Disease Severity and Proton Pump Inhibitor Use Impact Strongest on Faecal Microbiome Composition in Liver Cirrhosis

Vanessa Stadlbauer; Irina Komarova; Ingeborg Klymiuk; Marija Durdevic; Alexander Reisinger; Andreas Blesl; Florian Rainer; Angela Horvath

Disclosures

Liver International. 2020;40(4):866-877. 

In This Article

Discussion

Faecal microbiome composition has been associated with various diseases, but not only disease severity and comorbidities, but also other factors such as medication, diet or lifestyle habits may influence its composition. It is therefore important to discern the effect of different influencing variables as a first step from association to causality. We performed a system biology analysis of a well-characterized cohort of patients with liver cirrhosis and were able to show by multivariate RDA that severity and aetiology of liver disease, PPI use, nutritional status, age and CRP levels were significant explanatory variables for faecal microbiome composition. Although we find some taxonomic overlaps especially between severity and PPI use on network analysis, we could show that the effects of disease severity, aetiology, PPI use and age are independent factors influencing microbiome composition also in subgroup analyses.

Changes in faecal microbiome composition have been associated with liver disease since more than 35 years, long before sequencing techniques became available.[41] When 16S sequencing techniques emerged, stool microbiome composition in liver disease was studied showing compositional alterations throughout all levels of taxonomy in liver cirrhosis.[3,42] However both of these studies did not associate compositional changes with potentially important influencing factors such as aetiology of cirrhosis or drug intake in a systematic way. The strength of our cohort is the detailed characterization of the participants, that allows to fill this knowledge gap on the association of different factors with faecal microbiome composition in cirrhosis and the use of ANCOM and LEfSE as specialized bioinformatics methods to study microbiome composition.

Severity of liver disease was shown to impact microbiome composition. A positive correlation between Child-Pugh score and Streptococcaceae as well as a negative correlation with Lachnospiraceae was described in the study by Chen et al Several positive and negative correlations between liver function and species abundance were reported in the study by Qin et al without describing more details on these associations.[3,42] Data on concomidant drug intake is missing in both studies, leading to scientific discussions and the need for further reserach[43,44] In subsequent studies strong associations of microbiome changes with hepatic encephalopathy were shown.[8,45] Our analysis demonstrates that disease severity, measured by composite scores (Child-Pugh and MELD) as well as some of the individual parameters of both scores (albumin, bilirubin, creatinine, INR) are significant explanatory variables for microbiome composition in univariate analysis. Child-Pugh score also remained significant in multivariate RDA. Higher Child-Pugh classes (B and C) were associated with distinct changes in microbiome composition related to an increase in oral bacteria and potential pathogens. On family level we found a higher abundance of Lactobacillaceae and Veillonellaceae and a lower abundance of Micrococcaceae in Child-Pugh B/C patients which is in line with previously published data.[3,8,42,45] However, it is still not fully elucidated, whether these changes are driven by disease severity itself or by other influencing factors.

Cirrhosis is a complex disease requiring long-term drug treatment with several drug classes. Many medically approved drugs influence microbiome composition.[19] In liver cirrhosis, PPI use has been described to alter microbiome composition, increase the rate of complications and negatively impact prognosis.[5,46–49] We recently expanded this knowledge by describing the consequences of PPI-induced dysbiosis and oralization of the faecal microbiome on inflammation, intestinal permeability and outcome in cirrhosis.[11] In the present study PPI use also had a strong impact on the faecal microbiome, being associated with an increased abundance of oral bacteria and potential pathogens, such as Streptococcus species and Veillonella. Although PPI use and disease severity were partially linked in our cohort, both were still independent factors influencing microbiome composition in subgroup analyses, however low sample size in this analysis weakens the explanatory power.

Also other drugs commonly administered in cirrhosis, may impact the faecal microbiome. However, in our cohort, other drugs influenced microbiome composition to a lesser extent. We were interested in the effect of lactulose on the cirrhotic microbiome, since available data are conflicting, showing no major alterations of the faecal microbiome between lactulose user and non-user, but withdrawal of lactulose leads to the loss of beneficial species.[16,50,51] In our cohort only 11% of the cirrhotic patients used lactulose. On univariate analysis we found that lactulose had a weak, but significant, effect on microbiome composition, which did not remain significant in multivariate analysis. Therefore our data support the notion that lactulose has no major effect on the taxonomic composition of the faecal microbiome in cirrhosis and may therefore exert its function through functional microbiome changes. Other drug classes did not have a major impact on microbiome composition.

