Summary of Study ST004443
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002806. The data can be accessed directly via it's Project DOI: 10.21228/M8ZC3H This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004443 |
| Study Title | Profiling the small intestinal microbiota in vitro using 4 donors |
| Study Summary | Although there is clear evidence demonstrating the importance of the small intestinal microbiota (SIM) for nutrient utilization within the upper gastrointestinal tract, research is limited by difficulties accessing this community in vivo. Additionally, the high level of interindividual variability in taxonomic structure, which is well documented for the SIM, raises the question of how such divergent communities fill the same physiological roles. Here, we designed and evaluated an in vitro model of the terminal ileum representative of four unique donors and utilized it to interrogate interindividual variability. Shotgun sequencing was used to evaluate the in vitro community structure and untargeted metabolomics used to examine community function. |
| Institute | United States Department of Agriculture |
| Department | Agricultural Research Service |
| Last Name | Narrowe |
| First Name | Adrienne |
| Address | 600 East Mermaid Lane Wyndmoor, PA 19038, Wyndmoor, Pennsylvania, 19038, USA |
| adrienne.narrowe@usda.gov | |
| Phone | 2158366926 |
| Submit Date | 2025-12-03 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2026-01-21 |
| Release Version | 1 |
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Project:
| Project ID: | PR002806 |
| Project DOI: | doi: 10.21228/M8ZC3H |
| Project Title: | Profiling the small intestinal microbiota in vitro using 4 donors |
| Project Summary: | The small intestinal microbiota (SIM) has been proven critical for nutrient utilization within the upper gastrointestinal tract. Currently, research is limited due to 1) difficulties accessing this community in vivo and 2) the high level of interindividual variability in taxonomic structure. Here, an in vitro model of the terminal ileum representative of four unique donors was developed and then used to evaluate interindividual variability using Shotgun sequencing and untargeted metabolomics used to examine community function. The results of Shotgun sequencing showed that, in vitro, the communities were representative of their specific inocula and composed of taxa typical of the SIM, such as Klebsiella, Escherichia, Streptococcus, and Enterococcus. Untargeted metabolomics was used to generate metabolic profiles and found that there was a high degree of similarity between the different tested communities in terms of which metabolites were produced. A core set of genes, features, and metabolites were identified by combing the results from sequencing and metabolomics that were shared across all communities. Together, these results found that despite variability in the taxonomic structure of the SIM, there were similarities in functional outcome due to underlying shared gene representation. |
| Institute: | United States Department of Agriculture |
| Department: | Agricultural Research Service |
| Last Name: | Narrowe |
| First Name: | Adrienne |
| Address: | 600 East Mermaid Lane Wyndmoor, PA 19038, Wyndmoor, Pennsylvania, 19038, USA |
| Email: | adrienne.narrowe@usda.gov |
| Phone: | 2158366926 |
Subject:
| Subject ID: | SU004605 |
| Subject Type: | Other organism |
| Subject Species: | Human gut microbiome |
Factors:
Subject type: Other organism; Subject species: Human gut microbiome (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Day | Unit | Donor | Collection date |
|---|---|---|---|---|---|---|
| SA526416 | QC3 | pooled samples | N/A | N/A | N/A | Apr-24 |
| SA526417 | QC2 | pooled samples | N/A | N/A | N/A | Apr-24 |
| SA526418 | QC1 | pooled samples | N/A | N/A | N/A | Apr-24 |
| SA526419 | 35media | small intestine microbiome incubation | 0 | Brain heart infused media - modified | N/A | Jan-23 |
| SA526420 | 36pj | small intestine microbiome incubation | 0 | Pancreatic juice | N/A | Jan-23 |
| SA526421 | 31 D10 U1 | small intestine microbiome incubation | 10 | SIM1 | 1 | Feb-23 |
| SA526422 | 32 D10 U2 | small intestine microbiome incubation | 10 | SIM2 | 2 | Feb-23 |
| SA526423 | 33 D10 U3 | small intestine microbiome incubation | 10 | SIM3 | 3 | Feb-23 |
