#METABOLOMICS WORKBENCH cbeekman_20240117_122530 DATATRACK_ID:4592 STUDY_ID:ST003141 ANALYSIS_ID:AN005154 VERSION 1 CREATED_ON 03-27-2024 #PROJECT PR:PROJECT_TITLE Spatial analysis of murine GI reveals role of small intestinal bile acid PR:PROJECT_TITLE metabolism in amoxicillin-induced dysbiosis PR:PROJECT_SUMMARY Antibiotics cause collateral damage to resident microbes that is associated with PR:PROJECT_SUMMARY various health risks. To-date, studies have largely focused on impacts of PR:PROJECT_SUMMARY antibiotics on large intestinal and fecal microbiota. Here, we employ a GI-wide PR:PROJECT_SUMMARY integrated multiomic approach to reveal that amoxicillin (AMX) treatment reduces PR:PROJECT_SUMMARY overall bacterial abundance, bile salt hydrolase activity and unconjugated bile PR:PROJECT_SUMMARY acids in the small intestine (SI). An accompanying loss of fatty acids and PR:PROJECT_SUMMARY increase in acyl-carnitines in the large intestine corresponded with PR:PROJECT_SUMMARY spatially-distinct expansions of proteobacteria. Parasutterella PR:PROJECT_SUMMARY excrementihominis utilized fatty acid biosynthesis, becoming dominant in the SI PR:PROJECT_SUMMARY while multiple Klebsiella species employed fatty acid oxidation during expansion PR:PROJECT_SUMMARY in the large intestine. Depletion of bile acids and lipids may contribute to PR:PROJECT_SUMMARY AMX-induced dysbiosis in the lower GI. To test this, we demonstrate that PR:PROJECT_SUMMARY restoration of unconjugated bile acids can mitigate losses of commensals in the PR:PROJECT_SUMMARY large intestine while also inhibiting the expansion of Proteobacteria during AMX PR:PROJECT_SUMMARY treatment. PR:INSTITUTE Brown University PR:DEPARTMENT MMI PR:LABORATORY MMI, Belenky Lab PR:LAST_NAME Beekman PR:FIRST_NAME Chapman PR:ADDRESS BMC 613, 171 Meeting Street, Providence RI 02912 PR:EMAIL Chapman_Beekman@brown.edu PR:PHONE 4012071832 PR:DOI http://dx.doi.org/10.21228/M8DD95 #STUDY ST:STUDY_TITLE untargeted metabolomic analysis of whole blood from AMX-treated and untreated ST:STUDY_TITLE mice ST:STUDY_SUMMARY untargeted metabolomic analysis of whole blood from AMX-treated and untreated ST:STUDY_SUMMARY mice ST:INSTITUTE Brown University ST:LAST_NAME Beekman ST:FIRST_NAME Chapman ST:ADDRESS 171 Meeting Street, Providence, Rhode Island, 02912, USA ST:EMAIL Chapman_Beekman@brown.edu ST:PHONE 4012071832 ST:SUBMIT_DATE 2024-01-17 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Mouse1 CB73 Treatment:Amox GI_site=Blood; plate_nr=SPB83Z; position=H1 SUBJECT_SAMPLE_FACTORS Mouse2 CB74 Treatment:Amox GI_site=Blood; plate_nr=SPB83Z; position=H2 SUBJECT_SAMPLE_FACTORS Mouse3 CB75 Treatment:Amox GI_site=Blood; plate_nr=SPB83Z; position=H3 SUBJECT_SAMPLE_FACTORS Mouse7 CB79 Treatment:Amox GI_site=Blood; plate_nr=SPB83Z; position=H7 SUBJECT_SAMPLE_FACTORS Mouse8 CB80 Treatment:Amox GI_site=Blood; plate_nr=SPB83Z; position=H8 SUBJECT_SAMPLE_FACTORS Mouse9 CB81 Treatment:Amox GI_site=Blood; plate_nr=SPB83Z; position=H9 SUBJECT_SAMPLE_FACTORS Mouse4 CB76 Treatment:Control GI_site=Blood; plate_nr=SPB83Z; position=H4 SUBJECT_SAMPLE_FACTORS Mouse5 CB77 Treatment:Control