#METABOLOMICS WORKBENCH rachelpilla_20190904_133611 DATATRACK_ID:1813 STUDY_ID:ST001248 ANALYSIS_ID:AN002072 PROJECT_ID:PR000835 VERSION 1 CREATED_ON September 9, 2019, 1:18 pm #PROJECT PR:PROJECT_TITLE The cecal and fecal microbiomes and metabolomes of horses before and after PR:PROJECT_TITLE metronidazole administration PR:PROJECT_SUMMARY Metronidazole (15mg/kg BID PO) was given to horses (n=5) with in-dwelling cecal PR:PROJECT_SUMMARY cannulas. The study was suspended after the fifth dose (day 3) due to adverse PR:PROJECT_SUMMARY gastrointestinal effects. Cecal and fecal samples were obtained before and after PR:PROJECT_SUMMARY (Days days -52, -28, -14, 0, 7, 14, 28 and 52) metronidazole administration. DNA PR:PROJECT_SUMMARY was extracted from the cecal and fecal samples, and 16S rRNA genes were PR:PROJECT_SUMMARY sequenced. The bacterial diversity and composition of each sample was PR:PROJECT_SUMMARY determined. Richness and evenness indices were significantly decreased by PR:PROJECT_SUMMARY metronidazole administration in both cecal and fecal samples, but overall PR:PROJECT_SUMMARY composition was only significantly changed in fecal samples on Day 3 (ANOSIM, PR:PROJECT_SUMMARY p=0.008). The most dominant phyla were Bacteroidetes and Firmicutes in all PR:PROJECT_SUMMARY groups examined. In fecal samples, significant changes at the phyla PR:PROJECT_SUMMARY Actinobacteria, Spirochaetes, Lentisphaerae and Verrucomicrobia occurred on Day PR:PROJECT_SUMMARY 3, which correlated with clinical signs of gastrointestinal disease. Analysis of PR:PROJECT_SUMMARY the predicted microbial functions using PICRUSt indicated changes associated PR:PROJECT_SUMMARY with metronidazole in fecal samples. The metabolome was characterized by mass PR:PROJECT_SUMMARY spectrometry-based methods. Fecal, but not cecal metabolites were affected by PR:PROJECT_SUMMARY metronidazole. The fecal metabolites affected represented diverse metabolic PR:PROJECT_SUMMARY pathways such as nucleic acid metabolism, secondary bile metabolism, fatty acid PR:PROJECT_SUMMARY synthesis/degradation/elongation or metabolism and sugar metabolism. PR:INSTITUTE Texas A&M PR:DEPARTMENT Department of Small Animal Clinical Sciences PR:LABORATORY Gastrointestinal Laboratory PR:LAST_NAME Arnold PR:FIRST_NAME Carolyn PR:ADDRESS 4474 TAMU PR:EMAIL carnold@cvm.tamu.edu PR:PHONE 979 845 3541 #STUDY ST:STUDY_TITLE The cecal and fecal metabolomes of horses before and after metronidazole ST:STUDY_TITLE administration ST:STUDY_SUMMARY Metronidazole (15mg/kg BID PO) was given to horses (n=5) with in-dwelling cecal ST:STUDY_SUMMARY cannulas. The study was suspended after the fifth dose (day 3) due to adverse ST:STUDY_SUMMARY gastrointestinal effects. Cecal and fecal samples were obtained before and after ST:STUDY_SUMMARY (Days days -52, -28, -14, 0, 7, 14, 28 and 52) metronidazole administration. The ST:STUDY_SUMMARY metabolome was characterized by mass spectrometry-based methods. Fecal, but not ST:STUDY_SUMMARY cecal metabolites were affected by metronidazole. The fecal metabolites affected ST:STUDY_SUMMARY represented diverse metabolic pathways such as nucleic acid metabolism, ST:STUDY_SUMMARY secondary bile metabolism, fatty acid synthesis/degradation/elongation or ST:STUDY_SUMMARY metabolism and sugar metabolism. ST:INSTITUTE Texas A&M ST:DEPARTMENT Department of Small Animal Clinical Sciences ST:LABORATORY Gastrointestinal Laboratory ST:LAST_NAME Arnold ST:FIRST_NAME Carolyn ST:ADDRESS 4474 TAMU ST:EMAIL carnold@cvm.tamu.edu ST:PHONE 979 845 3541 #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Equus caballus SU:TAXONOMY_ID 9796 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Alvin AlvinD0_030 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD0_031 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD0_032 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD0_033 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD28_034 organ:fecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Alvin AlvinD3_031 organ:fecal | Day:D3 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Alvin AlvinD7_032 organ:fecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD0_017 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Billy BillyD-14_016 organ:fecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Billy BillyD14_020 organ:fecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD-28_015 organ:fecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Billy BillyD28_021 organ:fecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD3_018 organ:fecal | Day:D3 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD7_019 organ:fecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Ernie ErnieD0_024 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Ernie ErnieD-14_023 organ:fecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Ernie ErnieD14_027 organ:fecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Ernie ErnieD-28_022 organ:fecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Ernie ErnieD3_025 organ:fecal | Day:D3 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Ernie ErnieD7_026 organ:fecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD0_003 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Frank FrankD-14_002 organ:fecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Frank FrankD14_006 