Liver cirrhosis is also a heterogeneous disease from an aetiological perspective. Aetiology of liver disease as a factor to explain differences in microbiome composition has already been studied. Bajaj et al describe alcohol and non-alcoholic steatohepatitis driven changes in microbiome composition.[45] Hepatitis C alters faecal microbiome which has been implicated in the pathogenesis of HCV-induced chronic liver disease. However, Hepatitis C induced dysbiosis seems to be stable over different disease stages.[52] Chronic cholestatic diseases such as primary biliary and primary sclerosing cholangitis are also associated with distinct changes in microbiome composition.[53] Although aetiology of liver disease was identified as a significant factor by multivariate RDA in our data set, the taxonomic differences of the microbiomes from different aetiologies were surprisingly small in our cohort. Patients with alcoholic cirrhosis had a higher abundance of the genus Erysipelatoclostridium and the group of patients with "other" aetiologies of liver cirrhosis had a higher abundance of two yet uncultured bacteria of the family Lachnospiraceae and of the genus Blautia. Since liver cirrhosis aetiology is varying in different geographical regions[54] and geographical region itself is a factor that impacts diversity and composition of the microbiome,[55] also the origin of the study population has to be taken into account. Our patient cohort consists only of Caucasians living in the same region, which is a likely explanation for the relatively similar microbiome composition in different aetiologies. Bajaj et al have shown that cirrhotic patients from Turkey, compared to patients from the USA, differ in aetiology and dietary habits, which resulted in a higher microbial diversity in Turkish cirrhotic patients.[14] We also analysed dietary habits in our patient cohort and only found one significant difference of questionable relevance. Patients with hepatitis C virus infection were more likely to consume muesli on a regular basis compared to alcoholic cirrhotic patients (62% vs 32%, P < .01)[56]

Furthermore age impacts on microbiome composition, however, the specific changes of the ageing microbiome are unknown and inconsistent. Data on microbial diversity are conflicting and seem to depend on confounding factors and analysis techniques. A loss of Bifidobacteria, Lactobacilli, Clostridium Cluster XIVa, Akkermansia muciniphila and Faecalibacterium prausnitzii and an increase of Escherichia coli species has been observed.[57]Collinsella aerofaciens, Alistipes onderdonkii and the genus Slackia were differentially abundant between age groups in our cirrhosis cohort. However, none of these have been previously described to be associated with age related changes in microbiome composition. Collinsella aerofaciens has been associated with diet and intestinal inflammatory diseases,[58–60] Slackia with equol production,[61] whereas Alistipes onderdonkii has not been associated with any human condition yet.[62]

Nutritional status is an important prognostic factor in liver cirrhosis. Especially sarcopenia is a frequent complication of malnutrition in cirrhosis and is associated with adverse outcome.[63,64] Assessing nutritional status in cirrhosis can be challenging. We used the well-established SGA, that is also recommended by the European Association for the Study of the Liver[65] to differentiate between well nourished and moderately malnourished patients. In our cohort of mostly compensated cirrhotic patients we did not have any severely malnourished patients. We found a higher abundance of the order Campylobacterales in moderately malnourished cirrhotics. Campylobacterales have been associated with malnutrition in children.[66,67] The order Verrucomicrobiales was enriched in adequately nourished cirrhotic patients. So far only one species of this order has been described in human faeces: Akkermansia muciniphila. A loss of Akkermansia muciniphila has been associated with metabolic diseases and the abundance may be influenced by diet.[68]

Changes in microbiome composition in cirrhosis impact intestinal barrier function and lead to intestinal and systemic inflammation due to translocation of bacterial products to the liver and also to the systemic circulation. This concept of a crosstalk between gut, liver and immune system – the so called gut-liver axis—is widely implicated in the pathogenesis of liver disease and a promising therapeutic target.[69,70] We assessed a panel of biomarkers of the gut–liver axis in our cohort and CRP as a marker of inflammation was found to be a significantly explanatory variable in the multivariate analysis, however the consistency of the effect remains unclear in our dataset. CRP is a well-known biomarker in liver cirrhosis and is predictive for complications and outcome.[71,72] Increased abundance of Veillonella and Streptococcus species but also Faecalibacterium species were associated with higher CRP levels. While the former support the link between microbiome composition and inflammation in liver cirrhosis, Faecalibacterium species are usually associated with anti-inflammatory properties, making a firm conclusion difficult.

Interestingly, gender did not impact on microbiome composition in our study. In studies in obese individuals, gender seems to cause taxonomic differences, whereas data on changes on alpha and beta diversity are still conflicting.[73,74] In liver cirrhosis, gender differences have not been associated with changes in beta diversity so far. The male predominance in liver cirrhosis may be a reason for difficulties to detect consistent gender differences.

In summary our cross sectional system biology study shows that disease severity and PPI use are the main factors explaining variation of the faecal microbiome in cirrhosis. Aetiology of liver disease, age, nutritional status and inflammation (CRP levels) are further explanatory variables. The limitation of our study is the single-center design that does not allow to account for geographical differences in the microbiome composition, the cross sectional design that does not allow to draw any conclusions on causality as well as the low sample size in the subgroup analyses. The strength of our study is the thorough characterization of our study patients, that allows detailed analysis of influencing variables. For future studies, we strongly suggest to increase sample size and expand the minimal set of metadata as suggested by the IHMS consortium[75] to include detailed information on disease aetiology, disease severity, drug intake and also information on further essential biomarkers, depending on the disease studied. This will open up new paths to understand the crosstalk between the faecal microbiome and the human body in disease states.

Lay Summary

The composition of gut bacteria—the gut microbiome—is altered in many diseases. In chronic liver diseases, such as liver cirrhosis, the gut microbiome is severely disturbed. We were able to show in this study which factors explain this disturbed microbiome composition. These factors were: The cause of liver disease, the severity of liver disease, intake of acid-blockers (proton pump inhibitors), age and inflammation. Our study also shows the importance of collecting sufficient data on the disease and drug intake to be able to assess the effects of different factors on the gut microbiome.

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