E. Giertsen

Validation of an in vitro Biofilm Model of Supragingival Plaque

The study of biofilm structure and function mandates the use of model systems for which a host of environmental variables can be rigorously controlled. We describe a model of supragingival plaque containing Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus, and Streptococcus oralis wherein cells are cultivated anaerobically in a saliva-based medium on hydroxyapatite discs coated with a salivary pellicle, with material and pieces of apparatus common to all microbiology laboratories. After 0.5 hr, 16.5 hrs, 40.5 hrs, and 64.5 hrs, the composition of adherent biofilms was analyzed by culture techniques, live/dead fluorescence staining, and confocal laser scanning microscopy. Repeated independent trials demonstrated the repeatability of biofilm formation after 40.5 hrs and 64.5 hrs. Brief exposures of biofilms to chlorhexidine or Triclosan produced losses in viability similar to those observed in vivo. This biofilm model should prove very useful for pre-clinical testing of prospective anti-plaque agents at clinically relevant concentrations.

An in vitro Oral Biofilm Model for Comparing the Efficacy of Antimicrobial Mouthrinses

The ability of commercial mouthrinses to reduce total viable counts of mixed microbial populations was examined using a previously developed in vitro model of supragingival plaque. Exploratory experiments aimed at fine-tuning the model indicated that optimal correspondence between in vitro and clinical results for chlorhexidine-containing formulations were obtained at a saliva:medium ratio of 70:30 (v/v); moreover, expanding the microbial population from 5 bacterial species to 5 bacterial species + Candida albicans had no noticeable impact on overall results. The efficacies of 12 different mouthrinse proprietary products containing chlorhexidine, hexetidine, octenidine, Triclosan, plant extracts, or aminefluoride/stannous fluoride vis-à-vis biofilm clearance were compared. All mouthrinses promoted a statistically significant reduction in microbial load compared to distilled water. The herbal- and phenolic-based products were substantially less effective than most chlorhexidine-containing mouthrinses, or mouthrinses containing hexetidine or octenidine. No significant difference between the plaque-clearing plaque-clearing abilities of Listerine® and Meridol® was observed. This polyspecies biofilm model can be a valuable tool for preclinical testing of antiplaque formulations, particularly during the product development stage.

Application of the Zürich biofilm model to problems of cariology.

The term biofilm is increasingly replacing ‘plaque’ in the literature, but concepts and existing paradigms are changing much more slowly. There is little doubt that biofilm research will lead to more realistic perception and interpretation of the physiology and pathogenicity of microorganisms colonizing plaques in the oral cavity. There is clear evidence that the genotypic and phenotypic expression profiles of biofilm and planktonic bacteria are different. Several techniques are available today to study multispecies biofilms of oral bacteria, each having its particular advantages and weaknesses. We describe a biofilm model developed in Zürich and demonstrate a number of applications with direct or indirect impact on prophylactic dentistry: spatial arrangement and associative behavior of various species in biofilms; multiplex fluorescent in situ hybridization analysis of oral bacteria in biofilms; use of the biofilm model to predict in vivo efficacy of antimicrobials reliably; mass transport in biofilms; de- and remineralization of enamel exposed to biofilms in vitro. The potential of biofilm experimentation in oral biology has certainly not yet been fully exploited and dozens of possible interesting applications could be investigated. The overall physiological parameters of multispecies biofilms can be measured quite accurately, but it is still impossible to assess in toto the multitude of interactions taking place in such complex systems. What can and should be done is to test hypotheses stemming from experiments with planktonic cells in monospecies cultures. In particular, it will be interesting to investigate the relevance to biofilm composition and metabolism of specific gene products by using appropriate bacterial mutants.

Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque

Our aim was to develop a rapid fluorescent in situ hybridization (FISH) assay for the identification of different oral groups of streptococci in dental plaque and to combine it with digital image analysis for the automated enumeration of target cells. Cy3-labeled oligonucleotide probes specific for 16S rRNA gene sequences of the anginosus, mitis, mutans, and salivarius groups of streptococci were hybridized under stringent conditions with bacterial cultures or supragingival plaque samples that had been permeabilized with lysozyme. Probe specificity was determined with strains from 30 different species, mainly of oral origin. Results showed that probes ANG541, MIT447, SSP001, and SAL090 with specificity for the anginosus, mitis, mutans, and salivarius groups, respectively, the pan-reactive streptococcal probe STR405, the S. mutans specific probe MUT590, and the S. sobrinus specific probe SOB174 were well-suited for the identification of cultured streptococci. Probes STR405, MIT447 and SSP001 were then successfully applied to enumerate automatically bacteria of the recognized taxa in 144 supragingival plaque samples. On the average, total streptococci accounted for 8.2%, streptococci of the mitis and mutans groups for 3.9 and 1.7%, respectively, of the plaques. The combined application of FISH and automated image analysis provides an objective time-saving alternative to culture or PCR for the enumeration of selected oral streptococci in dental plaque.

Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro.

The aim of this study was to examine the influence of glucosyltransferase-gene-negative (gtf –) Streptococcus mutans strains unable to synthesize water-insoluble or soluble glucan on the structure and macromolecular diffusion properties of in vitro grown mixed oral biofilms. Biofilms modeling supragingival plaque consisted of Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2, Streptococcus oralis SK 248, Veillonella dispar ATCC 17748T and one of the S. mutans strains UA159, OMZ 966, OMZ 937 or OMZ 977. Biofilms were grown anaerobically on sintered hydroxyapatite disks for 64.5 h at 37°C. To perform confocal laser scanning microscopy analyses, microorganisms were stained with Syto 13 and extracellular polysaccharides (EPS) with Calcofluor. Macromolecular diffusion properties were measured following timed biofilm exposure to Texas-Red-labeled 70-kDa dextran. Results showed that replacing wild-type S. mutans by a gtfC – mutant led to an increase in the volume fraction occupied by cells from 29 to 48% and a decrease of the EPS volume fraction from 51 to 33%. No such changes were observed when the S. mutans wild-type strain was replaced by a gtfB – or gtfD – mutant. The diffusion coefficient of 70-kDa dextran in biofilms containing the gtfC – S. mutans was 16-fold higher than in biofilms with the wild-type strain indicating a strong macromolecular sieving effect of GTF C-generated glucans. Our data demonstrate the influence of EPS on the structure and macromolecular diffusion properties of an oral biofilm model and uncover our still limited knowledge of the function of EPS in biofilms and plaque.

In vitro quantitative light-induced fluorescence to measure changes in enamel mineralization

A sensitive, quantitative method for investigating changes in enamel mineralization of specimens subjected to in vitro or in situ experimentation is presented. The fluorescence-detecting instrument integrates a Xenon arc light source and an object positioning stage, which makes it particularly suitable for the nondestructive assessment of demineralized or remineralized enamel. We demonstrate the ability of in vitro quantitative light-induced fluorescence (QLF) to quantify changes in mineralization of bovine enamel discs that had been exposed in vitro to a demineralizing gel (n=36) or biofilm-mediated demineralization challenges (n=10), or were carried in situ by three volunteers during a 10-day experiment (n=12). Further experiments show the techniques value for monitoring the extent of remineralization in 36 specimens exposed in vitro to oral multispecies biofilms and document the repeatability of in vitro QLF measurements (n=10) under standardized assay conditions. The validity of the method is illustrated by comparison with transversal microradiography (TMR), the invasive current gold standard for assessing experimental changes in enamel mineralization. Ten discs with 22 measurement areas for comparison demonstrated a positive correlation between TMR and QLF (r=0.82). Filling a technological gap, this QLF system is a promising tool to assay in vitro nondestructively localized changes in mineralization of enamel specimens.

Plaque Inhibition by a Combination of Zinc Citrate and Sodium Lauryl Sulfate

Bacteriological tests demonstrated an additive inhibitory effect of ZnCl2 and sodium lauryl sulfate (SLS) on in vitro growth of Streptococcus sobrinus OMZ 176 and of Streptococcus sanguis ATCC 10556. As measured by atomic absorption spectrophotometry, the solubility of zinc citrate increased in the presence of SLS. After 48 h, the concentration of solubilized zinc from aqueous solutions of 5.0 mM zinc citrate was 12.0 mM versus 14.4 mM in the presence of 34.7 mM SLS. The plaque-inhibiting properties of aqueous solutions of 12.0 mM Zn2+ from zinc citrate, 34.7 mM SLS, and 14.4 mM Zn2+ from zinc citrate in combination with 34.7 mM SLS were examined in 7 volunteers. Plaque accumulations were assessed by using a method earlier described after 3 days of twice daily mouthrinses with 10 ml test solution, during which period no mechanical oral hygiene was performed. Compared to placebo, SLS and zinc citrate increased the frequency of plaque index score 0 by 52.9 and 98.3%, respectively, and SLS gave 36.9% and zinc citrate 55.7% less surfaces with scores 2 or 3 (p less than 0.05 in all cases). The combination of zinc citrate and SLS gave a threefold increase of plaque index score 0 and a 70.5% reduction of scores 2 or 3 (p less than 0.05).