GI_site=Blood; plate_nr=SPB83Z; position=H5 SUBJECT_SAMPLE_FACTORS Mouse6 CB78 Treatment:Control GI_site=Blood; plate_nr=SPB83Z; position=H6 SUBJECT_SAMPLE_FACTORS Mouse10 CB82 Treatment:Control GI_site=Blood; plate_nr=SPB83Z; position=H10 SUBJECT_SAMPLE_FACTORS Mouse11 CB83 Treatment:Control GI_site=Blood; plate_nr=SPB83Z; position=H11 SUBJECT_SAMPLE_FACTORS Mouse12 CB84 Treatment:Control GI_site=Blood; plate_nr=SPB83Z; position=H12 #COLLECTION CO:COLLECTION_SUMMARY Lumenal contents collected from multiple GI sites and immediately flash frozen CO:SAMPLE_TYPE Blood (whole) #TREATMENT TR:TREATMENT_SUMMARY Mice were treated for 24h with amoxicillin/vehicle in drinking water TR:TREATMENT_COMPOUND Amoxicillin TR:TREATMENT_ROUTE oral #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen GI contents were lyophilized and 10mg of dry contents were extracted SP:SAMPLEPREP_SUMMARY twice in 300µL in (2:1) acetone and isopropanol. Samples were maintained on dry SP:SAMPLEPREP_SUMMARY ice during extraction steps. Extracts were diluted 1:10 in a solution of 80% SP:SAMPLEPREP_SUMMARY methanol in water. All solvents were LC-MS grade purchased from Fisher SP:SAMPLEPREP_SUMMARY Scientific. #CHROMATOGRAPHY CH:INSTRUMENT_NAME none CH:COLUMN_NAME none CH:COLUMN_TEMPERATURE na CH:FLOW_GRADIENT none CH:FLOW_RATE na CH:SOLVENT_A none CH:SOLVENT_B none CH:CHROMATOGRAPHY_TYPE None (Direct infusion) #ANALYSIS AN:LABORATORY_NAME General Metabolics AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS A pooled study sample (pSS) was prepared by combining 5 µL of each sample. The MS:MS_COMMENTS pSS was injected every ten samples during data acquisition. Instrumental MS:MS_COMMENTS Analysis Metabolome profiles of the sample extracts were acquired using MS:MS_COMMENTS flow-injection mass spectrometry. The method described here is adapted from MS:MS_COMMENTS Fuhrer et al 2011. The instrumentation consisted of an Agilent 6550 iFunnel MS:MS_COMMENTS LC-MS Q-TOF mass spectrometer in tandem with an MPS3 autosampler (Gerstel) and MS:MS_COMMENTS an Agilent 1260 Infinity II quaternary pump. The running buffer was 60% MS:MS_COMMENTS isopropanol in water (v/v) with 1 mM ammonium fluoride. Hexakis (1H, 1H, MS:MS_COMMENTS 3H-tetrafluoropropoxy)-phosphazene) (Agilent) and 3-amino-1-propanesulfonic acid MS:MS_COMMENTS (HOT) (Sigma Aldrich) were added to the running buffer to serve as lockmasses. MS:MS_COMMENTS The isocratic flow rate was set to 0.150 mL/min. The instrument was run in 4GHz MS:MS_COMMENTS High Resolution, negative ionization mode. Mass spectra between 50 and 1,000 m/z MS:MS_COMMENTS were collected in profile mode. 5 µL of each sample were injected twice, MS:MS_COMMENTS consecutively, within 0.96 minutes to serve as technical replicates. The pooled MS:MS_COMMENTS study sample was injected periodically throughout the batch. Data Processing & MS:MS_COMMENTS Annotation Raw profile data were centroided, merged, and recalibrated using MS:MS_COMMENTS MATLAB software described by Fuhrer et al(doi/10.1021/ac201267k). MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST003141_AN005154_Results.txt UNITS:ion counts Has m/z:Yes #END