organ:fecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD-28_001 organ:fecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Frank FrankD28_007 organ:fecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD3_004 organ:fecal | Day:D3 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD7_005 organ:fecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Steve SteveD0_010 organ:fecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Steve SteveD-14_009 organ:fecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Steve SteveD14_013 organ:fecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Steve SteveD-28_008 organ:fecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Steve SteveD28_014 organ:fecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Steve SteveD3_011 organ:fecal | Day:D3 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Steve SteveD7_012 organ:fecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Alvin AlvinD0_058 organ:cecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD-14_057 organ:cecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD14_060 organ:cecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Alvin AlvinD-28_056 organ:cecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Alvin AlvinD28_061 organ:cecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Alvin AlvinD7_059 organ:cecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD0_047 organ:cecal | Day:D0 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD-14_046 organ:cecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Billy BillyD14_049 organ:cecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD-28_045 organ:cecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Billy BillyD28_050 organ:cecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Billy BillyD7_048 organ:cecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Ernie ErnieD0_053 organ:cecal | Day:D0 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Ernie ErnieD-14_052 organ:cecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Ernie ErnieD14_055 organ:cecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Ernie ErnieD-28_051 organ:cecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Ernie ErnieD7_054 organ:cecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD0_037 organ:cecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Frank FrankD-14_036 organ:cecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Frank FrankD14_039 organ:cecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD-28_035 organ:cecal | Day:D-28 | Treatment:No SUBJECT_SAMPLE_FACTORS Frank FrankD28_040 organ:cecal | Day:D28 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Frank FrankD7_038 organ:cecal | Day:D7 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Steve SteveD0_042 organ:cecal | Day:D0 | Treatment:No SUBJECT_SAMPLE_FACTORS Steve SteveD-14_041 organ:cecal | Day:D-14 | Treatment:No SUBJECT_SAMPLE_FACTORS Steve SteveD14_043 organ:cecal | Day:D14 | Treatment:Yes SUBJECT_SAMPLE_FACTORS Steve SteveD28_044 organ:cecal | Day:D28 | Treatment:No SUBJECT_SAMPLE_FACTORS Steve SteveD7_062 organ:cecal | Day:D7 | Treatment:Yes #COLLECTION CO:COLLECTION_SUMMARY Cecal and fecal samples were obtained before and after (Days days -52, -28, -14, CO:COLLECTION_SUMMARY 0, 7, 14, 28 and 52) metronidazole administration. CO:SAMPLE_TYPE Feces #TREATMENT TR:TREATMENT_SUMMARY Metronidazole (15mg/kg BID PO) was given to horses (n=5) with in-dwelling cecal TR:TREATMENT_SUMMARY cannulas. The study was suspended after the fifth dose (day 3) due to severe TR:TREATMENT_SUMMARY adverse gastrointestinal effects. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were stored at −80 °C until shipped on dry ice to the West Coast SP:SAMPLEPREP_SUMMARY Metabolomics Core (University of California, Davis, CA, USA) using an untargeted SP:SAMPLEPREP_SUMMARY approach.(20) Samples were homogenized and extracted. Following resuspension in SP:SAMPLEPREP_SUMMARY methanol/chloroform, samples were derivatized by methoxyamine hydrochloride in SP:SAMPLEPREP_SUMMARY pyridine and subsequently by N-methyl-N-trimethylsilyltrifluoroacetamide for SP:SAMPLEPREP_SUMMARY trimethylsilylation of acidic protons. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 6890N CH:COLUMN_NAME Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Leco Pegasus III GC TOF MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE MALDI MS:ION_MODE POSITIVE MS:MS_COMMENTS Raw data files are preprocessed directly after data acquisition and stored as MS:MS_COMMENTS ChromaTOF-specific *.peg files, as generic *.txt result files and additionally MS:MS_COMMENTS as generic ANDI MS *.cdf files. ChromaTOF vs. 2.32 is used for data MS:MS_COMMENTS preprocessing without smoothing, 3 s peak width, baseline subtraction just above MS:MS_COMMENTS the noise level, and automatic mass spectral deconvolution and peak detection at MS:MS_COMMENTS signal/noise levels of 5:1 throughout the chromatogram. Apex masses are reported MS:MS_COMMENTS for use in the BinBase algorithm. Result *.txt files are exported to a data MS:MS_COMMENTS server with absolute spectra intensities and further processed by a filtering MS:MS_COMMENTS algorithm implemented in the metabolomics BinBase database. MS:MS_RESULTS_FILE ST001248_AN002072_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END