Microbial Diversity and Genetic Characterization

拟议的研究预计将产生关于组成龋齿相关斑块生物膜的细菌生物群的重要新信息。这些信息的长期益处应该引导研究人员在解决其病因的基础上设计龋齿的诊断和预防策略。该提案将重点关注患有严重龋齿的儿童，即早期儿童龋齿（ECC）。使用一种强大的梯度电泳技术将被用于从斑块生物膜中的一系列细菌中分离16S rDNA标记。这些凝胶应该显示出ECC和无龋儿童之间的微生物区系差异。这种分析，反过来，将使我们能够识别或近似那些细菌，有些可能是不可培养的，与龋齿有关。另一个有待验证的假设是，变异链球菌菌株或整个龋齿生物膜在致病能力上是否存在差异。减法DNA杂交将用于发现存在于易患龋齿儿童的变形链球菌或牙斑中的独特遗传位点。减法DNA杂交的进一步发展将导致我们的总体目标，即从整个斑块中表征龋活性生物膜内的一系列遗传位点，而不考虑首次培养特定细菌的限制。这将为随后的研究奠定基础，在这些研究中，一组DNA探针可以被编译和测试，这将有助于预测特定儿童是否有患龋齿的风险。了解这些基因位点的功能和它们产生的细菌宿主将为龋齿的病因提供重要信息。此外，拥有疾病的遗传标记可以消除从牙菌斑中培养细菌的昂贵和不精确的做法。提出的研究可能会对这些更严重的龋齿形式产生影响，最终导致其预防。

The proposed studies are anticipated to yield important new information as to the composition of the bacterial biota that comprises the caries-associated plaque biofilm. The long-term benefit of such information should lead researchers to devising both diagnostic and preventative strategies for dental caries based on addressing its etiological agents. This proposal will focus on children with a severe forms of dental caries called Early Childhood Caries (ECC). Using a powerful technique of gradient electrophoresis will be used to separate 16S rDNA markers from an array of bacteria in plaque biofilms. These gels should show differences between the microfloras of ECC and caries-free children. This profiling, in turn, will allow us to identify or approximate those bacteria, some likely to be uncultivable, associated with caries. Another hypothesis to be tested is whether strains of mutans streptococci, or the entire caries-biofilm differ in their ability to cause disease. Subtraction DNA hybridization will be used to discover unique genetic loci present in mutans streptococci or dental plaques of caries-prone children. Further development of subtraction DNA hybridization will lead to our overall objective, i.e., to characterize from whole plaque a constellation of genetic loci within the caries-active biofilm, irrespective of the limitation of first cultivating specific bacteria. This will set the groundwork for subsequent studies in which a set of DNA probes can be compiled and tested, which will be useful for predicting whether a particular child is at risk for caries. Knowing the function of these genetic loci and the bacterial host from which they arise will give important information as to the causation of caries. Moreover, having genetic markers for disease may eliminate the costly and imprecise practice of cultivating bacteria from dental plaque. The research proposed will likely impact on these more serious forms of caries, leading to its eventual prevention.