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. 2023 Nov 28;24(23):16888.
doi: 10.3390/ijms242316888.

Bile Microbiome Signatures Associated with Pancreatic Ductal Adenocarcinoma Compared to Benign Disease: A UK Pilot Study

Nabeel Merali  1   2   3 , Tarak Chouari  2   3 , Julien Terroire  4   5 , Maria-Danae Jessel  3 , Daniel S K Liu  6 , James-Halle Smith  7 , Tyler Wooldridge  3 , Tony Dhillon  3 , José I Jiménez  8 , Jonathan Krell  6 , Keith J Roberts  7 , Timothy A Rockall  1 , Eirini Velliou  9 , Shivan Sivakumar  10 , Elisa Giovannetti  11   12 , Ayse Demirkan  4   5 , Nicola E Annels  3 , Adam E Frampton  1   2   3   6
Affiliations

Bile Microbiome Signatures Associated with Pancreatic Ductal Adenocarcinoma Compared to Benign Disease: A UK Pilot Study

Nabeel Merali et al. Int J Mol Sci. .

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a very poor survival. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance and, therefore, patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures that can effectively distinguish malignant from benign tumours in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease. Prospective bile samples were obtained from 31 patients who underwent either Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiogram (PTC). Variable regions (V3-V4) of the 16S rRNA genes of microorganisms present in the samples were amplified by Polymerase Chain Reaction (PCR) and sequenced. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign). The bile microbial beta diversity significantly differed between patients with PDAC vs. benign disease (Permanova p = 0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering of Aitchison distance. We found three genera to be of significantly lower abundance among PDAC samples vs. benign, adjusting for false discovery rate (FDR). These were Escherichia (FDR = 0.002) and two unclassified genera, one from Proteobacteria (FDR = 0.002) and one from Enterobacteriaceae (FDR = 0.011). In the same samples, the genus Streptococcus (FDR = 0.033) was found to be of increased abundance in the PDAC group. We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation.

Keywords: 16S rRNA gene; bile; biomarker; microbiome; pancreatic cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bar plot showing the relative abundance of different bacteria within each sample and cohort at the genus level. A total of 135 genera from 29 individuals (12 PDAC and 17 benign) were identified, and a relative abundance of 10% and above was included in the figure. Forty-one different taxa that were not mapped to any bacteria at the genus level were clustered under “others” for the simplicity of this figure.
Figure 2
Figure 2
No difference was observed in Alpha diversity using the Shannon and Simpson index. Mean alpha diversity is higher among the benign samples, with p-values of 0.31 for Shannon and 0.3 for Simpson indices.
Figure 3
Figure 3
Microbial beta diversity significantly differs between bile from individuals with PDAC vs. bile from individuals with benign samples (p = 0.0173). The clusters were visualised by plotting the first two components that explain up to 35.3% of the variation in the sample space. The large blue triangle and the large red dot represent the centroids of the PDAC and benign sample groups.
Figure 4
Figure 4
Unsupervised clustering of selected genera abundances separates PDAC from benign samples. The phylogenic tree clustering learned from 135 genera is shown in the x-axis. The y-axis shows the individual samples, which are separated into two major clusters, as shown by the dendrogram on the left. The colour key code is a graphic representation of centre-log ratio (CLR) abundance that uses all taxon read counts within a sample divided by this geometric mean, and the log fold changes in this ratio between samples are compared. If the abundance of a bacteria is lower than the mean CLR value, then it will be negative (blue), and if it is higher, then it will be positive (red).
Figure 5
Figure 5
Study Flowchart outlining aspiration of bile at the time of obstructive jaundice at ERCP and subsequent sequencing of the bile microbiome. Prospective bile samples were obtained from 31 patients who underwent either ERCP or PTC. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign).
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Grants and funding

This research was funded by the CRUK Development Fund Imperial Centre grant number PSM295_LCIB, Mason Medical Research Trust grant number RC3597 and Royal College of Surgeons of England Research Fellowship. The APC was funded by the University of Surrey.