Academic literature on the topic 'Biofilm cariogenicity'

To exert anticaries effects, probiotics are described to inhibit growth and biofilm formation of cariogenic bacteria such as Streptococcus mutans (SM). We screened 8 probiotics and assessed how SM growth or biofilm formation inhibition affects cariogenicity of probiotic-SM mixed-species biofilms in vitro. Growth inhibition was assessed by cocultivating probiotics and 2 SM strains (ATCC 20532/25175) on agar. Probiotics were either precultured before SM cultivation (exclusion), or SM precultured prior to probiotic cultivation (displacement). Inhibition of SM culture growth was assessed visually. Inhibition of SM biofilm formation on bovine enamel was assessed using a continuous-flow short-term biofilm model, again in exclusion or displacement mode. The cariogenicity of mixed-species biofilms of SM with the most promising growth and biofilm formation inhibiting probiotic strains was assessed using an artificial mouth model, and enamel mineral loss (ΔZ) was measured microradiographically. We found limited differences in SM growth inhibition in exclusion versus displacement mode, and in inhibition of SM 20532 versus 25175. Results were therefore pooled. Lactobacillus acidophilus LA-5 inhibited significantly more SM culture growth than most other probiotics. L. casei LC-11 inhibited SM biofilm formation similarly to other alternatives but showed the highest retention of probiotics in the biofilms (p < 0.05). Mineral loss from SM monospecies biofilms (ΔZ = 9,772, 25th/75th percentiles: 6,277/13,558 vol% × µm) was significantly lower than from mixed-species SM × LA-5 biofilms (ΔZ = 24,578, 25th/75th percentiles: 19,081/28,768 vol% × µm; p < 0.01) but significantly higher than from SM × LC-11 biofilms (ΔZ = 4,835, 25th/75th percentiles: 263/7,865 vol% × µm; p < 0.05). Probiotics inhibiting SM culture growth do not necessarily reduce the cariogenicity of SM-probiotic biofilms. Nevertheless, SM biofilm formation inhibition may be relevant in the reduction of cariogenicity.

Despite promising results using probiotics, evidence of the preventive effect on enamel demineralization is insufficient and the cariogenic potential of probiotics is still controversial. Probiotics could affect biofilm formation and interfere with adherence, growth or coaggregation with Streptococcus mutans in biofilms. However, most of the studies have been conducted using planktonic bacteria. Hence, the aim of the study was to assess the effect of probiotic bacteria on the cariogenicity of S. mutans using an in vitro biofilm caries model on enamel. Single-species biofilms (S. mutans UA159, SM or Lactobacillus rhamnosus LB21, LB) or dual-species biofilms simultaneously inoculated (SM + LB) or LB inoculated 8 h after SM (SM → LB) were grown for 96 h. Biofilms were formed on bovine enamel saliva-coated slabs of known surface hardness (SH) and immersed in culture media. Biofilms were exposed 8 times per day to 10% sucrose. Medium pH was monitored twice daily as a biofilm acidogenicity indicator. After 96 h, biofilms were collected to determine biomass and bacteria viability. Slab demineralization was calculated as percentage of SH loss (%SHL). Additionally, the model was tested with different concentrations of the initial inoculum (103, 106, 108 cells/ml) and different adhesion times (2 or 8 h). The dual-species biofilm revealed no LB effects on SM cariogenicity, without changes in acidogenicity or %SHL among groups (p > 0.05, n = 12). Lack of activity of LB on SM cariogenicity persisted even when 105 times higher concentration of the probiotic was tested. Coaggregation was not observed. In conclusion, findings suggest that LB does not reduce cariogenicity of SM in a validated experimental caries model.

