Summary of Study ST001244
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 PR000831. The data can be accessed directly via it's Project DOI: 10.21228/M88H5F 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 | ST001244 |
| Study Title | Uropathogenic versus Urocolonizing Escherichia coli |
| Study Summary | Urinary tract infections (UTIs) represent a major burden across the population, although key facets of their pathogenesis challenge physicians and investigators alike. Escherichia coli epitomizes these obstacles: this Gram-negative bacterial species is the most prevalent agent of UTIs worldwide and can also colonize the urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB). Unfortunately, at the level of the organism, the relationship between symptomatic UTI and ASB is poorly defined, confounding our understanding of microbial pathogenesis and strategies for clinical management. Unlike diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli (UPEC) remains phenomenologic, without conserved phenotypes and (known) genetic determinants that rigorously distinguish UTI- and ASB-associated strains. This manuscript provides a cross-disciplinary review of the current issues – from interrelated mechanistic and diagnostic perspectives – and describes new opportunities by which clinical resources can be leveraged to overcome molecular challenges. Specifically, we present our work harnessing a large collection of patient-derived isolates to identify features that do (and do not) distinguish UTI- from ASB-associated E. coli strains. Analyses of biofilm formation, previously reported to be higher in ASB strains, revealed extensive phenotypic heterogeneity that did not correlate with symptomatology. However, metabolomic experiments revealed distinct signatures between ASB and cystitis isolates, including species in the purine pathway (previously shown to be critical for intracellular survival during acute infection). Together, these studies demonstrate how large-scale, wild-type approaches can help dissect the physiology of colonization-versus-infection, suggesting that the molecular definition of UPEC may rest at the level of global bacterial metabolism. |
| Institute | Vanderbilt University |
| Last Name | Rutledge |
| First Name | Alexandra |
| Address | 7330 Stevenson Center Lane, NASHVILLE, TENNESSEE, 37235, USA |
| a.rutledge@vanderbilt.edu | |
| Phone | 6155046923 |
| Submit Date | 2019-08-14 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-03-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000831 |
| Project DOI: | doi: 10.21228/M88H5F |
| Project Title: | Uropathogenic versus Urocolonizing Escherichia coli |
| Project Summary: | Urinary tract infections (UTIs) represent a major burden across the population, although key facets of their pathogenesis challenge physicians and investigators alike. Escherichia coli epitomizes these obstacles: this Gram-negative bacterial species is the most prevalent agent of UTIs worldwide and can also colonize the urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB). Unfortunately, at the level of the organism, the relationship between symptomatic UTI and ASB is poorly defined, confounding our understanding of microbial pathogenesis and strategies for clinical management. Unlike diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli (UPEC) remains phenomenologic, without conserved phenotypes and (known) genetic determinants that rigorously distinguish UTI- and ASB-associated strains. This manuscript provides a cross-disciplinary review of the current issues – from interrelated mechanistic and diagnostic perspectives – and describes new opportunities by which clinical resources can be leveraged to overcome molecular challenges. Specifically, we present our work harnessing a large collection of patient-derived isolates to identify features that do (and do not) distinguish UTI- from ASB-associated E. coli strains. Analyses of biofilm formation, previously reported to be higher in ASB strains, revealed extensive phenotypic heterogeneity that did not correlate with symptomatology. However, metabolomic experiments revealed distinct signatures between ASB and cystitis isolates, including species in the purine pathway (previously shown to be critical for intracellular survival during acute infection). Together, these studies demonstrate how large-scale, wild-type approaches can help dissect the physiology of colonization-versus-infection, suggesting that the molecular definition of UPEC may rest at the level of global bacterial metabolism. |
| Institute: | Vanderbilt University |
| Last Name: | Rutledge |
