Synchrotron imaging of crystalline biofilms in urinary catheters

描述(由申请人提供)：一套基于同步加速器的X射线成像方法将用于评估奇异变形杆菌在导尿管中形成的结晶生物膜的发育情况。变形杆菌是尿路感染的一种非常常见的病原体，在长期导尿的患者中尤其有问题。奇异肺炎杆菌能分解尿素，导致尿液pH值升高，并在尿路内形成矿物质沉淀物。这些沉积物在膀胱和肾脏形成结石和结垢，并在插入的装置上形成结垢，如导管和输尿管支架。大约一半长期使用导尿管的患者会因为这些感染而出现导尿管堵塞。堵塞事件可导致严重的急性症状，是导尿管患者发病率和死亡率的一个促成因素。监测和更换导尿管的需要也给这些患者的长期管理带来了很大的负担。理解晶体生物膜的发展和相关的导管堵塞的一个主要挑战是，矿物沉积阻止了光学方法的使用，如共焦显微镜。为了克服这一限制，尿管内的变形杆菌生物膜将使用我们最近开发的基于同步加速器的复杂方法进行成像，这些方法用于矿物样品的非侵入性研究。同步加速器x射线计算机显微断层扫描将用于以近微米级的分辨率对Proteus生物膜的三维孔结构进行成像，同步加速器x射线衍射也将用于评估生物膜中不同矿物相的分布。此外，将使用环境扫描电子显微镜评估生物膜内的细胞分布，并将使用能量色散光谱和电子衍射进行分析电子显微镜以确定晶体沉积物中的化学分布。分析将使用模型膀胱系统对含有体外生长的晶体生物膜的完整导管进行，以及对从患者身上取出的堵塞的导管进行分析。将使用图像分析方法对生物膜孔结构进行定量描述，并将使用层析数据结合孔隙流体流动的格子Boltzmann模拟来评估每个生物膜内的内部传输环境。对不同的变形杆菌菌株和不同的生长时间所获得的结果进行比较，以评估孔结构、内部传输条件和晶体生物膜发展的相互作用，以及生物膜生长对导管堵塞的整体影响。公共卫生相关性：导管堵塞变形杆菌感染仍然是患者和长期护理机构面临的一个重大、持续和严重的问题。由于缺乏对晶体生物膜结构和组成的了解，阻碍了预防导管感染和控制导管堵塞的新疗法的开发。该项目的结果将表明晶体结构如何影响尿管上变形杆菌生物膜的发展，并提出治疗这些感染和防止导管堵塞的改进策略。

DESCRIPTION (provided by applicant): A suite of synchrotron-based x-ray imaging methodologies will be used to assess the development of crystalline biofilms formed by Proteus mirabilis in urinary catheters. Proteus is an extremely common pathogen in urinary tract infections, and is particularly problematic in patients subject to long-term urinary catheterization. P. mirabilis hydrolyzes urea, leading to an increase in the pH of urine and the formation of mineral deposits within the urinary tract. These deposits form stones and encrustations in the bladder and kidneys, as well as encrustations on inserted devices such as catheters and ureteral stents. Approximately half of patients with long-term urinary catheters develop catheter blockage as a result of these infections. Blockage incidents can cause serious acute symptoms and are a contributing factor to morbidity and mortality in catheterized patients. The need to monitor and replace urinary catheters also presents a substantial burden for long-term management of these patients. One major challenge in understanding the development of crystalline biofilms and the associated catheter blockage is that the mineral deposits prevent use of optical methods such as confocal microscopy. To overcome this limitation, Proteus biofilms within urinary catheters will be imaged using sophisticated synchrotron-based methods that we have recently developed for non-invasive investigations of mineral samples. Synchrotron x-ray computed micro-tomography will be used to image the pore structure of Proteus biofilms in three dimensions with near-micron-scale resolution, and synchrotron x- ray diffraction will also be used to assess the distribution of different mineral phases within the biofilms. In addition, environmental scanning electron microscopy will be used to assess cell distributions within biofilms, and analytical electron microscopy will be performed using energy-dispersive spectrometry and electron diffraction to determine chemical distributions within the crystalline deposits. Analyses will be performed on intact catheters containing crystalline biofilms grown in vitro using a model bladder system, as well as on blocked catheters retrieved from patients. Quantitative descriptions of the biofilm pore structure will be developed using image analysis methods, and the internal transport environment within each biofilm will be evaluated by using the tomographic data in conjunction with lattice Boltzmann simulations of pore fluid flow. Results obtained for different Proteus strains and different growth durations will be compared in order to assess the interplay of pore structure, internal transport conditions, and crystalline biofilm development, as well as the overall effects of biofilm growth on catheter blockage. PUBLIC HEALTH RELEVANCE: Catheter blockage from Proteus infections still represents a major, ongoing, and serious problem for patients and for long-term care facilities. A lack of understanding of the structure and composition of crystalline biofilms has hindered the development of new treatments to prevent catheter infections and control catheter blockage. The project results will indicate how the crystalline structure influences the development of Proteus biofilms on urinary catheters and suggest improved strategies for treating these infections and for preventing catheter blockage.