Summary of Study ST001823

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 PR001152. The data can be accessed directly via it's Project DOI: 10.21228/M8ST22 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001823
Study TitleAlterations in the fecal microbiome and metabolome of horses with antimicrobial-associated diarrhea compared to antibiotic-treated and non-treated healthy case controls
Study SummaryHorses receiving antimicrobials may develop diarrhea due to changes in the gastrointestinal microbiome and metabolome. This matched, case-controlled study compared the fecal microbiome and metabolome in hospitalized horses on antibiotics that developed diarrhea (AAD), hospitalized horses on antibiotics that did not develop diarrhea (ABX) and a healthy, non-hospitalized control population (CON). Naturally-voided fecal samples were collected from AAD horses (n=17) the day that diarrhea developed and matched to ABX (n=15) and CON (n=31) horses for diet, antimicrobial agent and duration of antimicrobial therapy (< 5 days or > 5 days). Illumina sequencing of 16S rRNA genes on fecal DNA was performed. Alpha and beta diversity metrics were generated using QIIME 2.0. A Kruskal-Wallis with Dunn’s post-test and ANOSIM testing was used for statistical analysis. Microbiome composition in AAD was significantly different from CON (ANOSIM, R= 0.568, p=0.001) and ABX (ANOSIM, R=0.121, p=0.0012). Fecal samples were lyophilized and extracted using a solvent-based method. Untargeted metabolomics using gas chromatography-mass spectrometry platforms was performed. Metabolomic data was analyzed using Metaboanalyst 4.0 and Graphpad Prism v 7. Principal component analysis plots (PCA) were used to visualize the distribution of metabolites between groups. Heat maps were used to identify the relative concentrations amongst the most abundant 25 metabolites. A one-way ANOVA was used to compare differences in metabolites amongst the three groups of horses. Only named metabolites were included in the analysis. The microbiome of AAD and ABX horses had significantly decreased richness and evenness than CON horses (p<0.05). Actinobacteria (q=0.0192) and Bacteroidetes (q=0.0005) were different between AAD and CON. Verrucomicrobia was markedly decreased in AAD compared to ABX and CON horses (q=0.0005). Horses with AAD have a dysbiosis compared to CON horses, and show minor differences in bacterial community composition to ABX horses. Metabolite profiles of horses with AAD clustered separately from those with AAD or CON. Ten metabolites were found to be significantly different between groups (P<0.05) and are listed according to their metabolic pathway: amino acid metabolism (R-equol, L-tyrosine, kynurenic acid, xanthurenic acid, 5-hydroxyindole-3-acetic acid ) lipid metabolism (docosahexaenoic acid ethyl ester), biosynthesis of secondary metabolites (daidzein, isoquinoline) and two metabolites with unidentified pathways (1,3-divinyl-2-imidazolidinone, N-acetyltyramine).
Institute
Texas A&M University
Last NameArnold
First NameCarolyn
Address4475 TAMU College of Veterinary Medicine and Biomedical Sciences College Station, Texas 77843-4475
Emailcarnold@cvm.tamu.edu
Phone979-412-3145
Submit Date2021-03-10
Analysis Type DetailLC-MS
Release Date2021-09-10
Release Version1
Carolyn Arnold Carolyn Arnold
https://dx.doi.org/10.21228/M8ST22
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001152
Project DOI:doi: 10.21228/M8ST22
Project Title:Alterations in the fecal microbiome and metabolome of horses with antimicrobial-associated diarrhea compared to antibiotic-treated and non-treated healthy case controls
Project Summary:Horses receiving antimicrobials may develop diarrhea due to changes in the gastrointestinal microbiome and metabolome. This matched, case-controlled study compared the fecal microbiome and metabolome in hospitalized horses on antibiotics that developed diarrhea (AAD), hospitalized horses on antibiotics that did not develop diarrhea (ABX) and a healthy, non-hospitalized control population (CON). Naturally-voided fecal samples were collected from AAD horses (n=17) the day that diarrhea developed and matched to ABX (n=15) and CON (n=31) horses for diet, antimicrobial agent and duration of antimicrobial therapy (< 5 days or > 5 days). Illumina sequencing of 16S rRNA genes on fecal DNA was performed. Alpha and beta diversity metrics were generated using QIIME 2.0. A Kruskal-Wallis with Dunn’s post-test and ANOSIM testing was used for statistical analysis. Microbiome composition in AAD was significantly different from CON (ANOSIM, R= 0.568, p=0.001) and ABX (ANOSIM, R=0.121, p=0.0012). Fecal samples were lyophilized and extracted using a solvent-based method. Untargeted metabolomics using gas chromatography-mass spectrometry platforms was performed. Metabolomic data was analyzed using Metaboanalyst 4.0 and Graphpad Prism v 7. Principal component analysis plots (PCA) were used to visualize the distribution of metabolites between groups. Heat maps were used to identify the relative concentrations amongst the most abundant 25 metabolites. A one-way ANOVA was used to compare differences in metabolites amongst the three groups of horses. Only named metabolites were included in the analysis. The microbiome of AAD and ABX horses had significantly decreased richness and evenness than CON horses (p<0.05). Actinobacteria (q=0.0192) and Bacteroidetes (q=0.0005) were different between AAD and CON. Verrucomicrobia was markedly decreased in AAD compared to ABX and CON horses (q=0.0005). Horses with AAD have a dysbiosis compared to CON horses, and show minor differences in bacterial community composition to ABX horses. Metabolite profiles of horses with AAD clustered separately from those with AAD or CON. Ten metabolites were found to be significantly different between groups (P<0.05) and are listed according to their metabolic pathway: amino acid metabolism (R-equol, L-tyrosine, kynurenic acid, xanthurenic acid, 5-hydroxyindole-3-acetic acid ) lipid metabolism (docosahexaenoic acid ethyl ester), biosynthesis of secondary metabolites (daidzein, isoquinoline) and two metabolites with unidentified pathways (1,3-divinyl-2-imidazolidinone, N-acetyltyramine).
Institute:Texas A&M University
Department:Department of Large Animal Clinical Sciences
Last Name:Arnold
First Name:Carolyn
Address:4475 TAMU College of Veterinary Medicine and Biomedical Sciences College Station, Texas 77843-4475
Email:carnold@cvm.tamu.edu
Phone:979-412-3145

