Elderly patients with long-term indwelling urinary catheters invariably develop polymicrobial bacteriuria that may result in serious complications such as fever, bacteremia, pyelonephritis, and urinary stones. In an ongoing study by our group, two bacterial species, Providencia stuartii and Proteus mirabilis, were found to be the most prevalent isolates. Certain strains can persist for weeks and months. In this proposal we outline a course of study that should reveal why they species enjoy a niche in the long term catheterized urinary tract, why these species are able to persist for long periods of time, and what bacterial virulence factors contribute to the pathogenesis of pyelonephritis. The following hypotheses will be tested: 1) In a mouse model of catheter-associated urinary tract infection, that pyelonephritis of Providencia stuartii is associated with a) expression of MR/K hemagglutinin, b) binding of Tamm-Horsfall protein, and c) urease production. 2) In the mouse model, that pyelonephritogenesis of Proteus mirabilis is associated with a) urease production, b) swarming motility, c) a specific adherence factor, and d) the ability to invade kidney epithelium. 3) That persistence of Proteus mirabilis in the urinary tract of long-term catheterized patients is related to the ability of the organism to adhere to a) uroepithelial cells, b) catheter surfaces, and/or c) Tamm-Horsfall protein. 4) In an in vitro model of urease-induced struvite crystal formation, that, a Proteus mirabilis is a more effective inducer of crystal formation than other urease-producing species, b) urease-deficient transposon mutants of Proteus mirabilis and Providencia stuartii are unable to Induce crystal formation, and c) the bacterial cell of Proteus mirabilis is the nidus for crystal formation. Strains of P. stuartii expressing MR/K and MS (THP-binding) hemagglutinins have been selected for animal challenge. Urease- deficient mutants of P. stuartii and p. mirabilis and a non-motile mutant of P. mirabilis will be identified by transposon mutagenesis. Isolates of P. mirabilis with a specific hemagglutinin correlated with persistence and those correlated with internalization by renal proximal tubular epithelium will also be selected for animal challenge. For the in vitro struvite crystallization model, encrustation on glass rods induced by P. mirabilis in synthetic urine will be quantiated for calcium, magnesium, ammonium, and phosphate ions and total struvite. These values will be compared to those attained for other urease producing species. Association of P. mirabilis and struvite crystals will be confirmed by electron microscopy.
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