Specimen Collection and Analysis of the Duodenal Microbiome

Summary

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

Abstract

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut microbiome has been typically studied through fecal specimen collections. The ease of obtaining fecal samples has resulted in many studies that have revealed information concerning the distal luminal gastrointestinal tract. However, few studies have addressed the importance of the microbiome in the proximal gut. Given that the duodenum is a major site for digestion and absorption, its microbiome is relevant to nutrition and liver disease and warrants further investigation. Here we detail a novel method for sampling the proximal luminal and mucosal gut microbiome in human subjects undergoing upper endoscopy by obtaining duodenal aspirate and biopsies. Specimen procurement is facile and unaffected by artifacts such as patient preparatory adherence, as might be the case in obtaining colonic samples during colonoscopy. The preliminary results show that the luminal and mucosal microbiomes differ significantly, which is likely related to environmental conditions and barrier functions. Therefore, a combination of duodenal aspirate and biopsies reveal a more comprehensive picture of the microbiome in the duodenum. Biopsies are obtained from the descending and horizontal segments of the duodenum, which are anatomically close to the liver and biliary tree. This is important in studying the role of bile acid biology and the gut-liver axis in liver disease. Biopsies and aspirate can be used for 16S ribosomal RNA sequencing, metabolomics, and other similar applications.

Introduction

The intestinal microbiome has become an area of increased interest in recent years. It is now understood that the diverse bacterial population in the gut can differ based on a variety of factors, including genetics, diet, medication, and environmental influences¹. Studies have also identified unique microbial profiles linked to varying gastrointestinal diseases, such as obesity, inflammatory bowel disease, and liver disease^2,3. The majority of studies focus on profiling the microbiome of the large intestine through the analysis of fecal and distal mucosal samples⁴. Although the highest concentration of intestinal bacteria resides in the colon (10¹² bacteria/gram), there nevertheless is a complex community of microbes residing in the duodenum (10³/g), jejunum (10⁴/g), and ileum (10⁷/g) that plays a key role in digestive metabolism and absorption⁵.

The small intestine serves as the primary site of nutrient breakdown and absorption in the gastrointestinal tract. Commensal bacteria lining the small intestine play a fundamental role in aiding in the chemical breakdown of food substrates and in the release of bioactive compounds that aid in nutrient absorption⁶. These interactions contribute to a complex environment of microbe-microbe and host-microbe activity in the small intestine⁷. A study observing the small intestine microbiota in murine models found that germ-free mice fed a high fat diet had impaired lipid absorption but, when colonized with jejunal microbiota, had a direct increase in lipid absorption⁶. A human pilot study profiling the duodenal microbiota of obese and healthy individuals found that the duodenal microbiota of obese individuals had alterations in fatty acid and sucrose breakdown pathways, likely induced in a diet-dependent relationship⁸. Furthermore, dysbiosis in the small intestine microbiota has been identified in several diseases including small intestinal bacterial overgrowth, short-bowel syndrome, pouchitis, environmental enteric dysfunction, and irritable bowel syndrome⁷.

We are interested in the relationship between the microbiome and different stages of chronic liver disease. Specifically, the duodenum serves as the first site of chemical breakdown and nutritional absorption in the small intestine. Additionally, portal hepatic circulation brings nutrients and metabolites to the liver, where they are processed and regulated into the bloodstream. The anatomical proximity between the gut and the liver creates an environment susceptible to pro-inflammatory responses that can arise due to failure in the gut barrier or alterations in the gut microbiome⁹. Studies investigating the microbiome and liver disease progression have identified microbial dysbiosis in patients with non-alcoholic fatty liver disease (NAFLD), steatohepatitis (NASH), alcoholic liver disease, and cirrhosis^10,11. While the majority of studies characterize the microbiome of the colon, we were interested in investigating the small intestine microbiome in relation to liver disease. By utilizing the novel method presented here, we have identified unique duodenal microbial profiles in patients with liver cirrhosis in relation to diet¹².

