Summary of Study ST001862

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 PR001175. The data can be accessed directly via it's Project DOI: 10.21228/M8TM5F This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST001862
Study Title	Cross-feeding between intestinal pathobionts promotes their overgrowth during undernutrition
Study Summary	Child undernutrition is a global health issue associated with a high burden of infectious disease. Undernourished children display an overabundance of intestinal pathogens and pathobionts, and these bacteria induce enteric dysfunction in undernourished mice; however, the cause of their overgrowth remains poorly defined. Here, we show that disease-inducing human isolates of Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic cross-feeding, resulting in synergistic growth of a mixed community in vitro. Growth synergy occurs uniquely under malnourished conditions limited in protein and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae are strongly correlated in undernourished children, but not in adequately nourished children, consistent with a diet-dependent growth synergy in the human gut. Together these data suggest that dietary cross-feeding fuels the overgrowth of pathobionts in undernutrition.
Institute	University of British Columbia
Department	Michael Smith Laboratories
Last Name	Huus
First Name	Kelsey
Address	3125 East Mall
Email	khuus@msl.ubc.ca
Phone	+1-604-822-2210
Submit Date	2021-07-11
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2021-11-06
Release Version	1

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Project:

Project ID:	PR001175
Project DOI:	doi: 10.21228/M8TM5F
Project Title:	Cross-feeding between intestinal pathobionts promotes their overgrowth during undernutrition
Project Summary:	Child undernutrition is a global health issue associated with a high burden of infectious disease. Undernourished children display an overabundance of intestinal pathogens and pathobionts, and these bacteria induce enteric dysfunction in undernourished mice; however, the cause of their overgrowth remains poorly defined. Here, we show that disease-inducing human isolates of Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic cross-feeding, resulting in synergistic growth of a mixed community in vitro. Growth synergy occurs uniquely under malnourished conditions limited in protein and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae are strongly correlated in undernourished children, but not in adequately nourished children, consistent with a diet-dependent growth synergy in the human gut. Together these data suggest that dietary cross-feeding fuels the overgrowth of pathobionts in undernutrition.
Institute:	University of British Columbia
Department:	Michael Smith Laboratories
Last Name:	Huus
First Name:	Kelsey
Address:	3125 East Mall, Vancouver, British Columbia, V6T 1Z4, Canada
Email:	khuus@msl.ubc.ca
Phone:	+1-604-822-2210

Subject:

Subject ID:	SU001939
Subject Type:	Bacteria
Subject Species:	Bacteroides spp. and Escherichia spp. (mixed communities)
Taxonomy ID:	B. fragilis 3_1_12; B. vulgatus 3_1_40A; B. ovatus 3_8_47; B. dorei 5_1_36; P. distasonis 2_1_33B; E. coli 3_1_53; E. coli 4_1_47

Factors:

Subject type: Bacteria; Subject species: Bacteroides spp. and Escherichia spp. (mixed communities) (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor
SA174375	27_Blank3	0h
SA174376	26_Blank2	0h
SA174377	blank_media1	0h
SA174378	blank_media2	0h
SA174379	blank_media3	0h
SA174380	25_Blank1	0h
SA174381	28_Blank4	0h
SA174382	BO5_16h	16h
SA174383	BO6_16h	16h
SA174384	E4_16h	16h
SA174385	09_E3-16h	16h
SA174386	B6_16h	16h
SA174387	01_B1-16h	16h
SA174388	B4_16h	16h
SA174389	B5_16h	16h
SA174390	E5_16h	16h
SA174391	E6_16h	16h
SA174392	04_BO1-16h	16h
SA174393	03_B3-16h	16h
SA174394	02_B2-16h	16h
SA174395	05_BO2-16h	16h
SA174396	BE6_16h	16h
SA174397	BE4_16h	16h
SA174398	BE5_16h	16h
SA174399	06_BO3-16h	16h
SA174400	BO4_16h	16h
SA174401	07_E1-16h	16h
SA174402	11_BE2-16h	16h
SA174403	12_BE3-16h	16h
SA174404	10_BE1-16h	16h
SA174405	08_E2-16h	16h
SA174406	E4_24h	24h
SA174407	BO6_24h	24h
SA174408	BO5_24h	24h
SA174409	E5_24h	24h
SA174410	BE5_24h	24h
SA174411	13_B1-24h	24h
SA174412	BE6_24h	24h
SA174413	BO4_24h	24h
SA174414	BE4_24h	24h
SA174415	E6_24h	24h
SA174416	B5_24h	24h
SA174417	21_E3-24h	24h
SA174418	20_E2-24h	24h
SA174419	22_BE1-24h	24h
SA174420	23_BE2-24h	24h
SA174421	24_BE3-24h	24h
SA174422	19_E1-24h	24h
SA174423	18_BO3-24h	24h
SA174424	14_B2-24h	24h
SA174425	B4_24h	24h
SA174426	15_B3-24h	24h
SA174427	16_BO1-24h	24h
SA174428	17_BO2-24h	24h
SA174429	B6_24h	24h