ABSTRACT Objectives Frequent consumption of sugars-containing carbonated beverages has been associated with caries, but the consequences on the dental biofilm remain unclear. The aim was to evaluate the effect of commercial carbonated beverages and their sugar-free version on enamel and dentine demineralization and on the cariogenic properties of Streptococcus mutans biofilms. Materials and Methods Biofilms of S. mutans UA159 were grown on enamel and dentin slabs and exposed 3 times/ day for 5 min, to a commercial cola or orange-flavored carbonated beverage or to their sugar-free version. Biofilms/slabs were recovered to assess biomass, viable microorganisms, protein content and polysaccharides. Demineralization was estimated by the variation of Knoop surface microhardness. Results Exposures to the biofilm with sugars-containing carbonated beverages resulted in similar biomass, viable microorganisms, proteins, and polysaccharides than sucrose (P < 0.05). The sugar-free cola and orange-flavored drink showed lower effect on the biofilm, as compared with sucrose or their sugared version (P < 0.05). All of the products tested, included the sugar-free, showed higher demineralization than the negative control (P < 0.05). Conclusions: Sugars-containing carbonated beverages enhance cariogenic activity of S. mutans biofilms, comparable with sucrose. Sugar-free carbonated beverages also have a high demineralizing potential, without affecting biofilm properties.

Streptococcus mutans are considered the most cariogenic bacteria, but it has been suggested that Candida albicans could increase their cariogenicity. However, the effect of this dual-species microorganisms’ combination on dentine caries has not been experimentally evaluated. Biofilms of C. albicans, S. mutans and C. albicans + S. mutans (n = 12/biofilm) were grown in ultra-filtered tryptone yeast extract broth culture medium for 96 h on root dentine slabs of known surface hardness and exposed 8 times per day for 3 min to 10% sucrose. The medium was changed 2 times per day (after the 8 cariogenic challenges and after the overnight period of famine), and aliquots were analyzed to determinate the pH (indicator of biofilm acidogenicity). After 96 h, the biofilms were collected to determine the wet weight, colony-forming units, and the amounts of extracellular polysaccharides (soluble and insoluble). Dentine demineralization was assessed by surface hardness loss (% SHL). The architecture of the biofilms was analyzed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Data were analyzed by ANOVA followed by Tukey’s test (α = 0.05). The dual-species C. albicans + S. mutans biofilm provoked higher % SHL on dentine (p < 0.05) than the S. mutans and C. albicans biofilm. This was supported by the results of biofilm acidogenicity and the amounts of soluble (6.4 ± 2.14 vs. 4.0 ± 0.94 and 1.9 ± 0.97, respectively) and insoluble extracellular polysaccharides (24.9 ± 9.22 vs. 18.9 ± 5.92 and 0.7 ± 0.48, respectively) (p < 0.05). The C. albicans biofilm alone presented low cariogenicity. The images by CLSM and TEM, respectively, suggest that the C. albicans + S. mutans biofilm is more voluminous than the S. mutans biofilm, and S. mutans cells interact with C. albicans throughout polysaccharides from the biofilm matrix. These findings show that C. albicans enhances the cariogenic potential of the S. mutans biofilm, increasing dentine demineralization.

Extracellular polysaccharides (EPS) could increase the penetration of fluoride through dental biofilm, reducing its cariogenicity. We measured the concentration of fluoride in EPS-containing (EPS+) or not-containing (EPS–) Streptococcus mutans bacterial pellets resembling test biofilms, before and up to 60 min after a 0.05% NaF rinse in situ. Fluoride penetration and clearance were higher in EPS+ bacterial pellets. The data suggest that EPS enhances fluoride penetration, but also accelerates fluoride clearance from dental biofilms.

ABSTRACT Objective: Although sucrose is considered the most cariogenic carbohydrate in the human diet, the question of how many exposures are needed to induce damage on the hard dental tissues remains unclear. To approach this question, different frequencies of daily sucrose exposure were tested on a relevant biological caries model. Materials and Methods: Biofilms of the Streptococcus mutans were formed on enamel slabs and exposed to cariogenic challenges with 10% sucrose for 5 min at 0, 1, 3, 5, 8, or 10 times per day. After 5 days, biofilms were retrieved to analyze biomass, protein content, viable bacteria, and polysaccharide formation. Enamel demineralization was evaluated by percentage of microhardness loss (percentage surface hardness loss [%SHL]). Results: Biomass, protein content, polysaccharide production, acidogenicity of the biofilm, and %SHL proportionally increased with the number of daily exposures to sucrose (P < 0.05). One daily sucrose exposure was enough to induce 20% more demineralization than the negative unexposed control. Higher frequencies induced greater demineralization and more virulent biofilms, but eight and ten exposures were not different between them in most of the analyzed variables (P > 0.05). Conclusions: Higher sucrose exposure seems to increase cariogenicity, in a frequency-dependent manner, by the modification of bacterial virulent properties.

