We found that cirrhotic subjects who required non-elective 90-day hospitalization had a different microbial profile that could add to the current models for this prediction. We also found that although DM in the presence of cirrhosis alters the mucosal and stool microbiota compared to cirrhotics without DM, it does not add to the 90-day hospitalization risk.

Hospital admissions are a growing healthcare burden in cirrhosis that requires urgent attention5,11,13. Clinical models of these admissions center on cirrhosis severity and complications, which may require refinement using further patho-physiological tools11. In our study, we found that gut microbiota alterations can independently add to this predictive capacity beyond cirrhosis severity and medication usage. We found significant differences in the stool and sigmoid mucosal microbiota composition at the family level between those with and without hospitalizations. Replicating prior studies, we found a significantly lower autochthonous bacterial relative abundance and increased potentially pathogenic microbiota in advanced cirrhotics, who in turn were more likely to be hospitalized14,15. An independent contribution of a reduced relative abundance of Bacteroidaceae and Clostridiales XIV towards this outcome was found. These results extend prior studies that have evaluated hospitalized cirrhotic patients for either 30-day mortality and organ failure or those with established acute-on-chronic liver failure with early mortality, into the outpatient cirrhosis realm14,16. These studies showed that different bacterial families, Lachnospiraceae, Clostridiales XIV, Ruminococcaceae and Pasteurellaceae were associated with short-term mortality compared to Bacteroidaceae and Clostridiales cluster XIV in the current study evaluating relatively longer-term events. Similar results were also found with saliva-related microbiota in the prediction of hospitalizations in a smaller set of patients17. However the greater sample size and quantum higher bacterial concentration in the stool compared to saliva would potentially make this a more robust observation.

Different bacterial functions and roles may be relevant in these differences over the short and long-term prognoses. Clostridiales XIV, Lachnospiraceae and Ruminococcaceae relative abundance has been shown to parallel liver disease severity and they have been potentially beneficial impacts on bile acids and short-chain fatty acids, which could reduce colonic pH and support the intestinal barrier18,19. The role of Bacteroidaceae may be more nuanced. Bacteroidaceae are a large family within Bacteroidetes phylum which usually form 20–30% of bacterial abundance in cirrhotic subjects8,14. Cirrhotics who were ultimately hospitalized had a significant reduction in only two families in Bacteroidetes, Bacteroidaceae and Porphyromonadaceae but not others such as Prevotellaceae or Rikenellaceae. This indicates that this is not simply a reduction in the whole phylum but specific families, especially since the Firmicutes/Bacteroidetes ratio was not significantly different between groups. Members of Bacteroidaceae are relatively resistant to antibiotics, produce a weak endotoxin and can protect commensal bacteria against antibiotics20. Indeed an environment low in Bacteroidetes has been shown to promote the growth of C.difficile21. Therefore this reduction in Bacteroidaceae may indicate a gut milieu prone towards development of further insults regardless of HE and MELD score. The relative increase in relative abundance of Bacteroidaceae in NASH and DM cirrhotics compared to alcoholics and other etiologies of cirrhosis could also explain the historically higher rate of infections in alcoholic cirrhotic subjects compared to NASH patients14,22.

The relationship between sigmoid mucosal Porphyromonadaceae reduction, increase in families belonging to Proteobacteria and subsequent hospitalizations is novel in this study. Porphyromonadaceae are usually of oral origin that have been associated with higher inflammation, progression of fatty liver disease and cognitive dysfunction in human and animal studies23,24. Members of Proteobacteria are usually increased in the stool of cirrhotic subjects and are linked with endotoxemia, but our study extended this onto the mucosa and linked them with clinically-relevant outcomes15. A recent study showed that in cirrhotics that have already been hospitalized ultimately achieve a microbial pattern i.e. significantly lower Bacteroidaceae, Porphyromonadaceae and Clostridiales XIV relative abundance compared to outpatients, indicating the complicity of these changes in promoting future adverse outcomes14.

