Mechanisms of antibiotic resistance in confined microcolonies

描述（由申请人提供）：细菌是社会性生物，在群体中表现出不同的表型，这种变化通常会导致毒性增强。对种群规模增加的表型反应包括耐抗生素的无根生物膜群落的形成，对扩散信号（群体感应）的基因表达模式的修改，以及群体运动性（蜂群）。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，这些细菌群落通常含有超过108个细胞。虽然在某些环境中，庞大种群的行为可能很重要，但自然界中的细菌通常以密集的微群形式存在，个体数量要少得多；重要的是，已经提出这些集群是许多病原体传播的主要途径，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群中社会表型的发生和维持，但由于缺乏将细菌组织成含有可调密度的特定数量的细胞的3D模式的技术，这些人群中群体行为的基本要求仍然很大程度上未知。在本提案中，我们描述了利用三维（3D）蛋白质微加工的策略，在某些情况下，动态地控制小细菌群体的空间排列，该技术将用于阐明细菌微集群中群体依赖性抗生素耐药性的基础。这项R21拨款的研究重点是临床重要的革兰氏阴性细菌P. aeruginosa，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，包括金黄色葡萄球菌。

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.