Streptococcus mutans is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense vicR RNA (AS vicR) associated with an adjacent RNase III–encoding ( rnc) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the AS vicR RNA mainly regulated carbohydrate metabolism. In particular, overproducing AS vicR demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from AS vicR presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by rnc-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an rnc-related messenger RNA, was verified to bind to the 5′-UTR regions of the vicR gene. Furthermore, AS vicR regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III–encoding genes. There was an increase in AS vicR transcript levels in clinical S. mutans strains from caries-free children, while the expression of AS vicR was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of AS vicR-mediated sponge regulation in S. mutans, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.

Dental caries is a biofilm-dependent oral disease, and fermentable dietary carbohydrates are the key environmental factors involved in its initiation and development. However, among the carbohydrates, sucrose is considered the most cariogenic, because, in addition to being fermented by oral bacteria, it is a substrate for the synthesis of extracellular (EPS) and intracellular (IPS) polysaccharides. Therefore, while the low pH environment triggers the shift of the resident plaque microflora to a more cariogenic one, EPS promote changes in the composition of the biofilms’ matrix. Furthermore, it has recently been shown that the biofilm formed in the presence of sucrose presents low concentrations of Ca, Pi, and F, which are critical ions involved in de- and remineralization of enamel and dentin in the oral environment. Thus, the aim of this review is to explore the broad role of sucrose in the cariogenicity of biofilms, and to present a new insight into its influence on the pathogenesis of dental caries.

Streptococcus mutans is an important factor in the etiology and pathogenesis of dental caries, largely owing to its ability to form a stable biofilm. Previous animal studies have indicated that rnc could decrease the amount of sulcal caries, and that the downregulation of cariogenicity might be due to its capacity to disrupt biofilm formation. However, the biofunctions by which rnc is involved in biofilm formation remain to be elucidated. In this study, we further investigate the role of rnc based on the study of mature biofilm. Scanning electron microscopy and the crystal violet assay were used to detect the biofilm forming ability. The production and distribution of exopolysaccharides within biofilm was analyzed by exopolysaccharide staining. Gel permeation chromatography was used to perform molecular weight assessment. Its adhesion force was measured by atomic force microscopy. The expression of biofilm formation-associated genes was analyzed at the mRNA level by qPCR. Here, we found that rnc could occur and function in biofilm formation by assembling well-structured, exopolysaccharide-encased, stable biofilms in S. mutans. The weakened biofilm forming ability of rnc-deficient strains was associated with the reduction of exopolysaccharide production and bacterial adhesion. Over all, these data illustrate an interesting situation in which an unappreciated regulatory gene acquired for virulence, rnc, most likely has been coopted as a potential regulator of biofilm formation in S. mutans. Further characterization of rnc may lead to the identification of a possible pathogenic biofilm-specific treatment for dental caries.

Extracellular polysaccharides (EPS), mainly the insoluble ones, increase the cariogenicity of dental biofilm, but whether they interfere with the binding and retention of fluoride is unknown. EPS-rich (EPS+) and EPS-poor (EPS–) pellets of <i>Streptococcus mutans</i> were formed and treated with increasing fluoride concentrations (0, 0.1, 1, or 10 mM). A concentration-dependent fluoride binding was observed in both EPS– and EPS+ pellets, but the presence of EPS did not affect the retention of fluoride in the pellets. In conclusion, the data suggest that a matrix of dental biofilm rich in EPS does not affect fluoride retention in the biofilm.

