"Development of a 3D, in vitro model of RUTI incorporating Escherichia " by Sadie Wyatt

Graduation Year

2025

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Human Biology

Reader 1

Dr. Claudia Marques

Reader 2

Dr. Jason Tor

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2024 Sadie J Wyatt

Abstract

Urinary Tract Infections (UTIs) are the most common outpatient infections, affecting more than half of women in their lifetime. Many of these women will experience what is known as a Recurrent Urinary Tract Infection (RUTI), defined by more than two UTIs in six months or three in a year. Researchers attribute RUTI’s recurrence to initial infections that were never fully treated, which may recur due to the lack of effectiveness of antibiotics against biofilm infections. Vaginal swabs, urine, and blood samples from 79 women experiencing RUTIs were collected. Sequencing of vaginal and urine samples found that the most prevalent bacterial species present was Escherichia coli. Analysis of blood indicated that 82% of patients carried mutations in the SERPINE1 gene, which codes for the plasminogen activator inhibitor type 1 (PAI-1) protein. PAI-1 is produced in the liver and is essential for blood clotting; it regulates the breakdown of fibrin. Mutations in PAI-1 result in systemic fibrin build-up. In this work it was hypothesized that a build-up of fibrin enhances the growth of bacterial biofilms and/or provides resistance to antibiotic treatments. To confirm this hypothesis, E. coli biofilms were cultured over a monolayer of HTB-5 bladder epithelial cells for 48 hours to establish an in vitro environment. The infections were then treated with ciprofloxacin (30 μg/L), a common antibiotic prescribed to treat RUTIs. Two experimental groups were tested with and without a fibrin hydrogel to evaluate fibrin’s role, where the fibrin overlaid the HTB-5 cells’ monolayer. It was found that in the presence of fibrin hydrogels E. coli biofilms had increased cell numbers, compared to the absence of fibrin. Furthermore, E. coli cells could fully penetrate the fibrin hydrogel and reach the cell monolayer, resulting in a loss of viability of bladder cells to the point of eradication (100% loss) by day 4. These findings highlight a new opportunity to investigate the possible pore-toxin-forming abilities of E. coli and their effect on bladder epithelial cells. Overall, this model could prove essential in investigating novel antimicrobial treatments for RUTIs that could lower the recurrence rate.

This thesis is restricted to the Claremont Colleges current faculty, students, and staff.

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