As characterization of the small intestine microbiome continues to become an area of increased interest, it is necessary to develop uniform techniques for obtaining samples that accurately represent the small intestine microbiota. However, there are challenges associated with specimen procurement that have complicated the study of the small intestine microbiome environment. Current sampling methods require invasive procedures that are often subject to contamination, as outlined by Kastl et al⁷. Here we detail a novel method for obtaining duodenal aspirate and biopsies for microbial analysis from patients with liver disease undergoing esophagogastroduodenoscopy.

Protocol

Duodenal samples were obtained at the Veteran Affairs Greater Los Angeles Healthcare System, Cedars-Sinai Medical Center, and the Ronald Reagan UCLA Medical Center after the clinical protocol for the Microbiome, Microbial Markers and Liver Disease (M3LD) study was accepted by the institutional review board of the local ethics review committee. Written informed consent was obtained from all participating patients.

1. Consent of participants

1. Approach subjects with a diagnosis of liver cirrhosis of any etiology with endoscopies scheduled for variceal screening or portal hypertension for recruitment. Exclude patients with hepatocellular carcinoma (HCC).
2. Introduce the study, carefully ensure participant understanding, and ask the participant if they would like to volunteer in the study. Complete an informed consent form if they agree.
3. Ensure that a properly trained and credentialed medical professional performs the endoscopy.

2. Specimen collection

1. Anesthetize the patient with 50 mg of fentanyl and 2 mg of midazolam. Increase the dosage as needed until the patient is moderately anesthetized and can tolerate the endoscope.
2. Insert an endoscope (e.g., Pentax EG29-i10) into the patient’s esophagus and progress toward the pylorus.
   NOTE: The endoscope size will vary based on the patient's physiology. Do not use the endoscope for any aspiration prior to sample collection to avoid contaminating the endoscope channel with gastric or oropharyngeal secretions.
3. Obtain a microscope slide and blunt 18 Gauge syringe needle to prepare for specimen procurement. Prepare a sterile pad which will be used to transfer biopsies from the microscope slide to cryovials.
4. When the scope arrives at the second portion of the duodenum distal to the ampulla of Vater, aspirate any fluid present through a sterile disposable aspiration catheter passed through the working channel of the upper endoscope and collect it into a 40 mL disposable specimen container. Flush up to 30 mL of additional sterile water by syringe into the duodenum and aspirate the fluid into a fluid specimen trap to collect a total of 15 mL.
5. Immediately remove and cap the aspirate container attached to the suction component of the catheter. Place the container into a thermos over ice and into a specimen transport bag for transfer to the processing lab.
6. Insert a 2.8 mm single-use biopsy forceps into the port on the side of the endoscope to remove tissue specimens for research. Perform 2 random passes of the forceps within the second portion of the duodenum, collecting two bites of tissue in each pass.
7. Use a blunt 18 Gauge syringe needle to extract tissue samples from the forceps and onto the microscope slide before transferring the samples to four separate empty 2 mL cryovials. Tightly screw on the caps.
8. Insert all 4 tightly capped cryovials into an empty container, such as a urine cup, and create a bath, consisting of 20 mL of ethanol and dry ice, to flash freeze the biopsies.
9. Place the biopsy cryovials in an insulated Styrofoam container on a bed of dry ice and transfer the specimens to a -80 °C freezer for long term storage to preserve the specimens.

3. Aspirate processing

1. Following biospecimen collection, immediately transfer the biospecimens to a BSL2 or higher laboratory for processing.
2. Gently invert the container of aspirate fluid four times, and then aliquot approximately 1.5 mL of the fluid into 2 mL cryovials.
3. Store specimens at -80 °C until batch analysis can be performed.

4. Questionnaire administration and collection of clinical data

1. Ask participants to complete questionnaires to elicit information on diet, demographics, smoking /alcohol habits, medical conditions/medications, and quality of life¹³.
2. Review medical charts to capture clinical data including clinical labs, imaging data, medications, and medical complications.

