Bacterial Community Shift Assessed by DGGE and Sequence Analysis

Objective: Change of bacterial community after a standard prophylactic treatment has been evidenced. The objective of this study was to determine and quantify the changes in total oral microbial population in a clinical study. Methods: Twenty-four healthy adults participated in a single-blind study using a commercial fluoride dentifrice (Colgate Total^®). Supra-gingival plaque samples were collected at baseline and 24-hours post-brushing. Total bacterial genomic DNA was isolated (Epicenter). Targeted 16S rRNA genes were amplified using a nested-PCR approach with two sets of universal 16S rRNA primers. Denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified 16S rDNA gene fragments were performed to analyze the changes in bacterial diversity of the plaque before and after using the dentifrice. Fingerprinting II Informatix program (Bio-Rad) was used to generate a dendrogram based on the average matrix with the unweighted pair group method by means of arithmetic averages. For identification and taxonomical classification, the sequences were compared by BLAST with known sequences listed in GenBank and RDP II Database. Results: The study found two distinguished clusters of DGGE profiles, suggesting that there was a difference in the overall microbial composition between the two groups of samples. The difference was statistically significant (p <0.01). Total of 371 clones were sequenced and 7 phyla and 29 genera were identified, including known and unknown bacterial species. An increased bacterial diversity was found in the clinical sample of 24 h after using the dentifrice, including S. sanguinis, Flavobacteriaceae, Prevotella, Corynebacterium, TM7 and other commensal bacteria. Conclusion: The results demonstrated the presence of highly diverse bacterial population in dental plaque and the use of a theraputic toothpaste like Colgate Total^® may provide beneficial effect on the bacterial community shift. Supported by the NIH/NIDCR Grant DE15706 and Colgate-Palmolive Company.