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In addition to occasional opportunistic colonization of the oral mucosa, Candida albicans is frequently found in carious dentin. The yeast's potential to induce dental caries as a consequence of its pronounced ability to produce and tolerate acids was investigated. Eighty caries-active Osborne-Mendel rats were raised on an ampicillin-supplemented diet and exposed to C. albicans and/or Streptococcus mutans, except for controls. Throughout the 28-day test period, the animals were offered the modified cariogenic diet 2000a, containing 40% various sugars. Subsequently, maxillary molars were scored for plaque extent. After dissection, the mandibular molars were evaluated for smooth surface and fissure caries. Test animals exposed to C. albicans displayed considerably more advanced fissure lesions (p < 0.001) than non-exposed controls. While S. mutans yielded similar results, a combined association of C. albicans and S. mutans had no effect on occlusal caries incidence. Substituting dietary sucrose by glucose did not modify caries induction by C. albicans. However, animals fed a diet containing 20% of both sugars showed no differences to non-infected controls. Smooth surface caries was not generated by the yeast. This study provides experimental evidence that C. albicans is capable of causing occlusal caries in rats at a high rate., (Copyright © 2011 S. Karger AG, Basel.)

Xylitol has been claimed to reduce mutans streptococci (MS) in dental plaque by energy-consuming futile metabolic cycles. This study aimed to investigate the effects of xylitol on MS in an in vitro 6-species oral biofilm model. Each multispecies biofilm contained either a laboratory reference strain, a fresh isolate, a xylitol-sensitive or a xylitol-resistant strain of Streptococcus mutans or Streptococcus sobrinus. Biofilms, grown on pellicle-coated hydroxyapatite discs, were fed with a glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in saliva between feedings. Before or after feeding, biofilms were exposed to either 7.5% xylitol, 7.5% sorbitol or to saliva (control) for 20 min. After 64.5 h, biofilms were harvested and the microbial composition was analysed by non-selective and selective culturing. Strain variability in the ability to colonize biofilms was observed. However, the response patterns in the biofilms to the 4 polyol treatments were similar. None of the MS were inhibited by xylitol provided either before or after feeding. Sorbitol given before feeding did not affect microbial growth whereas sorbitol provided after feeding showed a slight, albeit statistically significant increase in MS counts for some of the tested strains. It did so at the expense of Streptococcus oralis, which decreased in numbers. The present findings do not support the contention that xylitol reduces MS in plaque by futile metabolic cycles., (2010 S. Karger AG, Basel.)

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 degrees 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., (Copyright (c) 2006 S. Karger AG, Basel.)

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., (Copyright 2004 S. Karger AG, Basel)

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., (Copyright 2002 S. Karger AG, Basel)

Three animal studies were performed to investigate the influence of the macromolecular structure of milk casein on caries incidence and the possible ecological changes of the oral microbiota by such casein fractions. Towards this end, rats were infected with mixed bacterial suspensions of Streptococcus sobrinus OMZ 176 and Actinomyces viscosus Ny1. Various milk protein fractions were incorporated into carefully balanced powdered cariogenic diets to constitute the sole major protein component. Diets containing micellar casein had a pronounced and highly significant effect on almost all clinical and microbiological parameters examined. Both the formation of advanced dentinal fissure (B) and smooth surface (E) caries lesions was inhibited by diets containing micellar casein; this caries-inhibiting effect appeared to be due mainly to modifications within the plaque microbiota. The proportion of S. sobrinus in the oral cavity of rats was reduced (73-80%) by micellar casein-containing preparations, whereas the A. viscosus population was increased. Both these microbiological parameters were always negatively correlated. This appears to be the first example of a food component other than dietary sugars, selectively modifying the composition of the dental plaque microbiota of rats in such a way as to reduce its pathogenic potential. It also demonstrates the importance of establishing a molecular basis for the role of food components, which prove to be beneficial to oral health.

The microbiota of root caries lesions of different grades of severity were studied. Fourteen lesions were examined. The experimental design of the study allowed correlation of histopathologically distinguishable stages with specific and distinct microbial populations. Dentin samples were ground in a sterile mortar and cultured anaerobically on nonselective Columbia blood agar plates supplemented with 5% hemolyzed human blood and on media selective for Lactobacillus spp. and streptococci. The cultivable microbiota were quantitatively speciated using Rapid ID 32A, Rapid ID 32 Strep, API 20 Strep, APIZYM, and API50 CH tests and SDS-PAG electrophoresis. In initial as well as in advanced lesions gram-positive bacteria accounted for approximately 90% of the CFUt. The proportion of Actinomyces, and in particular A. naeslundii was significantly higher (p < 0.05) in initial lesions than in advanced lesions. In contrast, the percentage of Streptococcus and especially S. mutans was higher (p < 0.05) in advanced than in initial lesions. Surprisingly low (0.8% of the CFUt) was the percentage of lactobacilli in advanced lesions. Gram-negative bacteria formed a minor part of the microbiota in both initial and advanced lesions. Among the gram-negative isolates, Prevotella, Selenomonas, and Bacteroides spp. were most noticeable. In advanced lesions, only the outermost layer of 0.5 mm thickness was populated by a high number of bacteria; the following segments harbored a negligible number of bacteria only. It is concluded that root caries is a continuous destruction process which is restricted to a subsurface zone of limited depth. The necrotic dentin is successively worn away, leading to a saucer-shaped cavitation which is repopulated by plaque. The creation of cavitations favors an aciduric flora. This might explain the succession of bacterial populations observed during the destruction process.

