and correlations of the gut microbiome and immnunity in PBC patients28. The fecal microbiota of PBC patients were depleted of some potentially beneficial bacteria, such as Lachnobacterium and Acidobacteria, but were enriched in some bacterial taxa, such as Neisseriaceae and Klebsiella. Several altered gut bacterial taxa exhibited interactions with altered immunity and urine metabolism, such as Klebsiella with IL-2A and Neisseriaceae with urinary indoleacrylate. A close association between celiac disease and PBC has been extensively reported in literature29. In duodenum of adult celiac patients, members of Neisseria genus were significantly more abundant in active celiac disease patients than in controls30. Gemella has been found to be involved in pulmonary exacerbations of cystic fibrosis patients. The relative abundance of Gemella in airway microbiota increased in 83% of the patients with cystic fibrosis at exacerbation and was found to be the most discriminative genus between baseline and exacerbation samples31. It is hypothesized that specific species of Neisseria and Gemella may be involved in orchestrating inflammatory disease by promoting inflammation and remodeling normally benign microbiota into dysbiotic community. Recently, Sabino et al. found that primary sclerosing cholangitis is associated with alterations in intestinal microbiota independently of comorbidity with inflammatory bowel disease, which might indicate a potential effect of cholestatic and bile flow on gut microbiota32. Further studies are needed to confirm and assess these links and causality.

Our results showed that the duodenal microbiota is primary determined by cirrhosis itself. Only slight association can be observed between duodenal microbial alterations and endoscopy varices treatment or PPIs. An increase in the relative abundance of genus Dialister was observed in cirrhotic patients on PPIs. Certain Dialister species (D. pnermosintes, D. invisus) have been identified as pathogens, mainly in orthodontic infections33. It is hypothesized that PPIs could enhance gastrointestinal proliferation of these potential oral pathogen species by reduction in gastric acid. This hypothesis is supported by recent findings showing long-term PPIs use is associated with an increase of Holdemania filiformis34, which is also potential oral pathogens usually isolated from advanced periodontitis35. In the present study, genus SR1 genera incertae sedis and Staphylococcus were found increased and decreased in patients with endoscopic treatment, respectively. Endoscopic sclerotherapy and band ligation of esophageal varices have been showed to cause gastric hemodynamic changes, and thus increase the incidence and the severity of portal hypertensive gastropathy36. The duodenal microbiota alterations observed here might be related to mucosa changes in advanced portal hypertension. However, the present study was not designed to compare microbiota alterations before and after endoscopy treatment or PPIs use. Further large scale studies should be performed to confirm these links.

In conclusion, the study reported herein demonstrates that marked dysbiosis is associated with the duodenal mucosa of cirrhotic patients. The deviation of duodenum microbiota might be related to alterations of oral microbiota and duodenal microenvironment. Also, when considering the etiology of cirrhosis, PBC and HBV have slight difference of gut microbiota, indicating the effect mediated by immunity or bile acids between host and microbiota. We note that our study is only able to describe correlations between cirrhosis and duodenal microbiota, and causality cannot be inferred. Further studies, perhaps using experimental animals, are expected to shed light on the causal factors underlying this relationship.