| SA526424 | 34 D10 U4 | small intestine microbiome incubation | 10 | SIM4 | 4 | Feb-23 |
| SA526425 | 19 D5 U1 | small intestine microbiome incubation | 5 | SIM1 | 1 | Jan-23 |
| SA526426 | 20 D5 U2 | small intestine microbiome incubation | 5 | SIM2 | 2 | Jan-23 |
| SA526427 | 21 D5 U3 | small intestine microbiome incubation | 5 | SIM3 | 3 | Jan-23 |
| SA526428 | 22 D5 U4 | small intestine microbiome incubation | 5 | SIM4 | 4 | Jan-23 |
| SA526429 | 23 D6 U1 | small intestine microbiome incubation | 6 | SIM1 | 1 | Jan-23 |
| SA526430 | 24 D6 U2 | small intestine microbiome incubation | 6 | SIM2 | 2 | Jan-23 |
| SA526431 | 25 D6 U3 | small intestine microbiome incubation | 6 | SIM3 | 3 | Jan-23 |
| SA526432 | 26 D6 U4 | small intestine microbiome incubation | 6 | SIM4 | 4 | Jan-23 |
| SA526433 | 27 D7 U1 | small intestine microbiome incubation | 7 | SIM1 | 1 | Jan-23 |
| SA526434 | 28 D7 U2 | small intestine microbiome incubation | 7 | SIM2 | 2 | Jan-23 |
| SA526435 | 29 D7 U3 | small intestine microbiome incubation | 7 | SIM3 | 3 | Jan-23 |
| SA526436 | 30 D7 U4 | small intestine microbiome incubation | 7 | SIM4 | 4 | Jan-23 |
| Showing results 1 to 21 of 21 |
Collection:
| Collection ID: | CO004598 |
| Collection Summary: | The SIM was cultured using a bioreactor system (SHIME®, ProDigest, Belgium). Four glass bioreactors were connected to a source of fresh modified brain heart infused media (M-BHI), pancreatic juice (PJ), and waste collection. Acid (0.5M HCl) and base (0.5M NaOH) maintained pH at 7.2 ± 0.1 and gas provided continuously (5% oxygen, 95% nitrogen balance) at a flow rate of 5-10 mL/min. Ileostomy inoculums were cultures of ileostomy effluent harvested from healthy adult volunteers enrolled in a crossover feeding study (Enteral Nutritional Therapy and the Gut Microbiota and their Metabolites in Ileostomy Contents, EMMI, IRB 825368) with a diverting or end ileostomy for an indication other than Crohn’s disease. Each bioreactor was inoculated with an ileostomy inoculum from a single donor. After overnight growth, feeding cycles were initiated recapitulating a 4 hour transit time.12, 22 Samples were harvested from each bioreactor on days 1, 2, 3, 4, 5, 6, 7, and 10 and centrifuged at 5,000 x g for 10 mins at 4 °C. For metabolomics, a 15 mL volume of supernatant was filter-sterilized using a 0.22 µM PES filter and aliquoted. All samples were frozen at -80 °C. |
| Sample Type: | Small intestine microbiome incubation |
Treatment:
| Treatment ID: | TR004614 |
| Treatment Summary: | No treatment |
Sample Preparation:
| Sampleprep ID: | SP004611 |
| Sampleprep Summary: | Each sample was thawed and 1 mL of sample transferred into tube and lyophilized, adding 500 μL 80% methanol, vortexed 30 s and followed by sonication in ice-water bath for 30 min. Then sample was kept at -20°C for 1 h, vortexed for 30 s, and centrifuge 10 min at 12,000 rpm, 4°C. Finally, transfer 200 μL of supernatant and add 5 μL of 0.14 mg/mL DL-o- Chlorophenylalanine into vial, filtered through a 0.22 μm filter for LC-MS analysis. Pooled QC samples were generated by combining equal aliquots taken from each individual sample. The QC sample was injected once for every ten study sample injections to monitor and ensure instrument stability and analytical reproducibility throughout the run. |
Combined analysis:
| Analysis ID | AN007440 | AN007441 |
|---|---|---|
| Chromatography ID | CH005633 | CH005633 |
| MS ID | MS007132 | MS007133 |
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH005633 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| Column Temperature: | 40 |
| Flow Gradient: | 0-1 min, 5% B; 1-12.5 min, 5%-95% B; 12.5-13.5 min, 95% B; 13.5-13.6 min, 95%-5% B; 13.6-16 min, 5% B |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 100% water; 0.05% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS007132 |
| Analysis ID: | AN007440 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Heater Temp 300°C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 KV; Capillary Temp, 350°C; S-Lens RF Level, 30% |
| Ion Mode: | POSITIVE |
| MS ID: | MS007133 |
| Analysis ID: | AN007441 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Heater Temp 300°C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 KV; Capillary Temp,350°C; S-Lens RF Level, 60% |
| Ion Mode: | NEGATIVE |