Showing results 1 to 55 of 55

Showing results 1 to 55 of 55

Collection:

Collection ID:	CO001932
Collection Summary:	Bacteria were grown anaerobically at 37ºC in MAL-M medium. Culture supernatants at 0, 16 and 24h were collected by centrifugation at 16000 g for 20 minutes. Supernatants were filter sterilized at 0.22 µM and stored at -70ºC before analysis.
Sample Type:	Bacterial cells
Storage Conditions:	Described in summary

Treatment:

Treatment ID:	TR001951
Treatment Summary:	The groups differ by bacterial community composition as follows: B, Bacteroidales mix (B. fragilis, B. vulgatus B. ovatus, B. dorei, P. distasonis); E, E. coli mix (E. coli 3_1_53 and E. coli 4_1_47); BE, Bacteroidales-E.coli mix (all seven strains as above); BO, B. ovatus only. Strains were inoculated in equal proportions based on normalized O.D. from overnight input cultures.

Sample Preparation:

Sampleprep ID:	SP001945
Sampleprep Summary:	Sugars Analysis Low molecular weight sugars and NAc-sugar amines were quantified by LC-MRM/MS at The Metabolomics Innovation Centre (TMIC) commercial service (University of Victoria , Genome BC Proteomics Centre), according to previously published UPLC-MRM/MS methods (Han et al 2016, Electrophoresis), with necessary modifications. In brief, a mixed stock solution of 13 low-MW sugars and 4 N-acetyl sugar amines was prepared with the use of their standard substances in 80% methanol at 500µM for each compound. This solution was then serially diluted with the same solvent to prepare calibration solutions in a concentration range of 0.002 to 125µM. For chemical derivatization, 20µL of each medium sample or each calibration solution was mixed in turn with 20µL of an internal standard solution containing 13C6-glucose, 13C6-mannose, 13C6-fructose and 13C5-ribose in water, 40µL of 200-mM 3-nitrophenylhydrazine hydrochloride solution in 60% methanol and 40µL of 200-mM EDC.HCl solution prepared in a mixed solvent of methanol/water/pyridine (60:40:5, v/v/v). The mixture was allowed to react at 50ºC for 90 min. SCFA Analysis Quantification of short-chain fatty acids was performed in-house according to a method developed by Han et al., with minor modifications(Han et al 2015, Analytica Chimica Acta). Briefly, 500µL of supernatant were mixed with 500µL of 50 % acetonitrile, then the mixture was vortexed for 5 minutes and centrifuged at 7000 x g for 5 minutes. 50 µL of the organic phase were derivatized adding 20µL of 200mM 3NPH in 50 % acetonitrile and 20 µL 120 mM EDC solution of 6 % pyridine in 50 % acetonitrile. The mixture was left under agitation at 40ºC for 30 minutes. After this time reaction was stopped adding 100 µL of 0.1 % formic acid in 90 % acetonitrile.

