Molecular approaches for viable bacterial population and transcriptional analyses in a rodent model of dental caries.

Culturing methods are the primary approach for microbiological analysis of plaque biofilms in rodent models of dental caries. In this study, we developed strategies for the isolation of DNA and RNA from plaque biofilms formed in vivo to analyse the viable bacterial population and gene expression. Plaque biofilm samples from rats were treated with propidium monoazide to isolate DNA from viable cells, and the purified DNA was used to quantify total bacteria and the Streptococcus mutans population via quantitative polymerase chain reaction (qPCR) and specific primers; the same samples were also analysed by counting colony-forming units (CFU). In parallel, RNA was isolated from plaque-biofilm samples (from the same animals) and used for transcriptional analyses via reverse transcription-qPCR. The viable populations of both S. mutans and total bacteria assessed by qPCR were positively correlated with the CFU data (P < 0.001; r > 0.8). However, the qPCR data showed higher bacterial cell counts, particularly for total bacteria (vs. CFU). Moreover, S. mutans proportion in the plaque biofilm determined by qPCR analysis showed strong correlation with incidence of smooth-surface caries (P = 0.0022, r = 0.71). The purified RNAs presented high RNA integrity numbers (> 7), which allowed measurement of the expression of genes that are critical for S. mutans virulence (e.g. gtfB and gtfC). Our data show that the viable microbial population and the gene expression can be analysed simultaneously, providing a global assessment of the infectious aspect of dental caries. Our approach could enhance the value of the current rodent model in further understanding the pathophysiology of this disease and facilitating the exploration of novel anti-caries therapies.

Salivary glucosyltransferase B as a possible marker for caries activity.

Bacteria-derived glucosyltransferases (Gtf) (EC 2.4.1.5), through synthesizing glucan polymers from sucrose and starch hydrolysates, play an essential role in the etiology and pathogenesis of caries. We attempted to correlate the levels of Gtf in whole saliva with the prevalence of carious lesions in young children. We examined saliva from children who were either free of overt carious lesions, or had severe early childhood caries (mean dmfs = 18.72 +/- 9.0 SD), for Gtf by direct enzyme assay. The levels of GtfB, GtfC and GtfD from Streptococcus mutans in the saliva using monoclonal/specific antibodies in an enzyme-linked immunosorbent assay were determined. Multiple logistic regression analyses with model selection showed that GtfB levels correlated with dmfs values of the subjects (p = 0.006). There was no correlation between total Gtf activity as measured by direct enzyme assay and dmfs values. There was a strong correlation between mutans streptococci populations in saliva and caries activity. Collectively, these data show that GtfB levels in saliva correlate strongly with presence of clinical caries and with number of carious lesions in young children. It is also possible to measure different Gtfs, separately, in whole saliva. These observations may have important clinical implications, may lead to development of a chair side caries activity test and support the importance of GtfB in the pathogenesis of dental caries.

Comparison of cell sediment and surface grown "test plaque" using scanning electron microscopy.

Scanning electron microscopy was used to compare the morphology, integrity and distribution of bacterial cells in a test plaque grown on the surface of enamel with that of the cell sediment plaque routinely used in a short-term intraoral caries model. Cultures of S. mutans IB-1600 or S. sobrinus 6715-13 were grown in complex media supplemented with either 2.0% sucrose (glucan plaque) or 0.2% glucose (non-glucan plaque). Cell sediment (CS) plaque was prepared by centrifuging the cultures after incubation, recovering the cell sediment, and spreading it on Metricel membrane filter paper. Surface grown (SG) plaque was prepared by suspending saliva-coated bovine enamel in the culture medium, incubating, and recovering the enamel assembly with bacterial accumulations. Cell morphology and integrity, as well as the appearance of glucan-like material produced by the cells, was similar in both CS and SG test plaques. The cell distribution however, varied in the SG plaque from extremes of all cells to all glucan, whereas the cell sediment plaque was more uniform in cell distribution. A highly standardized test plaque minimizes variability in the intraoral caries model. These findings support the contention that the bacterial cells in a cell sediment plaque are similar in morphology, integrity and glucan production to surface grown plaque, and have the added advantage of uniform distribution, which makes the cell sediment plaque more appropriate for intraoral caries model studies.