Effects of mouthrinses with triclosan, zinc ions, copolymer, and sodium lauryl sulphate combined with fluoride on acid formation by dental plaque in vivo.

Bacteriological tests demonstrated a slight synergistic effect of triclosan and sodium lauryl sulphate (SLS) on the growth of Streptococcus mutans NCTC 10449 and Streptococcus sanguis ATCC 10556 in vitro. A single mouthrinse with SLS (17.4 mM) or SLS plus triclosan (3.5 mM) significantly decreased the number of salivary mutans streptococci in a group of 12 subjects up to 90 min after rinsing. The effect on plaque pH of a mouthrinse with either 12.0 mM NaF, NaF plus 10.0 mM zinc acetate, NaF plus 17.4 mM SLS, or NaF plus SLS plus 3.5 mM triclosan with or without the addition of zinc ions or 0.65% w/v of a polyvinylmethyl ether/maleic acid copolymer was investigated. The plaque pH responses to a 10% w/v sucrose mouthrinse were measured in 2-day-old plaque with microtouch pH electrodes in six groups of 10 subjects 90 min after a single mouthrinse with test solution. There was no significant difference in plaque pH between the various mouthrinses. In conclusion, triclosan enhanced the growth-inhibitory activity of SLS against oral streptococci in vitro but not against salivary mutans streptococci in vivo. Neither triclosan incorporated into a mouthrinse containing SLS plus fluoride, nor the addition of zinc ions or copolymer affected acid formation by dental plaque in vivo.

Effects of Mouth Rinses with Xylitol and Fluoride on Dental Plaque and Saliva

The aim of this study was to test the hypothesis that xylitol, alone and in combination with fluoride, affects the salivary flow rate and micro–biota, dental plaque accumulation, gingivitis development, and the acidogenic potential of plaque. Three groups, each of 10 subjects, rinsed for 1 min 3 times daily over two 4–week periods, first with 10 ml water (control), and thereafter with either 0.05% NaF, 40% xylitol, or with 0.025% NaF plus 20% xylitol according to a double–blind controlled design. They performed habitual mechanical tooth cleaning during the first 2 weeks of each period but abstained from interdental cleaning during the final 2 weeks. While mouth rinsing was continued, all mechanical oral hygiene was discontinued the last 2 days of each period to permit plaque accumulation. The last mouth rinse was administered in the clinic before the final examination. The following parameters were assessed: (1) unstimulated and paraffin–stimulated salivary secretion rates; (2) salivary micro–biota; (3) plaque index; (4) papillar bleeding; (5) plaque pH response to sucrose, and (6) lactate formation by dental plaque. No statistically significant differences in any of the parameters were found. In conclusion, three daily mouth rinses with fluoride and xylitol, separately or in combination, did not affect the salivary flow rate or micro–biota, dental plaque accumulation, gingivitis development, or the acidogenic potential of plaque.

Effects of Chlorhexidine-Fluoride Mouthrinses on Viability, Acidogenic Potential, and Glycolytic Profile of Established Dental Plaque

Inhibition of dental plaque acidogenicity by chlorhexidine (CHX) mouthrinses has been ascribed to a long-lasting bacteriostatic effect due to binding of CHX to oral surface structures combined with a slow release rate from the binding sites. The present aims were to study the effects of CHX-containing mouthrinses on the viability and glycolytic activity of established plaque in order to assess the bactericidal versus the bacteriostatic effects. Following 2 days of plaque accumulation, three groups of 10 students rinsed with either 12.0 mM NaF, 0.55 mM CHX plus NaF, or with 2.2 mM CHX plus NaF. Plaque samples were collected before and 90 min after mouthrinsing. The pH in pooled pre- and post-rinse plaque samples was recorded before and up to 10 min after the addition of D-[U-14C]glucose. Total colony-forming units in each sample were determined. High-performance liquid chromatography analyses showed lactate to be the major extracellular glycolytic metabolite in all samples. CHX-NaF markedly reduced the colony-forming units, the pH fall from fermentation of glucose, as well as glucose consumption and lactate formation, whereas NaF alone exhibited no such effects. The reduction of glucose consumption by the CHX-NaF mouthrinses corresponded to the reduction of colony-forming units, indicating no bacteriostatic effect. The plaque pH in vivo was monitored in each student 90 min after mouthrinsing with the test solutions prior to and up to 1 h after a sucrose mouthrinse using touch microelectrodes. The CHX-NaF mouthrinses reduced the fall in pH significantly (p < 0.05) as compared with the NaF mouthrinse.(ABSTRACT TRUNCATED AT 250 WORDS)