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ABSTRACT: Background
The human small intestine plays a central role in the processes of digestion and nutrient absorption. However, characterizations of the human gut microbiome have largely relied on stool samples, and the associated methodologies are ill-suited for the viscosity and low microbial biomass of small intestine samples. As part of the REIMAGINE study to examine the specific roles of the small bowel microbiome in human health and disease, this study aimed to develop and validate methodologies to optimize microbial analysis of the small intestine.Results
Subjects undergoing esophagogastroduodenoscopy without colon preparation for standard of care were prospectively recruited, and ~?2?ml samples of luminal fluid were obtained from the duodenum using a custom sterile aspiration catheter. Samples of duodenal aspirates were either untreated (DA-U, N =?127) or pretreated with dithiothreitol (DA-DTT, N =?101), then cultured on MacConkey agar for quantitation of aerobic gram-negative bacteria, typically from the class Gammaproteobacteria, and on blood agar for quantitation of anaerobic microorganisms. DA-DTT exhibited 2.86-fold greater anaerobic bacterial counts compared to DA-U (P =?0.0101), but were not statistically different on MacConkey agar. DNA isolation from DA-U (N =?112) and DA-DTT (N =?43) samples and library preparation for 16S rRNA gene sequencing were also performed using modified protocols. DA-DTT samples exhibited 3.81-fold higher DNA concentrations (P =?0.0014) and 4.18-fold higher 16S library concentrations (P ConclusionsThis study validates novel DTT-based methodology which optimizes microbial culture and 16S rRNA gene sequencing for the study of the small bowel microbiome. The microbial analyses indicate increased isolation of facultative and obligate anaerobes from the mucus layer using these novel techniques.
SUBMITTER: Leite GGS
PROVIDER: S-EPMC6824053 | biostudies-literature | 2019 Nov
REPOSITORIES: biostudies-literature
Leite Gabriela Guimaraes Sousa GGS Morales Walter W Weitsman Stacy S Celly Shreya S Parodi Gonzalo G Mathur Ruchi R Sedighi Rashin R Barlow Gillian M GM Rezaie Ali A Pimentel Mark M
BMC microbiology 20191101 1
<h4>Background</h4>The human small intestine plays a central role in the processes of digestion and nutrient absorption. However, characterizations of the human gut microbiome have largely relied on stool samples, and the associated methodologies are ill-suited for the viscosity and low microbial biomass of small intestine samples. As part of the REIMAGINE study to examine the specific roles of the small bowel microbiome in human health and disease, this study aimed to develop and validate metho ...[more]