Metabolic heterogeneity and antibiotic susceptibility in biofilms

生物膜的代谢异质性与抗生素敏感性 项目摘要 生物膜是附着在表面上生长的微生物群落。基于生物膜的感染经常发生，并且留置装置上的生物膜生长非常难以根除。生物膜内抗生素运输的低速率、生物膜基质的保护作用和生物膜内部代谢活性的低速率都被发现有助于这些感染的持续性，但目前对这些影响的过程了解甚少。虽然生物膜的空间异质性对于基于生物膜的感染的治疗选择显然是重要的，但关于局部环境条件影响生物膜空间模式发展的方式以及抗生素的有效性如何根据身体部位和留置器械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，并且这些相互作用导致生物膜复杂性随时间增加。我们还假设，流动环境不仅通过影响生物膜内细胞对抗菌剂的传递，而且通过决定群落内的代谢梯度来影响生物膜抗菌剂的敏感性。 我们建议通过以下具体目标来解决这些假设。 目的1：观察单物种生物膜在平面流动池中的生长，以评估生物膜形态、运输模式和生物膜的变化。 随着环境流动条件的空间变化，代谢活动增加。 目标二：观察抗生素治疗在消除具有不同程度的空间复杂性的生物膜中的有效性，并将局部杀伤效率的分布与运输条件和代谢活性的空间模式相关联。 目标3：开发一个改进的数值模型，以便对上述影响进行定量分析。目标4：使用该模型来阐明多尺度流动-生物膜相互作用，特别是评估有助于抗生素治疗下生物膜中细胞亚群存活的关键特征。 我们建议通过使用新的实验和数值模拟相结合来实现这些目标。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控制的空间变异程度。生物膜的生长，流量和氧气分布的变化，抗生素的运输，以及由此产生的细胞死亡都将直接在原位观察。我们将利用这些新的和独特的观察，以支持开发一个新的生物膜发展的数值模型，随后将用于模拟抗生素治疗在不同的局部生长条件下消除生物膜的有效性。测量和建模的这种组合将提供独特的见解，其中生物膜生长与外部流动相互作用并改变外部流动的方式，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微生物剂对驻留在生物膜基质中的细胞的影响。

Metabolic heterogeneity and antibiotic susceptibility in biofilms Project Summary 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 inwhich 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 hypothesizethat spatial patterns of metabolic activity within a biofilm are influenced by spatial patterns in the flowenvironment, and that these interactions cause biofilm complexity to increase over time. We also hypothesizethat the flow environment affects biofilm antimicrobial susceptibility not only by influencing delivery ofantimicrobials 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.