5. DNA extraction

1. Perform DNA extraction immediately upon removing the duodenal biopsies and aspirate from storage at -80 °C. No preparatory steps are required to extract DNA from duodenal biopsy specimens.
2. Conduct DNA extraction and sequencing for all of the samples on the same day to decrease the risk of a batch effect in analysis. Set up a library specific to the collected specimens before the samples can be sequenced.
3. Use a DNA microprep kit to extract DNA from duodenal biopsy specimens, per the manufacturer’s protocol¹⁴.
4. Add the maximum amount of sample per protocol guidelines to a lysis tube containing 750 µL of lysis solution and a 0.7 mL dry volume of 2 mm glass beads.
5. Secure the lysis tubes in a bead beater with a tube holder and run the beater at maximum speed for at least five minutes. The time will vary based on the bead beater and the sample being analyzed.
6. Centrifuge the lysis tubes in a microcentrifuge at 10,000 x g for 1 min.
7. Transfer up to 400 µL of supernatant through a filtered collection tube and centrifuge again at 8000 x g for 1 min.
8. Add 1200 µL of binding buffer to the filtrate in a microcentrifuge tube and mix thoroughly until homogenous.
9. Transfer 800 µL of the mixture to a spin column and tube assembly. Centrifuge at 10,000 x g for 1 min. Discard the flow-through.
10. Place the spin column on a new centrifuge tube with 400 µL of DNA wash buffer and mix thoroughly until homogenous. Centrifuge at 10,000 x g for 1 min. Discard the flow-through.
11. Repeat step 5.10 two times, with 700 µL and then 200 µL DNA wash buffer per wash.
12. Add 20 µL of elution buffer to the spin column in a new centrifuge tube. Incubate for one minute, then centrifuge at 10,000 x g for 1 min to elute the DNA. Discard the column.
13. Filter the DNA again. The DNA is now ready for PCR amplification.

6. DNA amplification

1. Perform PCR amplification of the V4 region of the 16S ribosomal RNA gene by 250 × 2 paired-end sequencing on aMiSeq, HiSeq or NovaSeq sequencer¹⁵.
2. Create a 96-well plate primer plate. Create a master mix of 165 μL of ILHS_515f conserved forward primer (100 mM stock) and 2970 μL of molecular biology grade water. Add 28.5 μL of this mix to each well.
3. Add 1.5 μL of corresponding reverse primer from the 96 well plate of barcoded IL_806r unique primers (100 μM stock). These barcodes will identify unique patient samples.
4. Create a master mix of water (23.1 μL x 3.3 x number of samples), PCR buffer (3 μL x 3.3 x number of samples), dNTPs (0.6 μL x 3.3 x number of samples), and JumpStart Taq DNA polymerase (0.3 μL x 3.3 x number of samples).
   NOTE: 16S DNA amplification should be performed in triplicate.
5. Add 81 μL of master mix to each well in the first 96 well plate. Add 6 μL of DNA and 3 mL of primer mix to each well. Use a P200 multichannel pipette to mix the wells, then pipette 30 μL into the corresponding well of each of the other two PCR plates. Seal the plate.
6. Use the following thermal cycler settings: 94 °C x 3 min; 35 cycles: 94 °C x 45 s, 50 °C x 1 min, 72 °C x 1.5 min. After 35 cycles: 72 °C x 10 min, 94 °C x 5 min, 4 °C indefinitely.

7. Cleanup and library setup

1. Employ a PCR or DNA cleanup kit to purify the PCR products. Elute the DNA with 30 μL of PCR grade (DEPC-treated) water.
2. Use a UV-visible spectrum spectrophotometer that has the capability to quantify PCR samples.
3. Open the spectrophotometer software on computer desktop. Click on Nucleic Acid.
4. Wipe the port with distilled water. Add 1 μL of 1x PCR buffer/water to port and calibrate.
5. Enter the sample ID and pipette 1 μL of sample onto the port.
6. Close the port and hit Measure.
   NOTE: Avoid air bubbles when adding samples onto the port.
7. Wipe off the sample with lab wipes and distilled water, and then prepare the next sample. Record values with each sample. The computer should also auto-save.
8. Combine 250 ng of each amplified PCR product in 1 tube. This is the DNA library.
9. Deliver the library, along with reads 1 and 2 and the index sequencing primers to a sequencing lab for sequencing.
   NOTE: If a local laboratory is not available, a commercial sequencing lab can be utilized for this step.