Sampleprep ID:

SP001945

Sampleprep Summary:

Sugars Analysis Low molecular weight sugars and NAc-sugar amines were quantified by LC-MRM/MS at The Metabolomics Innovation Centre (TMIC) commercial service (University of Victoria , Genome BC Proteomics Centre), according to previously published UPLC-MRM/MS methods (Han et al 2016, Electrophoresis), with necessary modifications. In brief, a mixed stock solution of 13 low-MW sugars and 4 N-acetyl sugar amines was prepared with the use of their standard substances in 80% methanol at 500µM for each compound. This solution was then serially diluted with the same solvent to prepare calibration solutions in a concentration range of 0.002 to 125µM. For chemical derivatization, 20µL of each medium sample or each calibration solution was mixed in turn with 20µL of an internal standard solution containing 13C6-glucose, 13C6-mannose, 13C6-fructose and 13C5-ribose in water, 40µL of 200-mM 3-nitrophenylhydrazine hydrochloride solution in 60% methanol and 40µL of 200-mM EDC.HCl solution prepared in a mixed solvent of methanol/water/pyridine (60:40:5, v/v/v). The mixture was allowed to react at 50ºC for 90 min. SCFA Analysis Quantification of short-chain fatty acids was performed in-house according to a method developed by Han et al., with minor modifications(Han et al 2015, Analytica Chimica Acta). Briefly, 500µL of supernatant were mixed with 500µL of 50 % acetonitrile, then the mixture was vortexed for 5 minutes and centrifuged at 7000 x g for 5 minutes. 50 µL of the organic phase were derivatized adding 20µL of 200mM 3NPH in 50 % acetonitrile and 20 µL 120 mM EDC solution of 6 % pyridine in 50 % acetonitrile. The mixture was left under agitation at 40ºC for 30 minutes. After this time reaction was stopped adding 100 µL of 0.1 % formic acid in 90 % acetonitrile.

Combined analysis:

Analysis ID	AN003018	AN003019
Analysis type	MS	MS
Chromatography type	Normal phase	Reversed phase
Chromatography system	Agilent 1290	Agilent 1200
Column	Phenomenex PFP UPLC (2.1 x 150mm,1.7um)	Agilent Zorbax 300 C18 (250x4.6mm)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6495 QQQ	Agilent 6460 QQQ
Ion Mode	NEGATIVE	POSITIVE
Units	µM	µM

Chromatography:

Chromatography ID:	CH002236
Chromatography Summary:	Sugars Analysis
Methods Filename:	methods_summary_ms.docx
Instrument Name:	Agilent 1290
Column Name:	Phenomenex PFP UPLC (2.1 x 150mm,1.7um)
Chromatography Type:	Normal phase

Chromatography ID:	CH002237
Chromatography Summary:	SCFA Analysis
Methods Filename:	methods_summary_ms.docx
Instrument Name:	Agilent 1200
Column Name:	Agilent Zorbax 300 C18 (250x4.6mm)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002807
Analysis ID:	AN003018
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The raw data was acquired using the Agilent MassHunter® 7.0 software. After data acquisitions, linearly regressed calibration curves of individual compounds were constructed with the analyte-to-internal standard peak area ratios measured from injection of the calibration curves. For those compounds without their isotope-labelling analogues as the internal standards, 13C6-fructose was used a common internal standard. Concentrations of the analytes were calculated by interpolating the calibration curves of individual compounds with their analyte-to-internal standard peak area ratios measured from injection of the sample solutions.
Ion Mode:	NEGATIVE

MS ID:	MS002808
Analysis ID:	AN003019
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	A collision energy of 10V was used for multiple reaction monitoring (MRM), and LC-MS/MS data were analysed by Mass Hunter Qualitative Analysis B.06.00 software (Agilent Technologies). The identification and quantification of the SCFAs were carried out based on the retention time and mass fragmentation pattern comparing with standards. Six-point calibration curves made by peak area vs concentration of the pure standards were used to quantify the different SCFA. The linearity of the curves was determined by the coefficient of determination (R2), being higher than 0.99 for all standards. Concentrations of the SCFAs were calculated by interpolating the calibration curves of individual compounds.
Ion Mode:	POSITIVE