Assessment of Microbial Diversity in Caries-active Filipinos using PCR-Dcnaturing Gradient Gel Electrophoresis

Abstract

Traditional studies on oral microbial communities relied mostly on the culturing and subsequent identification of microorganisms through an array of biochemical tests. However, this may underestimate microbial population since only 50% of oral bacterial species are cultivable on media. In this study, a molecular-based approach on oral microbial communities was employed. Four subjects (2 males, 2 females) assessed to be caries-active by a dental practitioner were recruited. PCR-Denaturing Gradient Gel Electrophoresis (DGGE) was employed to study the microbial diversity, as well as to identify dominant microorganisms present in the plaque, tongue and saliva of caries-active Filipinos. Microbial community DNA was extracted from the 3 oral sites using cetyltrimethyl ammonium bromide (CTAB) method and the obtained genomic DNA was amplified using specific primer pairs for eubacterial 16S ribosomal RNA (rRNA) gene. The obtained 585 base pair amplicons were then subjected to DGGE to separate the individual bacterial DNA in the sample. Relatedness of bacterial communities from the 3 oral sites was analyzed with Quantity One software (BioRad). Bacterial identities were determined through sequencing of excised DGGE bands and BLAST analysis. Results showed that bacterial communities present in saliva had greater similarity with microbial communities in plaque than with the microbial communities in the tongue. Fifteen dominant bacterial species were identified; seven among these were unculturable bacteria. This study demonstrated the efficiency of a molecular-based approach in the initial assessment of microbial diversity as well as the identification of dominant bacterial species residing within the oral cavity.