8. Data formatting

1. Sequencing data provides results in forward and reverse .fastq files. Convert this data into a data sheet and clean so that the information can be analyzed for downstream applications.
2. Open the R programming software. Follow the instructions and code listed in the supplemental coding file to proceed through the DADA2 pipeline and clean the DNA sequencing data¹⁶. Download the SILVA reference database for the taxonomy assignment after the sequences have been passed through the pipeline¹⁷.
3. Briefly, load the DADA2 package in R. Define a path in which all of the .fastq files are saved in the same directory and extract the files into R.
4. Read the names of the fastq files and match the lists of forward and reverse fastq files.
5. Inspect the quality of the forward reads and reverse reads.
   NOTE: Nucleotide sequences can be truncated to maintain high quality sequences.
6. Filter and trim the fastq files. Use the learnErrors method to learn the parametric error model that DADA2 uses. This will show the observed and expected error rates for each possible nucleotide substitution (A → C, A → T, A → G, etc.).
7. Apply the sample inference algorithm to determine the number of unique sequences in each sample and further inspect the dada-class object to arrive at the number of true unique variants per sample.
8. Merge the forward and reverse reads to obtain complete and denoised sequences.
9. Construct an amplicon sequence variant (ASV) table.
10. Remove chimeras from the ASV table. Chimeric sequences are a single sequence resulting from two more abundant parent sequences.
11. Track the total number of reads that have passed through the pipeline. DADA2 will use SILVA to assign taxonomy to each unique sequence. The accuracy can be analyzed by including a community of known bacteria sequences in the DADA2 pipeline and comparing DADA2 sequence variants to the expected composition of the community.
    NOTE: The DNA sequencing data is now cleaned and ready for analysis based on the specific scope and goals of the research project.

Results

Population differences between mucosal and luminal microbiome of proximal gut
Previous studies have found differences in the microbial populations of luminal and mucosal colon specimens^4,5,18. The preliminary results show that duodenal aspirate and biopsy specimens can measure for both luminal and mucosal microbiota in the proximal gut. Furthermore, we have found that these microbiome populations are disti...

Discussion

Studies of the microbiome are incredibly important, as this complex ecosystem has a critical role in energy homeostasis, immunologic responses, and metabolism¹⁹. Regional microbiome differences exist that may reflect the distinct physiological functions of various regions of the gastrointestinal tract, which may affect different disease states²⁰. The fecal microbiome is most commonly studied but more recently the small intestine microbiome has come under investigation

Materials

Name	Company	Catalog Number	Comments
2 mL cryovials	Corning	430659
96-well plates	Applied Biosystems	4306737
dNTPs	Sigma	D7295
Dry ice			Provided by institution
EG29-i10 endoscope	Pentax	N/A	Endoscope size may vary depending on patient physiology
Epoch microplate spectrophotometer	Biotek	N/A
Ethanol	Sigma Aldrich	676829
HiSeq 2500	Illumina	N/A
IL_806r reverse primer	IDT DNA technologies	custom	custom primers
ILHS_515f forward primer	IDT DNA technologies	custom	custom primers
JumpStart Taq DNA	Sigma	D4184
Mucus specimen trap	Busse Hospital Disposables	405	40 cc specimen trap with transport cap
Nanodrop Gen5 software	ThermoFisher Scientific
PCR buffer	Sigma	P2192
PCR cleanup kit	Zymo Research	D4204
Radial Jaw 4 Jumbo Forceps	Boston Scientific	M00513343	2.8mm Jaw OD
Vioscreen dietary questionnaire	VioCare	N/A
ZymoBIOMICS DNA Microprep Kit	Zymo Research	D4300	25 ug binding capacity