Orientador: Jaime Aparecido Cury
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-24T16:20:44Z (GMT). No. of bitstreams: 1 Botelho_JulianaNunes_D.pdf: 1920977 bytes, checksum: 99c53e9c14bfc15c6b8859cce1fe5dec (MD5) Previous issue date: 2014
Resumo: Sacarose é o carboidrato mais cariogênico da dieta e o amido é considerado não cariogênico para esmalte e moderadamente cariogênico para dentina. Por outro lado, a combinação de amido e sacarose (amido+sacarose) tem sido considerada mais cariogênica que apenas sacarose, mas esse ainda é um assunto em debate. Além do mais, o efeito do dentifrício fluoretado na cariogenicidade dessa combinação é desconhecido. Assim, com o objetivo de estudar esse assunto três experimentos foram conduzidos: (i) o primeiro avaliou efeito de amido+sacarose na desmineralização de esmalte e dentina, usando um modelo de biofilme de S. mutans modificado pela adição de saliva para simular a ação da amilase, (ii) o segundo avaliou in situ o efeito do dentifrício contendo 1.100 µg F/g (DF) na progressão da desmineralização da dentina radicular, e o terceiro (iii) avaliou in situ o efeito do fluoreto no potencial cariogênico de amido+sacarose na desmineralização de esmalte e dentina. In vitro, biofilmes de S. mutans foram formados sobre blocos de esmalte e dentina radicular, por 5 e 4 dias respectivamente, em meio de cultura contendo saliva e expostos a um dos seguintes tratamentos: amido a 1%, sacarose a 10% ou de sua combinação (8x/dia). Os biofilmes foram analisados quanto às suas composições bioquímicas e microbiológicas, e a desmineralização dos blocos foi avaliada. Biofilmes expostos à combinação foram mais acidogênicos (p<0,0001) e provocaram maior desmineralização (p<0,0001) no esmalte e dentina que o efeito dos carboidratos isolados. In situ, o efeito do DF foi testado em um estudo piloto, cruzado no qual sacarose a 10% foi aplicada extraoralmente 8x/dia em 2 fases de 14 dias. Após 10 e 14 dias em cada fase, a desmineralização da dentina foi avaliada. O efeito do dentifrício foi significativo (p<0,0001), mas o efeito do tempo não (p>0,05). Esses resultados sugerem que o DF com 1.100 µg F/g é capaz de diminuir a cárie dentinária mesmo sob alto desafio cariogênico de acúmulo de biofilme e exposição à sacarose. In situ, o efeito dos tratamentos (água, amido a 2%, sacarose a 10% e amido+sacarose) e o efeito do dentifrício (não fluoretado e fluoretado) foram testados em um estudo cruzado, cego, boca-dividida em 4 fases de 14 dias. Os voluntários usaram dois dos tratamentos 8x/dia e um dos dentifrícios 3x/dia. O efeito dos fatores (dentifrício e tratamentos) foram significativos (p<0,05) para esmalte e dentina, mas a interação não (p>0,05). Os resultados sugerem que, independente do desafio cariogênico provocado pelos diferentes carboidratos da dieta testados, o dentifrício fluoretado é efetivo na redução da desmineralização de esmalte e dentina. Em conclusão, os resultados sugerem que amido deve aumentar o potencial cariogênico da sacarose mas que fluoreto de dentifrício é capaz de reduzir a desmineralização tanto do esmalte quanto da dentina provocada pela combinação desses carboidratos
Abstract: Sucrose is the most cariogenic dietary carbohydrate while starch is considered non-cariogenic for enamel and slightly cariogenic for dentine. The combination starch and sucrose (starch+sucrose) has been considered more cariogenic than sucrose alone but this subject remains debatable. Also, the effect of fluoride toothpaste on the cariogenicity of this combination is unknown. The aims of this study were to evaluate: (i) the effect of starch+sucrose on enamel and dentine demineralization using an S. mutans biofilm model modified by adding human saliva to simulate amylase action; (ii) the in situ effect of fluoride toothpaste (FT) containing 1100 µg F/g on dentine demineralization progression; and (iii) the in situ effect of fluoride on the cariogenic potential of starch+sucrose on enamel and dentine demineralization. In vitro, S. mutans biofilms were grown on enamel and root dentine slabs for 5 and 4 days, respectively, in a saliva-containing medium and exposed to the following treatment: 1% starch; 10% sucrose; or starch+sucrose (8x/day). Biofilms were then analyzed for their biochemical and microbiological compositions, and dental demineralization was evaluated. Biofilms exposed to starch+sucrose were more acidogenic (p < 0.0001) and caused higher demineralization (p < 0.0001) on either enamel or dentine than those exposed to each carbohydrate alone. The in situ effect of FT on dentine demineralization was tested in a pilot crossover study, in which 10% sucrose was applied extra-orally to the slabs 8x/day in 2 phases of 14 days each. At days 10 and 14 of each phase, dentine demineralization was evaluated. The effect of toothpaste was significant (p<0.0001), but the effect of time was not (p>0.05). The results suggest that FT at 1100 µg F/g can reduce dentine demineralization even under high cariogenic challenges - biofilm accumulation and sugar exposure. The in situ effect of the treatments (water, 2% starch, 10% sucrose and starch+sucrose) and that of the toothpastes (non-FT and FT) were tested in a crossover, single-blind and split-mouth study conducted in 4 phases of 14 days each. The volunteers used two of the treatments 8 times/day and one of the toothpastes 3 times/day. The effect of the factors (toothpaste and treatments) was significant (p<0.05) for enamel and dentine, but not (p>0.05) for the interaction. The findings suggest that, regardless of the cariogenic challenge provoked by the different sources of the dietary sugars tested, fluoride toothpaste is effective in reducing enamel and dentine demineralization. In conclusion, the results suggest that starch may enhance the cariogenic potential of sucrose and fluoride from toothpaste reduces enamel and dentine demineralization caused by the combination of these carbohydrates
Doutorado
Cariologia
Doutora em Odontologia

