Mechanisms of antibiotic resistance in confined microcolonies

描述（由申请方提供）：细菌是社会性生物，当存在于群体中时，其表现出不同的表型，这些变化通常导致毒力增强。增加人口规模的表型反应，包括抗寄生虫的无柄生物膜社区的形成，基因表达模式的修改，以响应扩散信号（群体感应），和群体运动（群集）。我们对细菌社会反应的理解主要来自于对烧瓶和培养皿中细菌的体外研究，这些细菌群落通常包含超过108个细胞。虽然在某些环境中，非常大的种群的行为可能很重要，但自然界中的细菌通常居住在密集的微簇中，个体少得多;重要的是，已经提出这些簇是许多病原体的主要传播途径，包括铜绿假单胞菌，金黄色葡萄球菌和霍乱弧菌。尽管迫切需要了解细菌微簇内社会表型的发生和维持，但由于缺乏将细菌组织成3D模式的技术，这些群体中群体行为的基本要求在很大程度上仍然未知，这些模式包含可调密度的特定数量的细胞。在这个提议中，我们描述了利用三维（3D）基于蛋白质的微制造来控制的策略，在某些情况下，动态地，小细菌种群的空间排列，该技术将用于阐明细菌微簇中种群依赖性抗生素耐药性的基础。这项R21资助的研究重点是临床上重要的革兰氏阴性细菌铜绿假单胞菌，这是囊性纤维化死亡的主要原因，并将在未来的研究中扩展到其他病原体，包括S。金黄色。

DESCRIPTION (provided by applicant): Bacteria are social organisms that display distinct phenotypes when present in groups, changes that often result in heightened virulence. Phenotypic responses to increased population size include formation of antibiotic-resistant sessile biofilm communities, modification of gene expression patterns in response to diffusible signals (quorum sensing), and group motility (swarming). Our understanding of bacterial social responses derives primarily from in vitro studies of bacteria in flasks and on culture plates, communities that often contain greater than 108 cells. Although the behavior of very large populations may be important in some environments, bacteria in nature often reside in dense micro-clusters having far fewer individuals; importantly, it has been proposed that these clusters are the primary means of transmission of many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae. Despite the strong imperative to understand onset and maintenance of social phenotypes within bacterial micro-clusters, fundamental requirements for group behaviors in these populations remain largely unknown due to a dearth of technologies for organizing bacteria into 3D patterns that contain specified numbers of cells at tunable densities. In this proposal, we describe strategies for exploiting three-dimensional (3D) protein-based microfabrication to control, in some cases, dynamically, spatial arrangements of small bacterial populations, technology that will be used to elucidate the basis for population-dependent antibiotic resistance in bacterial micro-clusters. The studies in this R21 grant focus on the clinically important, Gram-negative bacterium P. aeruginosa, a primary cause of cystic fibrosis mortality, and will be extended in future studies to other pathogens, including S. aureus.