Optimizing microbiome sequencing for small intestinal aspirates: validation of novel techniques through the REIMAGINE study.
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 < 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.


CONCLUSIONS
This 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.
['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|0000-0002-0619-5115']
D000328:Adult D000368:Aged D000369:Aged, 80 and over D001419:Bacteria D058491:Bacterial Load D004269:DNA, Bacterial D004275:DNA, Ribosomal D004229:Dithiothreitol D016145:Endoscopy, Digestive System D005260:Female D000069196:Gastrointestinal Microbiome D006801:Humans D007421:Intestine, Small D008297:Male D008875:Middle Aged D010802:Phylogeny D011446:Prospective Studies D012336:RNA, Ribosomal, 16S D017422:Sequence Analysis, DNA D055815:Young Adult
Microbiome Small intestine 16S rRNA gene sequencing Microbial culture Mucus layer Methodology optimization