Social, Psychological, and Behavioral Predictors of Salivary Bacteria, Yeast in Caries-Free Children.

INTRODUCTION: Early childhood caries (ECC) is a complex, multifactorial oral disease that is a major public health concern because it is prevalent, profoundly alters a child's quality of life, is difficult to treat effectively, and has a distressing tendency to recur following treatment. OBJECTIVES: The purpose of the study was to examine social, psychological, and behavioral predictors of salivary bacteria and yeast in young children at risk for ECC. METHODS: A sample of 189 initially caries-free preschool children was assessed for child stress physiology from salivary cortisol, child and family stress exposure, diet, oral health behaviors, and sociodemographic risks. Multiple logistic regression analysis was implemented to examine the associations between these risk factors and cariogenic microorganisms: mutans streptococci (MS), lactobacilli (LB), and Candida species. RESULTS: Higher baseline salivary cortisol (odds ratio [OR] = 6.26; 95% confidence level [CL], 1.69-23.16) and a blunted response to an acute laboratory stressor (OR = .56; 95% CL, .37-.83) were associated with an increased likelihood of elevated salivary MS (≥105 colony-forming units/mL) in caries-free children. Sociodemographic risk for cariogenic microorganisms was also found. Specifically, lower education attainment of the parent/primary caregiver was associated with children being more likely to carry salivary Candida species and elevated salivary MS; in addition, children from households with an unemployed parent/primary caregiver were more likely (OR = 3.13; 95% CL, 1.2-8.05) to carry salivary Candida species and more likely (OR = 3.03; 95% CL, 1.25-7.33) to carry elevated levels of MS and/or salivary Candida and/or LB. CONCLUSIONS: The impact of sociodemographic risk and stress physiology on cariogenic disease processes are evident prior to ECC onset. The findings provide novel data on the early onset of cariogenic processes in children and the importance of considering sociodemographic, psychosocial, and behavioral factors when judging ECC risk. KNOWLEDGE TRANSFER STATEMENT: The findings provide valuable and novel findings that, pre-ECC onset, the caries disease process is explicable from a detailed assessment of behavioral, sociodemographic, and psychosocial stress variables.

Characterization of enamel caries lesions in rat molars using synchrotron X-ray microtomography.

Dental caries is a ubiquitous infectious disease with a nearly 100% lifetime prevalence. Rodent caries models are widely used to investigate the etiology, progression and potential prevention or treatment of the disease. To explore the suitability of these models for deeper investigations of intact surface zones during enamel caries, the structures of early-stage carious lesions in rats were characterized and compared with previous reports on white spot enamel lesions in humans. Synchrotron X-ray microcomputed tomography non-destructively mapped demineralization in carious rat molar specimens across a range of caries severity, identifying 52 lesions across the 30 teeth imaged. Of these lesions, 13 were shown to have intact surface zones. Depth profiles of fractional mineral density were qualitatively similar to lesions in human teeth. However, the thickness of the surface zone in the rat model ranges from 10 to 58 µm, and is therefore significantly thinner than in human enamel. These results indicate that a fraction of lesions in rat caries possess an intact surface zone and are qualitatively similar to human lesions at the micrometer scale. This suggests that rat caries models may be a suitable analog through which to investigate the structure of surface zone enamel and its role during dental caries.

Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

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.

Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo.

Cranberry crude extracts, in various vehicles, have shown inhibitory effects on the formation of oral biofilms in vitro. The presence of proanthocyanidins (PAC) in cranberry extracts has been linked to biological activities against specific virulence attributes of Streptococcus mutans, e.g. the inhibition of glucosyltransferase (Gtf) activity. The aim of the present study was to determine the influence of a highly purified and chemically defined cranberry PAC fraction on S. mutans biofilm formation on saliva-coated hydroxyapatite surface, and on dental caries development in Sprague-Dawley rats. In addition, we examined the ability of specific PAC (ranging from low-molecular-weight monomers and dimers to high-molecular-weight oligomers/polymers) to inhibit GtfB activity and glycolytic pH drop by S. mutans cells, in an attempt to identify specific bioactive compounds. Topical applications (60-second exposure, twice daily) with PAC (1.5 mg/ml) during biofilm formation resulted in less biomass and fewer insoluble polysaccharides than the biofilms treated with vehicle control had (10% ethanol, v/v; p < 0.05). The incidence of smooth-surface caries in rats was significantly reduced by PAC treatment (twice daily), and resulted in less severe carious lesions compared to the vehicle control group (p < 0.05); the animals treated with PAC also showed significantly less caries severity on sulcal surfaces (p < 0.05). Furthermore, specific A-type PAC oligomers (dimers to dodecamers; 0.1 mg/ml) effectively diminished the synthesis of insoluble glucans by GtfB adsorbed on a saliva-coated hydroxyapatite surface, and also affected bacterial glycolysis. Our data show that cranberry PAC reduced the formation of biofilms by S. mutans in vitro and dental caries development in vivo, which may be attributed to the presence of specific bioactive A-type dimers and oligomers.

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.

Apigenin and tt-farnesol with fluoride effects on S. mutans biofilms and dental caries.

Apigenin (Api) and tt-farnesol (Far) are two naturally occurring agents that affect the development of cariogenic biofilms. Fluoride (F) interferes physicochemically with caries development and also exhibits antibacterial activity. We examined whether the association of Api and Far enhance the anti-caries properties of F by acting cooperatively on the expression of virulence of Streptococcus mutans. The biological effects of each of the agents were greatly enhanced when used in combination with F. In general, biofilms treated with Api and/or Far in combination with F displayed less biomass and fewer insoluble glucans and iodophilic polysaccharides than did those treated with the test agents alone (P < 0.05). The combination of the test agents with F was highly effective in preventing caries development in rats, especially Api+Far+F, and results were comparable with those observed with chlorhexidine + F (positive control). Results from these studies showed that apigenin and tt-farnesol may enhance the cariostatic effectiveness of fluoride.

Interactions of Streptococcus mutans glucosyltransferase B with lysozyme in solution and on the surface of hydroxyapatite.

Several active enzymes have been identified as components of acquired enamel pellicle. In the present study, the interactions of Streptococcus mutans glucosyltransferase B (GtfB) with lysozyme in solution and on the surface of hydroxyapatite (HA) beads were studied. Experiments were also performed to investigate whether structural differences exist between glucans formed by GtfB enzyme in the presence or absence of lysozyme in solution and on the surface of HA. Hen egg-white lysozyme (HEWL) and saliva were used as the sources of lysozyme; lysozyme-depleted saliva was used as control. Lysozyme activity was significantly reduced when adsorbed onto HA beads compared with that in solution. The GtfB enzyme did not affect the activity of lysozyme in solution or that of adsorbed lysozyme onto HA. The presence of HEWL increased GtfB activity; bovine serum albumin had an even greater enhancing effect. Depletion of lysozyme from whole saliva increased GtfB activity in solution, but not on the surface of saliva-coated HA. The presence of lysozyme affected the amount of glucan formation by GtfB, but not the structure of glucans formed in solution and on the surface. Therefore, the interaction of lysozyme and GtfB enzymes on HA surface may modulate the formation of glucan and dental plaque.

Effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats.

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt-farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt-farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt-farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90-95%) and on the surface of saliva-coated hydroxyapatite beads (35-58%); it was devoid of antibacterial activity. tt-Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth-surface caries was significantly reduced by apigenin +tt-farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).

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.