In a recent article published in Nature, researchers developed an ingestible, single-use device to collect samples from different regions of the human intestine, including the duodenum, jejunum, ileum, and colon, during normal digestion.
The device has an expanding collapsible bladder capped by a one-way valve within a dissolvable capsule with an enteric coating.
Study: Profiling the human intestinal environment under physiological conditions. Image Credit: sdecoret/Shutterstock.com
Introduction
Since the human intestinal tract has four distinct regions, researchers asked the study participants to ingest four devices (as a set) after a meal. Each device opened at progressively higher pH levels, enabling luminal contents to be withdrawn through the one-way valve.
Note that the pH of the intestines rises from four to six in the duodenum up to eight in the ileum, and bacterial density and metabolites of interest are higher in the intestinal lumen than mucosa. Each device retrieved up to 400 µl of luminal contents, sufficient for multi-omic or ex vivo analyses.
Background
Studies of the human gut microbiome and metabolites have relied mainly on stool samples, which primarily contain waste products and downstream effluent. It does not provide a whole or accurate picture of the spatiotemporal and regional heterogeneity of the gut and its impact on local physiology.
Each gut region distal to the stomach varies profoundly in terms of nutrient availability, oxygen partial pressure, mucosal structure, flow rate, and pH, as described above, all of which influence their microbial composition, host proteome, pathogen activity, and bile acid content.
Each microbial community in the gut has specialized functions, metabolomes, immune niches, and proteomes.
A local sampling of gut microorganisms in natural, unperturbed states across spatial scales (each inch of the intestine) could uncover the secret of how they impact human physiology and vice versa.
All previous works used organ donors for these samples, typically treated with antibiotics, which often turn their gut ischaemic or necrotic, or did upper endoscopy of live individuals, which inadvertently contaminated their duodenal contents with oral, esophageal, or gastric contents. Previously used ingestible devices for sampling also had limitations, such as the inability to retrieve sufficient sampling volume for multi-omics analyses.
Given the dynamic environment of the human gut, there is a need for increased sampling from larger cohorts of healthy volunteers to enable robust assessments of differences in regional spatiotemporal variability and overall microbiota composition.
Additionally, there is a need to investigate how diet and disease affect the intestinal microbiota, metabolome, virome, and proteome to inform future clinical studies evaluating therapeutic interventions for gut disorders, e.g., inflammatory bowel disease (IBD).
About the study
The present study cohort comprised 15 healthy individuals, each of whom ingested at least 17 devices in three days. First, the researchers performed a feasibility study to visualize successful in vivo sampling in a human and confirm whether the device targeted expected intestinal regions and progressed through the intestinal tract without contamination.
Remarkably, the team retrieved all ingested devices, and no adverse events occurred during the study. Of 255 ingested devices, 22 contained gas or low sample volume; thus, they discarded those devices.
The final analysis set comprised 306 samples, of which 29, 218, and 59 were saliva, devices, and stool samples, respectively. These samples provided enough depth for 16S ribosomal ribonucleic acid (rRNA) gene sequencing.
Additionally, one participant provided samples for assessment of replicability and blooming analysis. For this, the team incubated all four devices retrieved from a single bowel movement in an anaerobic chamber at 37 °C for up to 87 hours.
Within an incubation time of ~58 hours, no notable changes occurred in the microbiota composition of any of the four devices.
In this limited experimental paradigm, the authors demonstrated that gut microbiota and metabolome exhibited longitudinal gradients – highly distinct from stool samples.
Study findings
Through multi-omics analysis, the researchers identified marked differences between bacteria, phages, host proteins, and metabolites in the intestines vs. stool.
For example, the Proteobacteria phylum, including a Bilophila wadsworthia amplicon sequence variant (ASV), was relatively more abundant in the intestines than in stool. Four other ASVs from the Escherichia/Shigella, Bacteroides, Enterococcus, and Romboutsia genera, were also more abundant in intestines than in stool.
Intra-individual microbiota variability was also higher in intestinal samples than in stool or saliva samples, confirming that the study devices collected a far more heterogeneous habitat.
Remarkably, this device preserved live viable bacteria to the same degree as a fresh stool. The team also recovered growing cells with different morphologies, perhaps, epithelial cells, from the device. Thus, this device could enable culturomics experiments to examine host cells in the intestinal lumen.
Metagenomic sequencing on all devices uncovered genetic variations across the human intestinal tract. The percentage of reads mapped to carbohydrate-active enzyme (CAZymes) gene abundance in devices displayed more variance than in stool.
The authors noted a positive correlation of CAZymes abundance with the relative abundance of five ASVs, two unnamed and two Bacteroides vulgatus species, and Parabacteroides merdae.
The metagenomics dataset also showed that prophage induction was more prevalent in the gut. Though viromes in stool and intestinal samples from the same participant were more similar than between stool or intestinal samples from different participants, their principal coordinate analysis (PCoA) showed comparable clustering as the microbiota qualitatively. Intriguingly, many induced prophages were unique to intestinal samples.
The host proteome along the intestines was highly distinct from stool, as assessed by liquid chromatography (LC) followed by tandem mass spectrometry (MS). Protein abundance varied most significantly between stool and type 1 device, suggesting longitudinal variation of the host proteome.
Bile acids, key chemical components of the human gut, are critical regulators of diverse physiological processes. Nearly 95% of bile acids reach the distal ileum, where bile salt hydrolases (BSHs), type of microbial enzymes, deconjugated their glycine or taurine or removed their hydroxyl group(s) from the steroid backbone.
Then, the gut transported these microbially transformed bile acids back to the liver, raising the possibility of longitudinal bile acid gradients along the human gut.
The team investigated 17 bile acids in stool and intestinal samples using LC–MS/MS metabolomics with multiple-reaction monitoring (MRM), which revealed highly variable bile acid concentrations in intestinal samples.
The authors noted a two-fold reduction in the total concentration of bile acids collected by type 4 devices than by type 1 devices, indicating reabsorption of bile acids along the intestinal tract.
Moreover, the dominant bile acid in intestinal samples was cholic acid (CA), whereas the secondary bile acid deoxycholic acid (DCA) was predominant in stool samples.
The authors noted a marked monotonic reduction in the percentage of liver-conjugated bile acids in samples from device type one to four, indicating a deconjugation trend along the gut and into the stool. Furthermore, microbially deconjugated bile acids displayed amino acid-dependent trends not observed in the stool samples.
Conclusions
The study demonstrated the utility of a safe and non-invasive device for retrieval and quantification of the luminal contents of the human gut, comprising microbiota, host metabolome, proteins, and bile acids during normal digestion.
When deployed at scale, this device could help get insights into the dynamicity of human metabolic pathways through which gut microorganisms influence various physiological states and disease conditions.
