Gut microbial RNA and DNA analysis predicts hospitalizations in cirrhosis

Abstract

Background: Cirrhosis is associated with gut microbial changes, but current 16S rDNA techniques sequence both dead and live bacteria. We aimed to determine the rRNA content compared with DNA from the same stool sample to evaluate cirrhosis progression and predict hospitalizations.

Methods: Cirrhotics and controls provided stool for RNA and DNA analysis. Comparisons were made between cirrhotics/controls and within cirrhosis (compensated/decompensated, infected/uninfected, renal dysfunction/not, rifaximin use/not) with respect to DNA and RNA bacterial content using linear discriminant analysis. A separate group was treated with omeprazole for 14 days with longitudinal microbiota evaluation. Patients were followed for 90 days for hospitalizations. Multivariable models for hospitalizations with clinical data with and without DNA and RNA microbial data were created.

Results: Twenty-six controls and 154 cirrhotics (54 infected, 62 decompensated, 20 renal dysfunction, 18 rifaximin) were included. RNA and DNA analysis showed differing potentially pathogenic taxa but similar autochthonous taxa composition. Thirty subjects underwent the omeprazole study, which demonstrated differences between RNA and DNA changes. Thirty-six patients were hospitalized within 90 days. In the RNA model, MELD score and Enterococcus were independently predictive of hospitalizations, while in the DNA model MELD was predictive and Roseburia protective. In both models, adding microbiota significantly added to the MELD score in predicting hospitalizations.

Conclusion: DNA and RNA analysis of the same stool sample demonstrated differing microbiota composition, which independently predicts the hospitalization risk in cirrhosis. RNA and DNA content of gut microbiota in cirrhosis are modulated differentially with disease severity, infections, and omeprazole use.

Trial registration: NCT01458990.

Funding: VA Merit I0CX001076.

Keywords: Bioinformatics; Clinical practice; Hepatology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. DNA comparison of controls versus cirrhosis.

Histograms of linear discriminant analysis (LDA) effect size (LEfSe) comparison between stool microbiota at the genus level between healthy controls (n = 26) and patients with cirrhosis (n = 154). Log-level changes in LDA score are displayed on the x axis. (A) DNA analysis comparison. (B) RNA analysis comparison. Green bars: taxa found in greater relative abundance in healthy controls. Red bars: taxa found in greater relative abundance in patients with cirrhosis.

Figure 2. DNA comparison of compensated versus decompensated cirrhosis.

Histograms of linear discriminant analysis (LDA) effect size (LEfSe) comparison between stool microbiota at the genus level between compensated-cirrhosis patients (n = 92) and patients with decompensated cirrhosis (n = 2). Decompensation in cirrhosis is defined as hepatic encephalopathy, ascites, jaundice, and variceal bleeding. Log-level changes in LDA score are displayed on the x axis. (A) DNA analysis comparison. (B) RNA analysis comparison. Green bars: taxa found in greater relative abundance in decompensated cirrhosis. Red bars: taxa found in greater relative abundance in patients with compensated cirrhosis.

Figure 3. DNA comparison of infected versus uninfected cirrhotic patients.

Histograms of linear discriminant analysis (LDA) effect size (LEfSe) comparison between stool microbiota at the genus level between infected cirrhotic patients (n = 54) and uninfected patients with cirrhosis (n = 100). Infected patients were those with clinically determined bacterial infections. Log-level changes in LDA score are displayed on the x axis. (A) DNA analysis comparison. (B) RNA analysis comparison. Red bars: taxa found in greater relative abundance in infected cirrhosis. Green bars: taxa found in greater relative abundance in uninfected patients.

Figure 4. DNA comparison of cirrhotic patients with and without renal dysfunction.

Histograms of linear discriminant analysis (LDA) effect size (LEfSe) comparison between stool microbiota at the genus level between cirrhotic patients with renal dysfunction (n = 20) and cirrhotic patients without renal dysfunction (n = 134). Renal dysfunction is defined as serum creatinine > 1.5 mg/dl on the day of sample collection. Log-level changes in LDA score are displayed on the x axis. (A) DNA analysis comparison. (B) RNA analysis comparison. Green bars: taxa found in greater relative abundance in those without renal dysfunction (low creatinine). Red bars: taxa found in greater relative abundance in patients with renal dysfunction (high creatinine) with compensated cirrhosis.

Figure 5. Comparison of cirrhotic patients on and not on rifaximin.

In the cirrhotic subgroup without infections or on other antibiotics (n = 85), histograms of linear discriminant analysis (LDA) effect size (LEfSe) comparison of stool microbiota at the genus level between patients on rifaximin (n = 19) and not on rifaximin (n = 66) are shown. Rifaximin is a nonabsorbable antibiotic used for hepatic encephalopathy. Log-level changes in LDA score are displayed on the x axis. (A) DNA comparison. (B) RNA comparison. Green bars: taxa found in greater relative abundance in patients on rifaximin. Red bars: taxa found in greater relative abundance in patients not on rifaximin.

Figure 6. Comparison of controls and cirrhotic patients before/after 14 days of omeprazole.

Healthy controls and compensated cirrhotic patients were administered 40 mg/day of omeprazole for 14 days. Stool microbiota were analyzed at baseline and after 14 days using Wilcoxon’s signed rank-sum test, focusing on relative abundance of oral-origin families Streptococcaceae and Veillonellaceae. (A) DNA comparison. (B) RNA comparison. Data shown as median and 95% CI. *P < 0.05, **P < 0.01. Ctrl, control; Cirr, compensated cirrhosis; Pre, before omeprazole; Post, after 14 days of 40 mg omeprazole daily.