Subject:

Subject ID:SU001900
Subject Type:Mammal
Subject Species:Equus caballus
Taxonomy ID:9796

Factors:

Subject type: Mammal; Subject species: Equus caballus (Factor headings shown in green)

mb_sample_id local_sample_id Group
SA16917151AAD
SA16917227AAD
SA16917355AAD
SA16917431AAD
SA16917518AAD
SA16917635AAD
SA16917743AAD
SA16917847AAD
SA16917939AAD
SA16918017AAD
SA1691811AAD
SA16918222AAD
SA16918359AAD
SA1691849AAD
SA16918513AAD
SA1691865AAD
SA16918732ABX
SA16918844ABX
SA16918914ABX
SA1691906ABX
SA16919136ABX
SA16919240ABX
SA16919328ABX
SA16919448ABX
SA1691952ABX
SA16919652ABX
SA16919760ABX
SA16919824ABX
SA16919910ABX
SA16920056ABX
SA16920119ABX
SA16920263CON
SA16920358CON
SA16920457CON
SA16920546CON
SA16920645CON
SA16920761CON
SA16920853CON
SA16920954CON
SA16921062CON
SA16921150CON
SA16921249CON
SA16921333CON
SA16921412CON
SA16921515CON
SA16921616CON
SA16921711CON
SA1692188CON
SA1692193CON
SA1692204CON
SA1692217CON
SA16922220CON
SA16922321CON
SA16922437CON
SA16922538CON
SA16922641CON
SA16922734CON
SA16922830CON
SA16922925CON
SA16923026CON
SA16923129CON
SA16923242CON
Showing results 1 to 62 of 62

Collection:

Collection ID:CO001893
Collection Summary:Fecal samples were collected from horses that were matched for diet and antimicrobial agent (including dose, route and duration of therapy).
Collection Protocol Filename:Collection_protocol.docx
Sample Type:Feces
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001913
Treatment Summary:Horses were prescribed antimicrobials as prophylaxis before elective surgery (excluding surgery of the gastrointestinal tract including colic) or to treat a suspected or confirmed infection.

Sample Preparation:

Sampleprep ID:SP001906
Sampleprep Summary:Five-hundred mg of feces was aliquoted into a 2ml tube, lyophilized overnight and vortexed with a 5mm stainless steel bead (Quiagen, Germantown, MD ) for 5 minutes. Samples were then extracted using a methanol:chloroform:water-based extraction method. Briefly 800 uL ice cold methanol:chloroform (1:1, v:v) was added to samples in a bead-based lysis tube (Bertin, Rockville, MD). Samples were homogenized for 30 seconds on a Precyllys 24 (Bertin, Rockville, MD) at a speed of 6000. The supernatant was collected and samples were homogenized a second time with 800 uL ice methanol:chloroform. 600 uL ice cold water was added to the combined extract, vortexed and centrifuged to separate the phases. The upper aqueous layer was passed through a 0.2 um nylon filter (Merck Millipore, Burlington, MA). 500 uL of the filtered aqueous phase was then passed through a 3 kDa cutoff column (Thermo Scientific, Waltham, MA) and the flow through was collected for analysis.

Combined analysis:

Analysis ID AN002959
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Xcalibur
Column Phenomenex Synergi Fusion (150mm x 2mm, 4um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode POSITIVE
Units peak intensity

Chromatography:

Chromatography ID:CH002192
Chromatography Summary:Samples were maintained at 4 °C before injection. The injection volume was 10 µL. Chromatographic separation was achieved on a Synergi Fusion 4µm, 150 mm x 2 mm reverse phase column (Phenomenex, Torrance, CA) maintained at 30 °C using a solvent gradient method. Solvent A was water (0.1% formic acid). Solvent B was methanol (0.1% formic acid). The gradient method used was 0-5 min (10% B to 40% B), 5-7 min (40% B to 95% B), 7-9 min (95% B), 9-9.1 min (95% B to 10% B), 9.1-13 min (10% B). The flow rate was 0.4 mL min-1.
Instrument Name:Thermo Xcalibur
Column Name:Phenomenex Synergi Fusion (150mm x 2mm, 4um)
Chromatography Type:Reversed phase

MS:

MS ID:MS002749
Analysis ID:AN002959
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Sample acquisition was performed Xcalibur (Thermo Scientific).
Ion Mode:POSITIVE
Analysis Protocol File:MS_protocol.docx
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