Dental caries results from an imbalance of the metabolic activity in the dental biofilm. The microbial communities of teeth have traditionally been studied by standard cultural approaches. More recently, cloning and sequencing of the 16S rRNA gene have been used to characterize the microbial composition of the oral biofilm, but the methodological limitations of this approach have now been recognized. Next-generation high-throughput sequencing methods have the potential to reveal the composition and functioning of the biofilm by means of metagenomic and metatranscriptomic analyses. Currently available high-throughput sequencing approaches are reviewed and discussed in relation to studying the biofilm associated with dental caries. Important in understanding the dynamic processes in caries is the metabolic activity of the biofilm; metabolome analysis is a new tool that might enable us to assess such activity. As caries is a localized disease, it is essential that biofilm samples are taken from precisely determined tooth sites; pooling samples is not appropriate. This paper presents the case that culture-based studies are important, but that the fullest understanding of the role of the biofilm in the caries process will only come from an integrated approach determining biological function and metabolic output.
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