The plaque microbiota covering sound or carious root surfaces were studied and compared with that covering arrested root caries lesions. From each of these categories five extracted teeth were examined. The experimental design of the study allowed us to relate the qualitative and quantitative microbial composition to the degree of integrity of the root surface. Plaque was sampled by a newly developed 'mowing' technique. Plaque samples were cultured anaerobically on nonselective Columbia blood agar plates supplemented with 5% hemolyzed human blood and on media selective for Lactobacillus spp. and streptococci of the mutans group. The cultivable microbiota were quantitatively speciated using Rapid ID 32A, Rapid ID 32 Strep, API 20 Strep, API ZYM, and API 50 CH tests and SDS-PAG electrophoresis. Regardless of the state of mineralization, the microbiota on all surfaces resembled marginal plaque associated with gingivitis. In addition to the gram-positive predominant facultative anaerobic genera Streptococcus, Staphylococcus, Lactobacillus and Actinomyces, gram-negative anaerobes, predominantly Bacteroides, Prevotella, Selenomonas, Fusobacterium, Leptotrichia, and Capnocytophaga, showed the highest isolation frequencies. On all surfaces Actinomyces spp. predominated, with streptococci and lactobacilli forming a minor part of the microbiota. With respect to the detected proportions of anaerobes, microaerophiles, Actinomyces naeslundii, Prevotella buccae and Selenomonas dianae, significant differences were observed between the three categories of root surfaces. The total CFU's on both caries-free and caries-active surfaces were significantly higher than on arrested lesions. In general, the results support a polymicrobial etiology for caries initiation on root surfaces, with A. naeslundii, Capnocytophaga spp., and Prevotella spp. making specific contributions to the processes of cementum and dentin breakdown.

The histopathology of arrested root caries lesions was examined in extracted human teeth. The main structural characteristics of arrested lesions were the completely mineralized surface area and the formation of a distinct sclerosis of the dentinal tubules. Intertubular dentin was, with the exception of the dentinal tubules, fully mineralized up to the surface. Dentinal tubules near the surface were either filled with ghosts of microorganisms or with crystals of different shapes. Sclerosis of the dentinal tubules was characterized by the presence of three different patterns of intratubular mineralization that occur in distinct regions of the zone of tubular sclerosis. The patterns were distinguishable by the type of crystals and their association with organic structures such as collagenous fibrils or odontoblast processes. It is suggested that arrested lesions are based on (1) the formation of an inner barrier that interrupts the diffusion of substrata from the pulp to invaded bacteria, (2) the formation of an outer barrier by a compact, highly mineralized surface region which blocks the diffusion of products of bacterial metabolism into dentin, and (3) an area of mineralization which extends from the outer barrier toward the root canal within demineralized dentin. The present study demonstrates the considerable potential of caries lesions in dentin to become arrested, and subsequently partially remineralized. These phenomena seem to depend on the severity of an active lesion and its location on the root surface. This should be taken into account when diagnosing root caries lesions. The potential of root caries lesions to become arrested indicates that the treatment concept of active root caries lesions should be reconsidered.

The cariogenicity of Ferrero Milchschnitte and apple was tested in rats using a frequency-controlled feeding machine. Twelve litters of Osborne-Mendel rats orally infected with Streptococcus sobrinus OMZ-176 and Actinomyces viscosus Ny-1 were used. Four treatment groups were tested: (1) cooked starch/sucrose 1:1 as positive control; (2) Ferrero Milchschnitte; (3) apple; (4) gastric intubation only as negative control. Groups 1-3 were additionally fed by gastric intubation with essential nutrients. Rats on Milchschnitte and apple had less fissure and smooth-surface caries than rats on a starch/sucrose diet, but significantly more than the negative controls. The Milchschnitte group exhibited less plaque extent than the positive and the negative control. The apple group showed a higher plaque extent and massively extended erosions on molar surfaces. The CFU means of S. sobrinus were decreasing from treatment 1-4. Although differences between treatments were not significant, they were associated by concomitantly decreasing caries incidence. The test foods were also assessed using intraoral plaque-pH telemetry in 6 human volunteers. Both Milchschnitte and apple were acidogenic in human telemetry. They can therefore not be regarded as safe for teeth.

The histopathology of human root caries was examined in extracted teeth. The present study details several stages of advanced carious lesions in root dentin. Lesions of different severities were assessed by different optical methods. Four major root caries patterns were defined and significant differences between coronal and root caries elucidated. The patterns were distinguishable by (1) their location at the exposed root surface, (2) their mode of spreading towards the root canal and over the root surface, (3) their mode of bacterial spreading in dentin and (4) the role of dentinal defense mechanisms. In addition, a comparison of clinical and histological classification of lesions revealed that the carious process extended much deeper towards the root canal and had a higher degree of both bacterial infection and dentin demineralization than could be predicted by clinical examination.