The fecal microbiota of patients with pancreatic ductal adenocarcinoma and autoimmune pancreatitis characterized by metagenomic sequencing

Abstract

Background: The fecal microbiota in pancreatic ductal adenocarcinoma (PDAC) and in autoimmune pancreatitis (AIP) patients remains largely unknown. We aimed to characterize the fecal microbiota in patients with PDAC and AIP, and explore the possibility of fecal microbial biomarkers for distinguishing PDAC and AIP.

Methods: 32 patients with PDAC, 32 patients with AIP and 32 age- and sex-matched healthy controls (HC) were recruited and the fecal microbiotas were analyzed through high-throughput metagenomic sequencing. Alterations of fecal short-chain fatty acids were measured using gas chromatographic method.

Results: Principal coordinate analysis (PCoA) revealed that microbial compositions differed significantly between PDAC and HC samples; whereas, AIP and HC individuals tended to cluster together. Significant reduction of phylum Firmicutes (especially butyrate-producing bacteria, including Eubacterium rectale, Faecalibacterium prausnitzii and Roseburia intestinalis) and significant increase of phylum Proteobacteria (especially Gammaproteobacteria) were observed only among PDAC samples. At species level, when compared with HC samples, we revealed 24 and 12 differently enriched bacteria in PDAC and AIP, respectively. Functional analysis showed a depletion of short-chain fatty acids synthesis associated KO modules (e.g. Wood-Ljungdahl pathway) and an increase of KO modules associated with bacterial virulence (e.g. type II general secretion pathway). Consistent with the downregulation of butyrate-producing bacteria, gas chromatographic analysis showed fecal butyrate content was significantly decreased in PDAC group. Eubacterium rectale, Eubacterium ventrisum and Odoribacter splanchnicus were among the most important biomarkers in distinguishing PDAC from HC and from AIP individuals. Receiver Operating Characteristic analysis showed areas under the curve of 90.74% (95% confidence interval [CI] 86.47-100%), 88.89% (95% CI 73.49-100%), and 76.54% (95% CI 52.5-100%) for PDAC/HC, PDAC/AIP and AIP/HC, respectively.

Conclusions: In conclusion, alterations in fecal microbiota and butyrate of patients with PDAC suggest an underlying role of gut microbiota for the pathogenesis of PDAC. Fecal microbial and butyrate as potential biomarkers may facilitate to distinguish patients with PDAC from patients with AIP and HCs which worth further validation.

Keywords: Autoimmune pancreatitis; Butyrate; Fecal microbiota; Metagenomic sequencing; Pancreatic ductal adenocarcinoma.

Fig. 1

Alterations in the gut microbiota among patients with PDAC and AIP. a Principal coordinate analysis (PCoA) based on Bray–Curtis distance at the species level. Each data point represents an individual sample. P-value was calculated by PERMANOVA. b Comparison of the gut microbiota among PDAC, AIP and HC groups at the phylum level and box and whisker plot of Firmicutes to Bacteroidetes ratios. c–e Species enriched in healthy controls and species enriched in case groups, respectively. The phylum, genus and species levels are colored as follows: purple, both enriched in PDAC and AIP; blue, both depleted in PDAC and AIP; red, enriched in PDAC only; green, depleted in PDAC only. Significance was set at adjusted p-value < 0.2 (FDR-corrected Kruskal–Wallis test) and p-value (Steel–Dwass test) < 0.05. Only taxa with adjusted p-value < 0.2 (except Bacteroidetes) are shown. Steel–Dwass test for pairwise comparisons following FDR-corrected Kruskal–Wallis test. PDAC: pancreatic ductal adenocarcinoma; AIP: autoimmune pancreatitis; HC: healthy controls

Fig. 2

Differentially abundant CAGs in the three groups. a Heatmap of CAGs with a p-value < 0.05determined by the Kruskal–Wallis testis shown in rows. Gene abundance is indicated by color gradient (white represents “undetected”, red represents “most abundant”). The p-value is shown on the right. b Abundance-based CAG co-abundance correlation network enriched in PC group (left), AIP group (media) and AIP group (right). The node size was proportional to the enrichment extent. The node shape was as follows: rectangle, PDAC enriched; rhombus, AIP enriched; diamond, case enriched; circle, HC enriched. Nodes are colored by family level. PDAC: pancreatic ductal adenocarcinoma; AIP: autoimmune pancreatitis; HC: healthy control

Fig. 3

Differentially enriched KO modules in the PDAC, AIP and HC groups. Modules with a reporter score > 2.3 (the former enriched) or < -2.3 (the latter were enriched) are shown. *reporter score > 2.3 or < − 2.3; **, reporter score > 3 or < 3. PDAC: pancreatic ductal adenocarcinoma; AIP: autoimmune pancreatitis; HC: healthy control

Fig. 4

Box-and-Whisker plot of four pathways for butyrate synthesis (a) and bar plot of involved genes in acetyl-CoA pathway (b). p-value shown in (a) was determined by Steel–Dwass test for pairwise comparison following FDR-corrected Kruskal–Wallis test. Pathways are shown as described by Vital et al. [14]

Fig. 5

Butyrate was downregulated only in feces of PDAC patients as revealed by gas chromatography analysis. Shown are relative content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and total acid in PDAC, AIP and HC groups. PDAC: pancreatic ductal adenocarcinoma; AIP: autoimmune pancreatitis; HC: healthy controls CI: confidence interval

Fig. 6

Microbial biomarkers classify PDACC patients from healthy controls and AIP patients. a, c, e ROC curves for testing cohort are shown. b, d, f Shown are the importance of the selected biomarkers. PDAC vs AIP (a and b), AIP vs HC (c and d), PDAC vs AIP (e and f). PDAC: pancreatic ductal adenocarcinoma; AIP: autoimmune pancreatitis; HC: healthy controls CI: confidence interval