Novel bacterial cluster “Prevotella, Bacteroides and Suterella” associated with mortality in Mexican patients with acute-on-chronic liver failure (ACLF) and clinical utility of systemic hs-CRP and IL-6: A frontier approach involving next-generation sequencing at the intestinal level in a cohort by alcoholic etiology.

Introduction and Objectives

ACLF is characterized by acute decompensation of cirrhosis, organ failure, and high short-term mortality. Several studies have demonstrated the relevance of intestinal microbiota (IM) in the pathophysiology of cirrhosis. To date, there are no studies in the Mexican population focused on IM in alcohol-associated ACLF and its relationship with mortality and inflammatory markers.

Aim

To analyze the composition and diversity of IM in patients with alcohol-associated cirrhosis and ACLF, healthy controls, and its correlation with inflammatory markers.

Materials and Patients

Cross-sectional study, which included 22 decompensated patients with ACLF, 16 decompensated patients without ACLF (CD) and 18 healthy individuals (HI), recruited at the Hospitales Civiles de Guadalajara. Fecal IM was characterized by NGS of the 16S-rRNA gene. Systemic levels of high-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6) were quantified by ELISA, and bioinformatics analysis of IM was performed using the QIIME2 package. Quality filtering, which includes removal of chimeras and non-biological sequences, was performed using the DADA2 algorithm. Resulting ASVs were taxonomically assigned through a self-trained naïve Bayesian classifier, against the SILVA database. Furthermore, α and β diversity analyses, relative abundances, and ANCOM-BC compositional analysis were performed in the QIIME2 package. Predictive values and associations were performed using ROC curves and Spearman correlations, respectively.

Results

ACLF and CD patients showed significantly lower α-diversity compared to CS. The comprehensive bacterial taxonomy profile in ACLF was significantly dominated by pathogenic/inflammatory genera such as Escherichia/Shigella, Enterobacter and Prevotella. In contrast, we observed a depletion of Bacteroides compared to CD. Interestingly, the subanalysis of MI in ACLF patients categorized at 7 and 90 days of mortality showed consistency with the enrichment of the Prevotella, Bacteroides and Suterella cluster. The Proteobacteria/Firmicutes ratio as a potential marker of dysbiosis, was significantly elevated in ACLF patients. Serum levels of hs-CRP and IL-6 were potentially increased in ACLF, in comparison to CD and CS. hs-CRP correlated positively with IL-6 and the Proteobacteria/Firmicutes ratio and negatively with α-diversity. IL-6 levels were positively correlated with MELD-Na. Finally, ROC curve analyses showed that hs-CRP allows discrimination of infections in patients with CD with a cut-off point >70.7 mg/L (AUROC: 0.75, with 90% sensitivity and 68.9% specificity). IL-6 allows discrimination of hepatic encephalopathy (HE) in patients with CD and ACLF with a cut-off point >7051.1 pg/mL (AUROC: 0.67, with 81.4% sensitivity and 45.8% specificity).

Conclusions

The dysbiotic/proinflammatory profile of IM in ACLF correlated with the potential increase in systemic inflammation. The bacterial cluster “Prevotella, Bacteroides and Suterella” represents a hallmark of mortality within 7 and 90 days. IL-6 and hs-CRP allow discrimination of HE and infections in patients with alcohol-associated cirrhosis.

Ethical statement: The study was conducted in accordance with the latest update of the Declaration of Helsinki and the Regulations on Human Studies in Health Matters of the Mexican Republic.

The protocol was approved by the ethics committees of the Civil Hospitals of Guadalajara (010/20 and 00012) and the Ethics, Research and Biosafety Committee of the University Center for Health Sciences of the University of Guadalajara (22-96).

Declaration of interests: None.

Funding: Institutional Funds PROSIN 2023 and Fortalecimiento a la Investigación y Posgrado (P3E 271879).

Figure 1.Analysis of fecal microbiota from patients with ACLF and its association with mortality. a) and b) Differential bacterial taxonomy (at the level of bacterial family and genera) at 7 and 90 days of patients who died, compared to those who did not die. The bars represent the log-fold change (LFC) between both groups. The blue bars indicate the characteristic taxa of the group that died, while the brown bars represent the survivor group. A cut-off of p<0.05 and q<0.05 was used. Analyzed by means of ANCOM-BC algorithm (Analysis of Compositions of Microbiomes with Bias Correction).

Figure 2. High-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6): biomarkers associated with bacterial infections and the presence of hepatic encephalopathy. a) ROC curve of hs-CRP, which demonstrates the prediction of bacterial infections in patients with decompensated cirrhosis without ACLF (cut-off point >70.7 mg/L (AUROC: 0.75, with 90% sensitivity and 68.9% specificity); b) ROC curve of IL-6, that demonstrates the prediction of hepatic encephalopathy in patients with decompensated cirrhosis with ACLF and without ACLF (cut-off point >7051.1 pg/ml (AUROC: 0.67, with 81.4% sensitivity and 45.8% specificity).