Combined NMR/Optical Microscopy of Oral Biofilm Physiology Studies

描述(申请人提供)：细菌生物膜在自然界中几乎随处可见，并已成为公认的持续性感染的原因。微生物在生物膜内的代谢和表型变化很快--这至少是它们对抗菌剂产生抗药性的部分原因。龋病是人类最普遍、最昂贵的细菌感染之一。龋齿和相关的牙齿腐烂与有机酸的口腔生物膜(口腔细菌膜，即牙菌斑)的产生引起的持续的pH下降密切相关。关于这些有机酸的分布和涉及的代谢过程，缺乏详细的知识。这在一定程度上是由于：[i]生物膜的复杂性，其中包含基质、氧气、有机酸等随时间演变的空间浓度梯度，以及[ii]当前微生物代谢方法的不足，这些方法具有破坏性，通常不产生空间分辨率。这个为期两年的R21项目的目标是适应、改进和应用独特的组合核磁共振和荧光共聚焦(核磁共振/光学)显微镜仪器和技术，用于活体、致龋性口腔生物膜的时间和生物膜深度分辨代谢研究。两步培养和测量程序将为相关(口腔)生长环境中可重复的深度分辨新陈代谢测量提供可重复的生物膜样本。一种和两种生物膜将以这种方式进行研究。核磁共振光谱成像和相关方法将被用来绘制10微米分辨率的生物膜代谢随深度的函数图。具体目标是：1)适应和改进核磁共振/光学显微镜相结合的方法来研究口腔生物膜生理学，以及2)使用新的能力来实时监测与龋病相关的动态过程。由此产生的生物膜技术和代谢数据将显著提高我们对口腔疾病过程的理解。由此产生的技术将普遍适用于各种混合物种、与医学相关的生物膜，从而为抗击细菌疾病提供关键的代谢信息。

DESCRIPTION (provided by applicant): Bacterial biofilms are found nearly everywhere in nature and are becoming recognized as the cause of persistent infections. Microbial metabolism and phenotype vary rapidly within a biofilm - this is at least partly responsible for their resistance to antimicrobial agents. Dental caries disease is one of the most prevalent and costly bacterial infections in humans. Caries and associated tooth decay are strongly correlated with a sustained pH decrease induced by oral biofilm (oral bacterial films, i.e. dental plaque) production of organic acids. Detailed knowledge is lacking about these organic acid distributions and the metabolic processes involved. This is due in part to: [i] the complexity of biofilms, which contain temporally evolving spatial concentration gradients for substrate, oxygen, organic acids, etc., and [ii] the inadequacy of current microbiological-metabolism methods which are destructive and typically yield no spatial resolution. The objectives of this two-year R21 project are to adapt, improve and apply unique combined nuclear magnetic resonance and fluorescent confocal (NMR/optical) microscopy instrumentation and techniques for time- and biofilm-depth-resolved metabolism studies of live, cariogenic oral biofilms. A two-step cultivation and measurement procedure will provide reproducible biofilm samples for repeatable depth-resolved metabolism measurements in a relevant (oral) growth environments. One and two-species biofilms will be studied in this fashion. NMR spectroscopic imaging and related methods will be employed to map biofilm metabolism as a function of depth with 10-micron resolution. Specific Aims are to: 1) adapt and improve combined NMR/optical microscopy to study oral biofilm physiology, and 2) employ the new capability to monitor dynamic processes relevant to caries in real time. The resulting biofilm technology and metabolic data will significantly improve our understanding of oral disease processes. The resulting technology will generally be applicable to a variety of mixed-species, medically- relevant biofilms, thus providing key metabolic information for battling bacterial diseases.