It is also interesting that despite being on medications that improve overall outcomes by altering gut microbiota composition and function i.e. lactulose and rifaximin, patients were still prone to development of hospitalizations that were predicted by microbial changes25,26,27. A recent study has found that the probiotic VSL#3 reduced overall hospitalizations but not specifically HE episodes, compared to placebo in patients who had recovered from HE but were not on lactulose28. This randomized trial clearly sets the standard for beneficial microbial manipulation but did not study the probiotics in the context of lactulose, the standard of care and did not evaluate the microbiome. However, our underlying microbial differences between those who were hospitalized or not but not within those who were hospitalized for HE compared to other conditions, could partly explain their results7. Therefore the impact of the microbiota (decreased Clostridiales XIV and Bacteroidaceae) may prime the milieu for future insults that are result in admission regardless of the proximate cause. Future research into therapies that can beneficially alter the microbiota and prevent these outcomes in cirrhotics already taking standard of care treatment (lactulose and rifaximin) is required.

In cirrhotics with concomitant DM compared to those without it, we found a significantly different microbial composition at the stool and mucosal level. Specifically families in stool showed an increased relative abundance of Bacteroidaceae, Veillonellaceae, Streptococcaceae and Eubacteriaceae with a decrease in autochthonous Ruminococcaceae. This pattern has been shown in prior NASH cirrhosis experience, which was over-represented in this population, as well as in non-cirrhotic DM studies and studies of obesity10,14,29,30. The modulation of the microbiome with NASH, DM and obesity, can now be interpreted in the context of concomitant cirrhosis. Interestingly, this pattern is different from advancing cirrhosis and those who ultimately required hospitalization; it is likely a DM-related change in microbial composition31. However despite an altered microbiota composition in the sigmoid mucosa and the stool, DM in itself did not predispose to higher 90-day hospitalizations. However, the subgroup on insulin was indeed associated with a higher hospitalization rate, which could possibly due to a worse DM control and accompanying dysbiosis. The lack of effect on hospitalization overall in all DM patients may be due to relatively shorter follow-up compared to prior studies that did show an impact of DM on prognosis2. We limited our follow-up to 90 days to minimize variability within the microbiota from the baseline and because that is the validity of the MELD score32.

Replicating prior studies, we found that PPI use was a significant predictor of admissions and were more likely used in those with more advanced liver disease33,34. In addition to the generalized dysbiosis, there was a significant increase in Streptococcacae relative abundance, presumably of a salivary origin, with PPI use as prior studies have also shown12,35. This specific increase in Streptococcacae in PPI-using subjects also highlights the exceedingly complex gut milieu that is influenced differently by each medication. Interestingly this trend persisted even in the presence of HE therapy. However, despite controlling for all other important variables, PPI use remained significantly predictive of admissions.

Although this is the largest experience of mucosal microbiota to date in cirrhosis, changes in mucosal microbiota were not as predictive as stool for hospitalizations. As expected families from Proteobacteria had a higher relative abundance in the mucosa of those who were hospitalized, that demonstrates a different pattern of dysbiosis from that seen in the stool. This could be due to a relative stability of mucosal microbiota compared to changes in stool over time with factors such as diet or could be due to the relatively smaller sample of patients who underwent sigmoid biopsies. However from a practical standpoint, the relative non-invasiveness of stool collection compared to sigmoid mucosa, is encouraging towards using these samples, rather than the mucosal ones, for prediction of hospitalizations.

The study is a descriptive and cross-sectional analysis of microbiota to predict outcomes over 90 days, which did not study variations over time. However, in a prior study we found that gut microbiota track the underlying disease process and are stable over time14. There are also several other factors, including genetic variations and changes in microbial functionality, that could also impact the development of further complications in cirrhosis, that were not specifically assessed36. While the changes in bacterial subgroups are not as striking as those found in studies comparing cirrhotics with non-cirrhotic groups or with healthy controls8,14,15, it is important to realize that these were found in the context of our population of only cirrhotic subjects and were independently related to poor clinical outcomes despite controlling for available biomarkers. The use of MTPS also limited us to a relative smaller depth compared to metagenomic sequencing37; future studies are needed to evaluate these for long-term clinical outcomes. Despite these limitations, we were able to define a distinct microbial pattern in concomitant DM and in cirrhotics who were ultimately hospitalized.

The results demonstrate that gut and mucosal microbiota are altered in cirrhotic subjects who are non-electively hospitalized within 90 days regardless of the cause of hospitalization. This pattern is different from that induced by concomitant DM. Stool microbiota changes can enhance the predictive capability of current traditional biomarkers in the prediction of 90-day hospitalizations. Further studies into beneficial microbial modulation in cirrhotic patients to prevent hospitalizations are needed on the background of standard of care treatments.