| First Name: | Alexandra |
| Address: | 7330 Stevenson Center Lane, NASHVILLE, TENNESSEE, 37235, USA |
| Email: | a.rutledge@vanderbilt.edu |
| Phone: | 6155046923 |
Subject:
| Subject ID: | SU001312 |
| Subject Type: | Bacteria |
| Subject Species: | Escherichia coli |
| Taxonomy ID: | 562 |
| Gender: | Not applicable |
Factors:
Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)
| mb_sample_id | local_sample_id | Group |
|---|---|---|
| SA090764 | SC_20180803_RPLCp_FMS_Sup_S3_C1 | ASB_1 |
| SA090765 | SC_20180803_RPLCp_FMS_Sup_S2_B1 | ASB_1 |
| SA090766 | SC_20180803_RPLCp_FMS_Sup_S1_A1 | ASB_1 |
| SA090767 | SC_20180803_RPLCp_FMS_Sup_S8_H1 | Cystitis_1 |
| SA090768 | SC_20180803_RPLCp_FMS_Sup_S7_G1 | Cystitis_1 |
| SA090769 | SC_20180803_RPLCp_FMS_Sup_S6_F1 | Cystitis_1 |
| SA090770 | SC_20180803_RPLCp_FMS_Sup_S5_E1 | Cystitis_1 |
| SA090771 | SC_20180803_RPLCp_FMS_Sup_S9_I1 | Cystitis_1 |
| SA090772 | SC_20180803_RPLCp_FMS_Sup_S10_J1 | Cystitis_1 |
| SA090773 | SC_20180803_RPLCp_FMS_Sup_S12_L1 | Cystitis_1 |
| SA090774 | SC_20180803_RPLCp_FMS_Sup_S11_K1 | Cystitis_1 |
| SA090775 | SC_20180803_RPLCp_FMS_Sup_S4_D1 | Cystitis_1 |
| SA090776 | SC_20180803_RPLCp_DDA2_Sup_QC_06 | Qcpool_1 |
| SA090777 | SC_20180803_RPLCp_DDA4_Sup_QC_02 | Qcpool_1 |
| SA090778 | SC_20180803_RPLCp_FMS_Sup_QC_05 | Qcpool_1 |
| SA090779 | SC_20180803_RPLCp_FMS_Sup_QC_04 | Qcpool_1 |
| SA090780 | SC_20180803_RPLCp_DDA6_Sup_QC_10 | Qcpool_1 |
| SA090781 | SC_20180803_RPLCp_FMS_Sup_QC_07 | Qcpool_1 |
| SA090782 | SC_20180803_RPLCp_FMS_Sup_QC_09 | Qcpool_1 |
| SA090783 | SC_20180803_RPLCp_DDA4_Sup_QC_08 | Qcpool_1 |
| SA090784 | SC_20180803_RPLCp_DDA2_Sup_QC_01 | Qcpool_1 |
| SA090785 | SC_20180803_RPLCp_FMS_Sup_QC_11 | Qcpool_1 |
| SA090786 | SC_20180803_RPLCp_DDA6_Sup_QC_03 | Qcpool_1 |
| Showing results 1 to 23 of 23 |
Collection:
| Collection ID: | CO001306 |
| Collection Summary: | Urine-associated E. coli isolates were collected in accordance with an approved IRB |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR001327 |
| Treatment Summary: | For each isolate, a single colony from an agar dish was inoculated in 5 mL LB and shaken overnight at 37°C under ambient atmospheric conditions. Cultures were then diluted 1:1000 in the combined human urine and grown for 6 hours to mid-log phase (37°C, shaking), under 4% oxygen to emulate the bladder environment. After 6 hours, OD600 of each isolate was measured – and cultures were normalized by volume to yield equal number of organisms from each strain – prior to pooling into groups of 8 isolates each. CFUs were enumerated for each pool to confirm bacterial denisty (~109 total E. coli per pool). Each pool was then centrifuged to separate the cellular (pellet) and supernatant fraction. Pellets and supernatants were flash frozen and stored at -80°C until for metabolomic analysis. |
Sample Preparation:
| Sampleprep ID: | SP001320 |
| Sampleprep Summary: | Global untargeted metabolomic analyses were performed on the supernatant-fraction of ASB and cystitis pools. Aliquots of each pool (200µL) were added to individual Eppendorf tubes containing 200µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0)) (LC-MS grade). Labeled creatinine-D3 and lysine-D4 were added to each sample to assess the metabolite extraction (sample preparation) step. Samples were first subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samples were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing supernatant was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis. The pellet-fraction of each sample pool prepared as described above was lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0) (LC-MS grade), followed by sonication in an ice bath for 10 min. Sample volume for each pool was adjusted such that all samples have the same cell number in each vial. Samples were first subjected to protein precipitation by addition of 1000µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samplwere were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing extract was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis. |
Combined analysis:
| Analysis ID | AN002067 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Thermo Hypersil Gold |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | abundance |
Chromatography:
| Chromatography ID: | CH001505 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Hypersil Gold |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001918 |
| Analysis ID: | AN002067 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Progenesis QI |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | ARutledge_MS_Protocol.docx |
