부산대학교 도서관

Indwelling urethral catheters are the most commonly used medical devices and catheter associated urinary tract infections (CAUTIs) are one of the most common hospital acquired infections. Over 40% of CAUTIs in long-term catheterised patients may be caused by the bacterium Proteus mirabilis. Urease produced by this bacterium generates alkaline conditions by breaking down urea, leading to the formation of dense crystalline biofilm structures on catheter surfaces. This crystalline biofilm makes infections hard to treat and causes the blockage of the catheter lumen, resulting in the retention of infected urine leading to episodes of ascending urinary tract infections. The aim of this study was to identify genes and pathways involved in crystalline biofilm formation by P. mirabilis, in order to inform the development of novel strategies for prevention of catheter blockage. To accomplish this, a bank of random mini-Tn5 transposon mutants was constructed in the clinical isolate P. mirabilis B4. A total of 3840 transposon mutants were screened for phenotypic alterations in biofilm formation. A total of 575 mutants isolated exhibited altered biofilm formation, but comparable rates of growth to P. mirabilis B4 under assay conditions (310 biofilm enhanced; 265 biofilm deficient). The disrupted genes of a subset of 35 transposon mutants were successfully identified. After further phenotypic characterisation 12 transposon mutants were selected and their ability to encrust and block urethral catheters analysed using an in vitro model of the catheterised urinary tract (the bladder model). The bladder models yielded 4 transposon mutants with significant differences in the time taken to block catheters when compared to P. mirabilis B4. Two blocking deficient mutants were further analysed because these types of mutations are most likely to give insights relevant to the prevention of crystalline biofilm formation. Mutants STS8.1D7 and NHBFF9 were disrupted in aspects of the nitrogen metabolism and MFS family transport systems respectively. Timed bladder model experiments and chemical analysis of catheters of these mutants and the wild type B4 were then carried out to further evaluate the differences in crystalline biofilm formation. Overall, transposon mutants that took longer to block catheters displayed a lower level of encrustation after 10 h bladder model experiments. This was confirmed quantitatively by a significant reduction in calcium and biomass deposited onto catheters. Scanning electron microscopy (SEM) and environmental SEM (ESEM) further substantiated the quantitative methods illustrating clear differences in crystalline biofilm distribution for mutants that took longer to block catheters when compared to P. mirabilis B4. ESEM analysis optimized for this purpose allowed the examination of the crystalline biofilm ultrastructure in fine detail in its native, hydrated state and identified delicate calcium based crystal sheets which had not been visualised before. Additional flow chamber experiments confirmed that the ability of the two mutants to adhere to catheter biomaterials was not impaired, highlighting that the initial stages of biofilm formation were not associated with the genes disrupted for these mutants. Overall, the research conducted during this study identified 4 mutants differing in the time taken to block catheters, elucidating 4 genes that are involved in this complex phenotypic trait. Mutants with significant reductions in the ability to block urinary catheters displayed disruptions of the nitrogen metabolism and efflux systems which are believed to be involved in waste management in this bacterium. The inhibition of efflux systems in particular could be of potential value in the treatment or prevention of P. mirabilis crystalline biofilm formation by increasing its susceptibility to antimicrobials, and further investigation of these genes in the future could lead to the development of novel treatments for P. mirabilis CAUTIs.