The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.

植入的医疗设备上细菌生物膜的生长会导致有害感染和设备故障。当细菌附着并感知表面时，生物膜就会开始形成。对于常见的医院病原体铜绿假单胞菌和许多其他病原体，向生物膜表型的过渡受细胞内信号和第二信使环-di-GMP（c-di-GMP）控制。尚不知道如何调整生物医学材料以阻止c-di-GMP信号传导，目前尚无进行这种研究的现存方法。在这里，我们开发了这样一种方法。我们允许铜绿假单胞菌附着在聚（乙二醇）二丙烯酸酯（PEGDA）水凝胶的表面上。这些细菌包含用于c-di-GMP的绿色荧光蛋白（GFP）报告基因的质粒。我们使用激光扫描共聚焦显微镜在将细菌引入水凝胶后1小时开始测量3小时的GFP报告分子动力学。我们使用无启动子的GFP质粒控制细菌代谢变化的影响，并通过使用荧光塑料珠作为“标准蜡烛”来校准通过不同水凝胶的光的衰减效果。我们证明该方法可以测量与不同PEGDA凝胶类型和表面暴露的蛋白PilY1相关的c-di-GMP信号的统计学显着差异。