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What's In That Urine? Guanosine and Adenosine Levels Define Molecular Signatures of Uropathogenic versus Urocolonizing Escherichia coli in Metabolomic Analyses
Allison R. Eberly, PhD1, Connor J. Beebout, BS1, Gerald T. van Horn, PhD1, Ching Man Carmen Tong, DO2, Hamilton D. Green, BS1, Madison J. Fitzgerald, BS1, Shuvro De, MD2, Emily K. Apple, BSN1, Alexandra C. Schrimpe-Rutledge, PhD3, Simona G. Codreanu, PhD3, Stacy D. Sherrod, PhD3, John A. McLean, PhD3, Charles W. Stratton, MD1, Jonathan E. Schmitz, MD/PhD1, Douglass B. Clayton, MD2, Maria Hadjifrangiskou, PhD1.
1Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA, 2Division of Pediatric Urology, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, USA, 3Center for Innovative Technologies, Department of Chemistry, Vanderbilt University, Nashville, TN, USA.

BACKGROUND: Urinary tract infections (UTIs) represent a major burden across the population, although key facets of their pathogenesis challenge physicians and investigators alike. Currently, there are no reliable molecular tools that can distinguish strains of E. coli that are isolated from cases of asymptomatic bacteriuria (ASB) versus UTI. Herein, we present our data using patient-derived isolates to identify potential phenotypic and metabolic features of ASB-associated E. coli strains, hypothesizing that metabolite usage may play a role in differentiating ASB- and cystitis-associated E. coli strains. METHODS: A retrospective, single-site study was performed with 300 clinical urinary E. coli isolates and corresponding demographics and clinical infection statuses such as ASB and cystitis were mined from electronic health records. Individual strains were assessed for biofilm formation by crystal violet assay and colony biofilm formation. Isolates were also subjected to a global metabolomics workflow using reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). Chemometrics and hierarchical cluster analyses were performed on peak intensity values using MetaboAnalyst 4.0 to generate principal component analysis (PCA) plots and heat maps, respectively. RESULTS: Extensive heterogeneity was observed in biofilm formation in both crystal violet and colony biofilm assays in both ASB and UTI isolates, consistent with observations from environmental and other extra-intestinal E.coli isolates. In particular, biofilms show that in addition to inter-strain heterogeneity, strains also demonstrated intra-strain variability, with sub-populations evolving in real-time for many isolates. Since phenotypic assays do not differentiate ASB from cystitis isolates, global untargeted metabolomics were performed. On LC-MS/MS, a total of 6,711 compounds were detected, which showed distinct clustering of the supernatant from ASB and cystitis samples in principal component analysis (PCA) (fig a). Filtering by a p-value <0.05 and a q-value of <0.1, 582 significant compounds were detected, with heat map visualizations also revealing differences in the metabolic profile of ASB compared to cystitis. Interrogation of known metabolites in the purine pathway demonstrates a difference in usage of the purine salvage and de novo pathways. Guanosine levels are found at lower levels in the supernatant of ASB isolates, while adenosine levels are found at lower levels in the supernatant of cystitis isolates (fig b). CONCLUSIONS:
In our study, a comparison of biofilm formation between UTI- and ASB-associated strains demonstrated extensive phenotypic heterogeneity, proving that there are no reliable bacterial markers that can distinguish uropathogens from urocolonizers. However, global, untargeted metabolomic analyses revealed distinct signatures between ASB and cystitis isolates, including metabolites in the purine pathway, which was previously shown to be critical for intracellular survival during acute infection. Collectively, these metabolomics studies point towards the potential use of these metabolites as urinary biomarkers in the clinical setting to aid in predicting colonization by a cystitis versus ASB strain.


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