Indiana University-Purdue University Indianapolis (IUPUI)
Dental biofilm is a main contributing factor in the initiation and progression of dental caries. The maturation of dental biofilms is expected to alter the anti-caries efficacy of fluoride compounds. In the first aim, we conducted a series of modeldevelopment experiments to test different variables to standardize a reproducible in-vitro microbial caries model. We evaluated: surface conditioning using saliva; sucrose concentrations and caries lesion severity; growth media conditions and mineral saturation; dental substrate types; pH cycling protocol characteristics. In the second aim, we used the developed model to evaluate the changes in the anti-caries efficacy of three fluoride compounds (Sodium fluoride (NaF); Stannous fluoride (SnF2); Amine fluoride (AmF); and deionized water (DIW- negative control)) at increasing maturation of a microcosm biofilm. We continued the pH cycling protocol for 4 days, 8 days, and 12 days. We tested biofilm cariogenicity and carious lesion severity at each maturation stage. In the third aim, we used the developed model to test the effect of different exposure periods (early vs. late exposure) of the biofilm to three fluoride compounds (NaF, SnF2, AmF, DIW) in comparison to DIW. We also evaluated the recovery of biofilm cariogenicity with each exposure period. We evaluated, for each exposure period and recovery stage, biofilm cariogenicity and carious lesion severity. We analyzed the relationships between different variables (biofilm age, fluoride compound type, exposure period) using ANOVA models. In conclusion: 1. The present model allows testing the effect of biofilm maturation on the anti-caries efficacy of fluoride compounds. 2. Biofilm maturation plays an important role in increasing biofilm tolerance against fluoride treatment; it could also influence the selection of fluoride compounds to achieve optimum cariostatic effect. 3. Exposure period, and type of fluoride compound, both influence the biofilm tolerance to fluoride anti-caries effect; they may also result in a sustainable release of fluoride over time.
2021-08-21