Bile microbiome analysis of recurrent CBD stone patients vs. Non-recurrent CBD stone patients
We conducted bile fluid microbiome analysis on patients that underwent ERCP at our institution from February 2019 to January 2021 to identify differences between the microbiome compositions of SR (recurrent CBD stone) and NSR (non-recurrent CBD stone) patients. Eligible patients were aged 18 years or older, required ERCP for bile duct decompression, and had developed recurrence of CBD stones confirmed by computed tomography (CT) or ultrasound after at least six months of stone removal. Patients with an intrahepatic duct stone, hemolytic anemia, inflammatory bowel disease, or severe liver disease were excluded. Patients with problematic endoscopic approaches due to esophageal stenosis, gastric outlet obstruction, or duodenal stenosis were also excluded, as were patients with a medical history of conditions associated with thrombocytopenia or coagulopathy (PT-INR > 1.5; normal 0.85-1.25, platelet count < 60,000/mm3).
Bile fluid and Duodenal microbiomes of Recurrent CBD stone patients:
Duodenum tissues were obtained from SR patients, recruited as described above, to analyze the microbiome compositions of bile fluid and duodenum tissues. Microbiome compositions were then analyzed to identify differences with the duodenum microbiome composition of controls (gastric ulcer patients without recurrent CBD stones).
Bile fluid collection and Duodenal biopsy under ERCP: sample collection
Bile fluid collection during ERCP:
Prior to ERCP, all patients were administrated prophylactic antibiotics (ciprofloxacin 400 mg IV over 60 minutes) to prevent bacteremia. ERCP was performed using a conventional side-viewing duodenoscope (TJF-260; Olympus Corporation, Tokyo, Japan) and a straight standard injection catheter. After achieving the therapeutic aim of ERCP, an endoscopic nasobiliary drainage (ENBD) tube was inserted such that its proximal end was located in the proximal CBD. Bile samples (20-30 cc) were aspirated 24 hours after endoscopic procedures from CBDs via ENBDs to prevent contamination of the upper gastrointestinal tract, including the oral cavity. Samples were immediately placed in germ-free sputum cups and stored at −80°C until required.
Duodenal tissue collection during ERCP:
Duodenal biopsy was performed prior to biliary access to avoid contamination with duodenal mucosa during ERCP. The duodenum was entered using the conventional side-viewing scope, and two duodenal tissue samples were obtained using biopsy forceps. Duodenal biopsy samples were collected on the opposite side of the ampulla of Vater (AoV) to minimize the risk of bile fluid contamination. Biopsies were performed using disposable biopsy forceps.
Duodenal tissue collection during Endoscopy for gastric ulcer patients:
Esophagogastroduodenoscopy (EGD) procedures were performed using the GIF-H290 duodenoscope (Olympus Co., Ltd., Tokyo, Japan). Two biopsies were obtained from the 2nd portion of the duodenum. Duodenal biopsy specimens were also obtained from the opposite side of the AoV to minimize the risk of bile contamination. Specimens were taken using disposable biopsy forceps.
DNA extraction from bile fluid and duodenum tissue
The total bacterial genomic DNA was extracted from 10 mL of bile fluid or duodenal tissue using a Maxwell® RSC PureFood GMO and Authentication Kit (Promega, Madison, WI, USA). Samples were initially centrifuged at 5,000 g for 5 minutes at room temperature and then resuspended in 500 µL of cetyltrimethylammonium bromide buffer, according to the manufacturer’s protocol. DNA concentrations were determined using a UV-vis spectrophotometer (NanoDrop 2000c; Thermo Fisher Scientific, Waltham, MA, USA). QuantiFluor® ONE dsDNA System (Promega), and samples were stored at −20°C until required.
PCR amplification of the V3–V4 region of the bacterial 16S ribosomal RNA (rRNA) gene
The V3 and V4 variable regions of the bacterial 16S rRNA gene were amplified using a two-step PCR protocol. Briefly, PCR was conducted using F319 (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACG-GGNGGCWGCAG) and R806 (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA-GGACTACHVGGGTATC- TAATCC-3′) primers. Amplified products were distinguished by 2% agarose gel electrophoresis, and 16S rRNA libraries were purified using AMPure XP magnetic beads, according to the manufacturer’s instructions (Beckman Coulter, Wycombe, UK). A Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) was used for the sample quality control. For second-round PCR, Illumina Nextera barcodes (Illumina, Inc., San Diego, CA, USA) were attached to first step PCR products using i5 forward primer and i7 reverse primer. Amplified products were purified as described for first-round PCR. DNA quantitation was performed using the QuantiFluor® ONE dsDNA System (Promega). A Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) was used for sample quality control. 16S rRNA gene amplification and library preparation (using a two-step PCR protocol) were used to perform 16S rRNA sequencing using a MiSeq v3 Reagent Kit (Illumina, Inc.).
Sequencing data were processed using the mothur software package (v1.39.4). DNA sequences were clustered into operational taxonomic units (OTUs) by reference-based OTU clustering using SILVA rRNA database (http://www.mothur.org/wiki/Silva_reference_files), release 102 (http://www.mothur.org/w/images/9/98/Silva.bacteria.zip). Chimera reads were detected and removed using Chimera UCHIME. Each OTU was assigned taxonomically using the Ribosomal Database Project reference database. Species richness and differences in microbial profiles were demonstrated by alpha and beta diversities calculated using mothur. Microbial diversities, evaluated using OTUs richness, were used to evaluate alpha diversities using Chao1, Shanon, and Simpson indices. Beta diversity refers to compositional dissimilarity between OTUs on phylogenetic trees. To compare group beta diversities, we used non-metric multidimensional scaling (NMDS) and the phyloseq package for R. Linear discriminant analysis with effect size estimation (LEfSe) was conducted to determine differences between relative abundances of taxa between groups. A linear discriminant analysis score > 2 with a p-value < 0.05 was considered statistically significant. Data visualization was performed using R (version 3.6.0) and the graphics packages MASS, ggplot2, and reshape2.
The study protocol was approved by the Institutional Review Board of Inha University Hospital (INHAUH 2019-02-015) and written informed consents were obtained from all participants before the procedures began. The study was conducted in accordance with the Declaration of Helsinki.