Microbial Diversity and Genetic Characterization

预计拟议的研究将产生重要的新信息，包括龋齿相关菌斑生物膜的细菌生物群的组成。 这些信息的长期利益应导致研究人员制定诊断和预防策略的基础上解决其病因代理龋齿。 该提案将重点关注患有严重龋齿的儿童，称为幼儿龋齿（ECC）。 使用梯度电泳的强大技术将被用来从菌斑生物膜中的细菌阵列中分离16S rDNA标记。 这些凝胶应该显示出ECC和无龋儿童的微生物群之间的差异。 反过来，这种分析将使我们能够识别或近似那些与龋齿相关的细菌，其中一些可能是不可培养的。 另一个有待检验的假设是，变异链球菌菌株或整个龋生物膜是否在致病能力上存在差异。DNA差减杂交将用于发现存在于易患龋儿童的变形链球菌或牙菌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，从整个菌斑中表征龋活性生物膜内的一系列遗传基因座，而不考虑首先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，可以编译和测试一组DNA探针，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提供有关龋齿病因的重要信息。 此外，拥有疾病的遗传标记可能会消除从牙菌斑中培养细菌的昂贵和不精确的做法。 这项研究可能会对这些更严重的龋齿产生影响，从而最终预防龋齿。

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.