Microbial and Environmental Genomics, The J Craig Venter Institute, San Diego, California, United States of America.
PLoS One. 2012;7(3):e32219. doi: 10.1371/journal.pone.0032219. Epub 2012 Mar 5.
Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.
METHODOLOGY/PRINCIPAL FINDINGS: Supragingival plaque samples from caries-free children incubated with (13)C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by Lactobacillus and Propionibacterium species, both of which have been previously found within carious lesions from children.
CONCLUSIONS/SIGNIFICANCE: Our approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.
许多人类微生物传染病，包括龋齿，本质上都是多微生物的。这些复杂的多物种群落如何从健康状态演变为疾病状态尚不清楚。尽管已经在体外对许多与健康或疾病相关的口腔细菌进行了描述，但在当前的方法中，很难确定它们在复杂口腔微生物组中的生理学特性。此外，迄今为止，这些物种中约有一半仍然无法培养，除了它们的 16S rRNA 序列之外，人们对它们知之甚少。由于缺乏基于培养的生理分析，即使这些物种与疾病明显相关，未培养物种的功能作用也仍然是个谜。为了开始解决这些知识空白，我们应用了磁共振光谱（MRS）与基于 RNA 和 DNA 的稳定同位素探测（SIP）相结合的方法，对来自健康儿童的口腔斑块群落进行体外时间监测，以研究代谢物，并鉴定代谢活跃和不活跃的细菌物种。
方法/主要发现：从无龋儿童的龈上斑块样本中，用（13）C 底物孵育，在人工诱导的健康（缓冲，pH7）和疾病状态（pH5.5 和 pH4.5）下，葡萄糖代谢产生乳酸作为主要有机酸。在 pH7 条件下，葡萄糖耗尽后迅速利用乳酸。SIP 分析显示，有许多属含有在 pH5.5 下具有代谢能力的培养和未培养的类群。在 pH4.5 下，活跃物种的多样性显著下降，主要由乳酸杆菌和丙酸杆菌属的物种组成，这两种菌以前都在儿童龋齿病变中发现过。
结论/意义：我们的方法允许鉴定在不同 pH 条件下代谢碳水化合物的物种，并支持乳酸杆菌和丙酸杆菌在儿童龋齿发展中的重要性。在健康受试者中鉴定在低 pH 下活跃的物种，可以更好地了解口腔龋齿的发病机制，并为早期预防措施提供适当的目标。
