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 Approach subjects with a …

Representative 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 specimens4,5,18. 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<sup c…

Materials

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