Inhibition of Streptococcus mutans Growth and Biofilm Formation by Probiotics in vitro.

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.

Penetration of Streptococcus sobrinus and Streptococcus sanguinis into dental enamel.

The aim of this pilot study was to assess the difference in virulence of acidogenic and aciduric oral streptococci in an in vitro caries model using their penetration depths into dental enamel. 30 caries-free extracted molars from 11- to 16-year-olds were cleaned ultrasonically for 1 min with de-ionized water and, after air-drying, embedded in epoxy resin. After 8-h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13-16 µm). Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 µm. 15 specimens each were incubated anaerobically under standardized conditions with 24 h-cultures of Streptococcus sanguinis 9S or Streptococcus sobrinus OMZ 176 in Balmelli broth at 37 ± 2 °C; the pH-values of the broths were measured at the beginning and end of each incubation cycle. After 2, 4, 6, 8, and 10 weeks 3 teeth each were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3× and dehydrated 30-60min by sequential washes through a series of 30-100% graded ethanol. The teeth were cut in half longitudinally; afterward, two slits were made to obtain fracture surfaces in the infected area. After critical-point-drying the fragments were gold-sputtered and viewed in a scanning electron microscope at magnifications of ×20-20,000. After 10 weeks of incubation, penetration of S. sanguinis of 11.13 ± 24.04 µm below the break edges into the enamel was observed. The invasion of S. sobrinus reached depths of 87.53 ± 76.34 µm. The difference was statistically significant (paired t test: p = 0.033). The experimental penetration depths emphasize the importance of S. sanguinis versus S. sobrinus in the context of the extended ecological plaque hypothesis.

A randomized clinical trial comparing the impact of different oral hygiene protocols and sealant applications on plaque, gingival, and caries index scores.

OBJECTIVE: This prospective randomized clinical trial investigated the impact of different tooth brushing strategies and sealant application on patients with fixed appliances: plaque, gingival, caries index scores, periodontal parameters, microbial, and molecular biological parameters were assessed. MATERIALS AND METHODS: Fifty-five male and 63 female patients aged 11-15 years were enrolled in this 12-week, four-arm parallel-group trial. Patients of group 1 used a Sonicare FlexCare electric brush, patients of group 2 used a manual (elmex interX short head) plus interdental (Curaprox CPS 15) brush, and patients of group 3 and 4 used a manual brush only. The teeth of patients in groups 1, 2, and 3 were sealed with a filled resin (ProSeal). Patients were advised to brush twice daily and measured time spent brushing mornings and evenings [tooth brushing duration (TBD)]. Plaque (PIB, TQHI, MAPI) and gingival index (PBI) as well as caries index (DMFT/DMFS) scores were assessed at baseline and after 4, 8, and 12 weeks. RESULTS AND CONCLUSIONS: TBD did not differ significantly between patients using the electric or manual brush only (between 197 and 209 seconds) but was longer when using the combination of two manual brushes. TBD was slightly longer in the evenings. There was no gender difference. Although TBD was longer for the combination group, we failed to demonstrate any beneficial effect on outcome parameters for this group. No differences between sealed or unsealed tooth surfaces or for use of a manual or electric brush were observed.

Identification of Lactobacilli from deep carious lesions by means of species-specific PCR and MALDI-TOF mass spectrometry.

BACKGROUND: The aim of the present study was to compare MALDI-TOF results for the identification of 87 lactobacilli, isolated from soft or hard carious dentin from 70 first molars of 7- to 8-year-old children with those obtained by species-specific PCR. METHODS: The 87 isolates were analyzed by MALDI-TOF MS (Microflex LT, MALDI Biotyper 3.0, Bruker Daltonik, Bremen, Germany), using a reference data base of 4110 strains including > 90 lactobacillus species. For the identification with species-specific PCR, oligonucleotide primers (16S rRNA) specific for L. casei, L. paracasei, L. rhamnosus, L. gasseri, L. plantarum, and L. acidophilus were used; type strains served as controls. The PCR-products were separated electrophoretically on a 1.5% agarose gel and identified by their position on the gel. RESULTS: For 93% of the strains both methods produced concordant results: 40 strains were identified as L. rhamnosus, 16 as L. paracasei subsp. paracasei, 15 as L. paracasei subsp. tolerans, 4 as L. paracasei, 3 as L. gasseri, 2 as L. plantarum, and 1 as L. casei. In 4.5% of the cases the results were discordant. Of the 3 strains, not identified by species-specific PCR, 1 strain was identified by MALDI-TOF MS as L. spec. and 1 as L. parabuchneri. One strain could not be identified by either method. CONCLUSIONS: Both methods are highly sensitive. Limitations can be the precision of the primers (PCR) or the scarcity of strains from a certain habitat in the data base. Additional information is necessary for the strains without or with discordant identification.

Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI-MS.

A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI-MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level.

Fluorescence-controlled Er:YAG laser for caries removal in permanent teeth: a randomized clinical trial.

The aim of this randomized clinical study was to compare the efficacy of a fluorescence-controlled erbium-loaded yttrium aluminum garnet (Er:YAG) laser with conventional bur treatment for caries therapy in adults. Twenty-six patients with 102 carious lesions were treated using either the Er:YAG laser, at threshold levels of 7, 8, 9, and 10 [U], or rotary burs. Both techniques were applied to each lesion at separate locations. After treatment, dentine samples were obtained using a carbide bur. The viable counts of Streptococcus mutans (SM) and lactobacilli (LB) [expressed as colony-forming units (log10 CFUs)], treatment time, pain, vibration, and sound intensity were determined. The median numbers of CFUs for SM and LB were not statistically different between laser and bur treatment at threshold levels 7 and 8 [U]. At threshold levels 9 and 10 [U], the median number of CFUs for LB [1.11 (range: 0.00-2.04)] were significantly higher following laser treatment than following bur treatment [0.30 (range: 0.00-0.60)]. The results indicate that treatment with a fluorescence-controlled Er:YAG laser at threshold levels of 7 and 8 removed caries to a level similar to that achieved using conventional bur treatment, with clinically irrelevant amounts of remaining bacteria. Although more time consuming, laser treatment provided higher patient comfort than bur treatment.

A triclosan-containing compomer reduces Lactobacillus spp. predominant in advanced carious lesions.

OBJECTIVES: To assess the antimicrobial effect of acid etching or a triclosan-containing compomer on the cultivable microflora of incompletely excavated dentinal carious lesions. METHODS: Thirty detinal lesions were opened with a diamond burr. Subsequent to removal of the softened biomass a sample of dentin was taken from the cavity floor with a round bur. Ten cavities each were treated with 36% phosphoric acid (PH) for 15s, covered with a triclosan-containing compomer (TC) or received no treatment as control (CO). All lesions were restored with a compomer composite. Sampling was performed directly after etching in the PH group and at re-entry after 6 weeks in all groups. Aliquots were plated on blood agar and selective media for Lactobacilli (Rogosa) and mutans Streptococci (MSB). Cultures were incubated anaerobically for 7 days at 37 degrees C prior to quantitative assessment and biotyping of the isolates. RESULTS: Application of phosphoric acid resulted in initial reduction of the totally cultivable microflora (p=0.006). Evaluation of the total number of cultivable microflora after 6 weeks revealed no differences between the groups (p>0.05). Lactobacilli counts were significantly lower in the TC group compared to the PH and CO groups (p<0.05). No difference was detected between the PH and CO groups after 6 weeks. SIGNIFICANCE: Phosphoric acid initially reduces the number of microorganisms in carious dentin but not in the longer term. The experimental triclosan composite suppresses Lactobacilli species over a period of 6 weeks.

Effects of heat-inactivated Bifidobacterium BB12 on cariogenicity of Streptococcus mutans in vitro.

