| Summary: | Urinary tract infections (UTI) are among the most prevalent infectious diseases worldwide, leading to significant morbidity and mortality and wreaking a substantial economic cost. Uropathogenic Escherichia coli (UPEC) has been shown to invade the urothelium in murine models of acute UTI, forming intracellular reservoirs that are thought to evade conventional antibiotic treatment and the immune response, allowing recurrence at a later date. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the growing elderly population, is not well understood. Moreover, the species of bacteria involved remains largely unknown, the model systems used to study them need improvement, and treatment options are not currently optimal. This thesis addresses these important research aims with a view towards improving the situation for LUTS patients who have an underlying UTI. In the first research section, we found strong evidence of intracellular Enterococcus faecalis harboured within urothelial cells shed via an innate immune response from the bladder of LUTS patients. Furthermore, these patient-isolated strains of E. faecalis showed robust invasive properties in a bladder cell line. However, E. coli only formed surface biofilms in these patients, suggesting that the murine UPEC model may not apply to patients with chronic LUTS. In the second section, we addressed the issue that the murine and human urinary bladder differ structurally and functionally, which may be hindering our understanding of UTI pathogenesis in humans. We therefore 4 designed and characterised a human three-dimensional (3D) bladder mimetic differentiated from primary urothelial progenitors, and showed that it closely resembles human tissue. Moreover, infection in this organoid model resulted in outcomes similar to those seen in LUTS patients. In the future we aim to use this 3D culture as a platform for modelling chronic infection and tissue regeneration in the presence of novel therapeutic agents. Finally, in the third section we tackled the issue that traditional oral antibiotic regimens for UTI fail in a high proportion of cases. This recurrence of disease post-treatment could be explained in part by the lack of cellular penetration of orally administered antibiotics, which are not able to accumulate to an effective concentration within intracellular bacterial niches. Meanwhile, oral antibiotics may also lead to antimicrobial resistance and systemic side effects. Using our human urothelial organoid, we tested the ability of novel liposome-coated ultrasound-activated lipid microbubbles to deliver drugs into the cortex of the apical cell layer. Ultrasound-activated intracellular delivery of gentamicin using microbubbles was over twice that achieved by liposomes alone. Moreover, little cell damage was detected and this therapeutic technology exhibited very encouraging antimicrobial activity, showing great promise as a more efficacious alternative to traditional oral antibiotic regimens. In conclusion, these collective results have implications for both the diagnosis and treatment of chronic UTI.
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