This research aims to investigate four novel approaches to the detection and prevention of catheter encrustation and blockage following infection by the Gram-negative, motile bacterium Proteus mirabilis (P. mirabilis). Expression of a potent bacterial urease elevates urinary pH, leading to the local supersaturation and precipitation of mineral deposits from the urine that occludes urine-flow through the catheter. Vesico-ureteric reflux following blockage by crystalline biofilms may result in serious symptomatic episodes such as pyelonephritis, endotoxic shock and septicaemia.Three of the four described approaches for the management of catheter-associated urinary tract infection employ a dual-layered polymeric delivery system, in which a lower poly(vinyl alcohol) reservoir layer contains either a diagnostic (5(6)-carboxyfluorescein) or therapeutic (lytic bacteriophage) cargo. The hydrogel layer is capped and sealed by an upper layer of the pH-responsive polymer poly(methyl methacrylate-co-methacrylic acid) (Eudragit S100®). Alkalinisation of local urine media (pH >7) induces degradation of the Eudragit layer, thus releasing the contained cargo. Release of 5(6)-carboxyfluorescein results in a clear and unambiguous colour change to give advance warning (up to 14.5 hours) of impending catheter blockage, whilst bacteriophage release increased time to catheter blockage by 100% (13 hours to 26 hours).The final approach describes the formulation and development of a novel small-molecule urease inhibitor, 2-mercaptoacetamide (2-MA), for the prevention of crystalline biofilm formation. Inhibition of bacterial urease without inducing bacterial cell death allows for the ‘disarming’ of P. mirabilis without directing population evolution via initiation of a selective pressure. Comparison of 2-MA with the approved drug acetohydroxamic acid (AHA) allows for direct evaluation against the current ‘gold standard’ in treatment of chronic urea-splitting infection.All approaches were evaluated for clinical efficacy within a physiologically representative model of the catheterised tract. In vitro bladder models were formulated to emulate late-stage infection by uropathogenic clinical isolates (initial inoculum 108 CFU/mL), such that preventative strategies were evaluated under ‘worst-case scenario’ conditions. Performance within in vitro systems inoculated with P. mirabilis was directly compared to urease-negative Escherichia coli (E. coli), to assess uropathogenic specificity. Translation of these approaches into a clinical environment may allow a multifaceted approach to the detection and prevention of catheter blockage; a problem for which there is currently no effective control method.
|Date of Award
|3 Apr 2019
|Steven Bull (Supervisor) & Toby Jenkins (Supervisor)