OBJECTIVE: Since some probiotic bacteria are cariogenic themselves, their suitability for caries management is questionable. Inactivated bacteria or their supernatants have been found to exert probiotic effects, whilst having several advantages compared with living bacteria. We hypothesized that viable and heat-inactivated Bifidobacterium animalis BB12 reduces the cariogenicity of Streptococcus mutans (SM) in vitro. DESIGN: We assessed mono- and mixed species biofilms of SM and viable or heat-inactivated BB12. Biofilms were grown in a continuous-culture-system under cariogenic conditions on smooth proximal enamel or cavitated dentine. For each of eight experimental subsets (4 biofilms×2 hard-tissue conditions), a total of 32 specimens was used. After 10 days, bacterial numbers of 12 biofilms per group were analysed, and all specimens submitted to transversal microradiography. RESULTS: Mineral loss was higher in cavitated dentine than smooth enamel for all biofilms (p<0.001, t-test). BB12-monospecies biofilms induced significantly less mineral loss than SM in both enamel (p<0.05) and dentine (p<0.001). Viable BB12 did not significantly reduce cariogenicity of SM (p>0.05), whilst heat-inactivated BB12 decreased cariogenicity of SM in dentinal cavities (p<0.01). Bacterial numbers were higher on dentine than enamel (p<0.05), but not significantly influenced by biofilm species (p>0.05). CONCLUSIONS: Heat-inactivated BB12 reduced the cariogenicity of SM in dentinal cavities in vitro. Inactivated probiotics might be suitable for caries control.

Caries and its risk factors in young children in five different countries.

The state of oral health plays an essential role in human comprehensive health. Nevertheless, although considerable improvement in oral health caries has been noted in both developed and newly developing countries, caries is still widespread among children. Although it can be monitored, caries cannot be properly eliminated. MATERIALS AND METHODS. This investigation, performed in cooperation with Jena Hospital in Germany, was conducted in five countries from 2002-2008. The cities Riga (Latvia), Ouro Preto (Brazil), Erfurt (Germany), Volgograd (Russia) and Minsk (Belorus) were engaged in this investigation. Children at the age of 26-34 months were surveyed. Consistent with the research design, the mothers filled out questionnaires about the children's health, and an examination of the children's oral health was performed. The statistics program SPSS 15.0 was used to analyze the obtained data, and the correlations between changing findings were expressed by the Spearmen rank correlation coefficient (r). RESULTS. In this study, 472 children were observed: 179 from Riga, 152 from Erfurt, 62 from Ouru Preto, 116 from Minsk and 84 from Volgograd. A direct correlation existed between the country and plaque (r=0.16) and caries (dmft) (r=-0.11). The direct correlation between dmft and Streptococcus mutans (r=-0.36) was characteristic of children from Erfurt. The lowest dmft index (0.62) was present in children from Erfurt, and the highest (1.57) in children from Ouro Preto. CONCLUSIONS. The frequency and prevalence of caries in young children in Riga is high; it was the lowest in Erfurt and the highest - in Brazil. Plaque and dt were one of the indices with a direct relationship in Riga, Brazil and Minsk. Poor oral hygiene, irregular tooth brushing and the consumption of cariogenic foods and drinks are the most important caries risk factors among children at the age of 2-3 years. The mother's knowledge and attitude affect the child's oral health.

Modeling of S mutans and A naeslundii acid production in vitro with caries incidence of low- and high-risk children.

OBJECTIVES: Nowadays, the extended ecological plaque hypothesis equates dental health with the occurrence of acidogenic generalists in the plaque flora and explains disease?the irreversible demineralization of the dental hard tissue?by an ecologic shift in plaque composition, favoring acidogenic and aciduric specialists, following repeated conditions of high sugar. In the present study, aspects of the extended ecological plaque hypothesis were examined in retrospect. METHOD AND MATERIALS: Serving as a basis were data on (1) the caries incidence of children, classified according to their DMFT as having low or high caries risk; (2) the qualitative plaque composition of the children with the prevalence of mutans streptococci and actinomyces; (3) the amounts of acid and acid tolerance, determined by pH state titration (glucose), of representatively isolated strains of mutans streptococci and actinomyces; (4) the extrapolation of the acid amount to the occurrence of mutans streptococci and actinomyces in the plaque of children; and (5) the relationship between the extrapolated acid amount in plaque and caries incidence and the respective classification of the children into low and high caries risk. RESULTS: The synoptic consideration of the numbers of A naeslundii and S mutans in plaque of children, with their capacity for acid production in vitro, reflected the caries risk classification. CONCLUSION: The analysis of the clinical, microbiologic, and in vitro findings concerning the virulence of the plaque isolates supports the extended ecological plaque hypothesis. Children with low caries risk had developed one new decayed surface within 4 years; children with high caries risk developed four within the same time period.

