Abstract
BackgroundThe 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.ResultsSubjects 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 < 0.0001) then DA-U samples. 16S rRNA gene sequencing revealed increases in the detected relative abundances of obligate and facultative anaerobes in DA-DTT samples, including increases in the genera Clostridium (false discovery rate (FDR) P = 4.38E-6), Enterococcus (FDR P = 2.57E-8), Fusobacterium (FDR P = 0.02) and Bacteroides (FDR P = 5.43E-9). Detected levels of Gram-negative enteropathogens from the phylum Proteobacteria, such as Klebsiella (FDR P = 2.73E-6) and Providencia (FDR P < 0.0001) (family Enterobacteriaceae) and Pseudomonas (family Pseudomonadaceae) (FDR P = 0.04), were also increased in DA-DTT samples.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.
Highlights
The human small intestine plays a central role in the processes of digestion and nutrient absorption
127 duodenal aspirates (DA) were not pretreated with DTT prior to microbial culture (DA-U, Fig. 1 Workflow for pretreatment and microbial culture, including the number of subjects in each group the untreated group), and 101 were pretreated with DTT prior to microbial culture
For the purposes of statistical analysis only, no growth was designated as 1 bacterial colony-forming units (CFU)/mL of aspirate
Summary
The human small intestine plays a central role in the processes of digestion and nutrient absorption. Stool sequencing may reveal the microbial signature of the distal colon, it is well known that stool does not adequately represent the entire gastrointestinal tract, given the multiple environments that exist as one travels from the stomach to the small bowel and the colon [3]. Conditions such as acidity and transit time vary tremendously along the intestine with likely significant effects on microbes by area of exam [3]. Characterizing the microbial populations of the small intestine is of central importance, but efforts to date have been hampered both by the difficulty of obtaining samples, and by challenges associated with adapting sample processing for DNA isolation techniques that were designed for stool
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