Metabolic heterogeneity and antibiotic susceptibility in biofilms

描述(申请人提供)：生物膜是附着在表面上生长的微生物群落。以生物膜为基础的感染经常发生，留置设备上的生物膜生长很难根除。抗生素在生物膜内的低转运速度、生物膜基质的保护作用以及生物膜内部的低代谢活动率都被发现是导致这些感染持续存在的原因，但目前对导致这些影响的过程了解很少。尽管生物膜的空间异质性对生物膜感染的治疗方法的选择很重要，但关于当地环境条件如何影响生物膜空间模式的发展，以及抗生素的有效性如何随身体部位和留置装置的类型而变化的信息很少。我们假设生物膜内代谢活动的空间模式受到流动环境中空间模式的影响，这些相互作用导致生物膜的复杂性随着时间的推移而增加。我们还假设，流动环境不仅通过影响抗菌剂向生物膜内细胞的输送，而且还通过决定群落内的代谢梯度来影响生物膜对抗生素的敏感性。我们建议通过以下具体目标来解决这些假设。目的1：观察单种生物膜在平面流动细胞中的生长，以评估在环境流动条件下，随着空间变异性的增加，生物膜形态、运输方式和代谢活性的变化。目的：观察抗生素治疗对不同空间复杂程度生物被膜的清除效果，并将局部杀灭效率的分布与运输条件和代谢活动的空间格局联系起来。目的3：开发一种改进的数值模型，以便对上述影响进行定量分析。目的4：利用该模型来阐明多尺度流-生物膜相互作用，特别是评估在抗生素处理下生物膜中细胞亚群存活的关键特征。我们建议使用新颖的实验和数值模拟相结合的方法来实现这些目标。我们将在一个新的实验系统中进行生物膜生长和处理的实验，该系统能够在流入和流出模式中施加精确可控的空间变异性程度。生物膜的生长、流动和氧气分布的变化、抗生素的运输以及由此导致的细胞死亡都将直接在现场观察到。我们将利用这些新的和独特的观察结果来支持开发一个新的生物被膜发展的数值模型，该模型随后将被用来模拟在不同局部生长条件下抗生素治疗在根除生物被膜方面的有效性。这种测量和建模的结合将提供独特的见解，了解生物膜生长与外部流动相互作用和修改的方式，以及最终这种复杂的相互作用如何控制生物膜的整体形成，以及引入的抗菌剂对生物膜基质中细胞的影响。 公共卫生相关性：生物被膜中的代谢异质性和抗生素敏感性概述基于生物被膜的插入和植入的医疗设备，如导管、神经外科设备和整形外科设备的感染很难治疗。抗生素在生物膜内的低传输率、生物膜基质的保护作用以及生物膜内部的低代谢活动率都被发现是导致这些感染持续存在的原因，但目前对这些影响的过程了解很少。拟议的工作将促进对当地环境条件如何影响生物膜生长的了解，并将开发改进的工具，用于评估抗生素对生物膜感染的有效性。

DESCRIPTION (provided by applicant): Biofilms are microbial communities that grow attached to a surface. Biofilm-based infections occur frequently, and biofilm growth on indwelling devices is very difficult to eradicate. Low rates of antibiotic transport within biofilms, protective effects of the biofilm matrix, and low rates of metabolic activity within the biofilm interior have all been found to contribute to the persistence of these infections, but there is currently little understanding of the processes responsible for these effects. While spatial heterogeneity in biofilms is clearly important to selection of therapy for biofilm-based infections, little information is available on the way in which local environmental conditions influence the development of spatial patterns in biofilms, and hence how the effectiveness of antibiotics varies depending on the body site and type of indwelling device. We hypothesize that spatial patterns of metabolic activity within a biofilm are influenced by spatial patterns in the flow environment, and that these interactions cause biofilm complexity to increase over time. We also hypothesize that the flow environment affects biofilm antimicrobial susceptibility not only by influencing delivery of antimicrobials to cells within the biofilm but also by dictating metabolic gradients within the community. We propose to address these hypotheses through the following specific aims. Aim 1: Observe growth of mono- species biofilms in a planar flow cell in order to assess changes in biofilm morphology, transport patterns, and metabolic activity with increasing spatial variability in environmental flow conditions. Aim 2: Observe the effectiveness of antibiotic treatment in eradicating biofilms having different degrees of spatial complexity, and relate the distribution of local killing efficiency to spatial patterns in transport conditions and metabolic activity. Aim 3: Develop an improved numerical model to allow quantitative analysis of the effects described above. Aim 4: Use the model to clarify multi-scale flow-biofilm interactions, and particularly to evaluate the key features that contribute to the survival of subpopulations of cells in biofilms under antibiotic treatment. We propose to achieve these aims by using a combination of novel experiments and numerical modeling. We will conduct experiments on biofilm growth and treatment in a new experimental system that provides the ability to impose a precisely controlled degree of spatial variability in inflow and outflow patterns. Biofilm growth, changes in flow and oxygen distributions, transport of antibiotic, and the resulting cell death will all be observed directly in situ. We will utilize these new and unique observations to support development of a new numerical model for biofilm development, which will subsequently be used to simulate the effectiveness of antibiotic treatment in eradicating biofilms under different local growth conditions. This combination of measurements and modeling will provide unique insight into the way in which biofilm growth interacts with and modifies the external flow, and ultimately how this complex interaction controls the overall formation of the biofilm and the effects of introduced antimicrobial agents on cells residing in the biofilm matrix. PUBLIC HEALTH RELEVANCE: Metabolic heterogeneity and antibiotic susceptibility in biofilms Summary Narrative Biofilm-based infections of inserted and implanted medical devices such as catheters, neurosurgical devices, and orthopedic devices are difficult to treat. Low rates of antibiotic transport within biofilms, protective effects of the biofilm matrix, and low rates of metabolic activity within the biofilm interior have all been found to contribute to the persistence of these infections, but there is currently little understanding of the processes responsible for these effects. The proposed work will advance understanding of how local environmental conditions influence biofilm growth, and will develop improved tools for assessing the effectiveness of antibiotics against biofilm-based infections.