Sampling for the study of dental caries has been mainly based on saliva (e.g. McInnes P and Cutting M, 2010; Prasanthi B et al., 2014; Bardow A et al., 2014), since it is the oral fluid that envelops all tissues, assumed to be reproducible in different patients, and for its ease and accessibility in the sampling. In recent years, methodological techniques have been developed that allow analyzing in more detail the microbial composition (Wade WG, 2013). In this Thesis, by using molecular methodologies based on high-throughput sequencing, we have studied the composition of different oral samples in relation to the main microorganisms that cause tooth decay. This taxonomic
43
approach will determine which oral sample is more appropriate to be analyzed in etiological and/or epidemiological studies of dental caries.
Methodologies based on culture have allowed isolating some bacteria that are present in carious lesions, such as S. mutans (Clarke JK, 1924) and
Lactobacillus spp. (Bunting RW, 1937), in chronological order. Subsequently, by
the use of cloning techniques, several authors identified in tooth decay other microorganisms that could not get by traditional culture techniques, including (Dewhirst FE et al., 2001) or Bifidobacterium spp. (Mantzourani M et al., 2009).
However, these two approaches are insufficient in nature. It has been recognized that traditional bacterial culture is insufficient to cover the full spectrum of microorganisms and their metabolic requirements (Marsh PD et al., 2011; Wade WG, 2002). Although the cloning technique of PCR products represented a step forward in the advancement of knowledge since it avoids cultivation-associated bias (Aas JA et al., 2005), but given that only allows analyzing an average of 50-100 clones, it is not a definitive methodological solution to study dental caries.
For this reason, massive sequencing techniques have opened the way to deepen our knowledge of bacterial populations that are involved in health and disease. In this Thesis, Second Generation Techniques were applied for obtaining thousands of sequences by sample, which allowed us to cover a large portion of the bacterial diversity related to dental caries.
The knowledge of microorganisms present in dental caries is a link that is required to understand the onset and progression of the disease. However,
44
the taxonomic study of bacterial populations does not reflect what role is played by each member of this complex ecosystem. In this Thesis, by using metagenomics and metatranscriptomics techniques, I studied not only the composition, but also the functionality of each microorganism in the initiation and progression of dental caries.
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