Evaluation of selective caries removal in deciduous teeth by a fluorescence feedback-controlled Er:YAG laser in vivo.

This study investigated the ability and efficacy of an Er:YAG laser with a fluorescence feedback system for caries removal in deciduous teeth. Seventy-nine carious lesions were excavated using a fluorescence-controlled Er:YAG laser. Endpoint of treatment was defined by emission of fluorescence from the dentine surface below the pre-selected threshold level of 7 units and the subsequent termination of Er:YAG laser radiation. Dentine samples were obtained from the cavity floor, and viable counts of both Streptococcus mutans and Lactobacilli, expressed as colony forming units (log CFU), were evaluated. Preparation time was recorded to assess efficacy of the treatment procedure. S. mutans and/or Lactobacilli were found in 25 out of 79 lesions. Regarding the counts for S. mutans and Lactobacilli, the median log CFU was 0 (min, 0; max, 5.5) and 0 (min, 0; max, 6), respectively, with 2.4% of all samples yielding more than 100 CFU S. mutans and 4.8% yielding more than 100 CFU Lactobacilli. In 8 out of 79 cases, laser excavated cavities were not judged being caries-free using the conventional tactile criterion for assessing caries tissue. Focussing on these teeth, the median log CFU was 0 (min, 0; max, 0.5) for S. mutans and 0 (min, 0; max, 1.6) for Lactobacilli. The mean time for treatment was 2.3+/-1.2 min. Of the children, 93.8% rated the laser treatment to be comfortable. The study indicates that the fluorescence feedback-controlled Er:YAG laser might be an appropriate device for caries removal in children using the suggested threshold level of 7 units.

Oral Actinomyces isolates forming red colonies on brain heart blood agar can bee unambiguously classified as A odontolyticus by macroscopic examination.

The accurate classification of oral Actinomyces isolates as one species is difficult. Out of 18 Actinomyces isolates forming red colonies on brain heart blood agar, 12 could be straightforwardly assigned as Actinomyces odontolyticus by biochemical, morphological, and chemotaxonomic characteristics. For the remaining six isolates, the results of the different identification methods were inconsistent. By sequencing a 16S ribosomal DNA fragment by a rapid mass spectrometric method, all isolates could be identified unambiguously as A. odontolyticus. This result proves the importance of red colony pigmentation on brain heart blood agar together with the characteristic cell morphology for unequivocal assignment of oral Actinomyces isolates to the species A. odontolyticus.

Suppression of caries-related microorganisms in dentine lesions after short-term chlorhexidine or antibiotic treatment.

This study investigated the efficiency of a chlorhexidine varnish and an antibiotic paste in suppressing the cultivable microflora of deep dentine cavities in a stepwise excavation procedure. Subsequent to enamel preparation and removal of the central biomass, infected dentine was sampled from the cavity floor. Ten cavities each were either covered with the 1% chlorhexidine- and 1% thymol-containing varnish Cervitec (CE), the demeclocycline hydrocortisone-containing ointment Ledermix (LE) or received no treatment as control (CO). A compomer composite was used as intermediate restorative. Cavities were reassessed after 6 weeks and again dentine samples were microbiologically investigated for total viable counts, mutans streptococci and lactobacilli. After 6 weeks a significant reduction of the total viable counts was observed in the LE group (p = 0.011) compared to the control, whereas no differences were found in the CE group (p > 0.05). Mutans streptococci were rarely recovered at baseline and after 6 weeks. Compared to the CO group counts of lactobacilli were significantly reduced in the CE and LE groups (p < 0.05). Lactobacillus species were frequently recovered at baseline and after 6 weeks of observation. Lactobacillus rhamnosus was the predominant species in all samples investigated. Application of CE or LE resulted in reduced counts of lactobacilli after a period of 6 weeks. Although none of the materials completely eliminated the viable microorganisms, the use of LE was more effective than CE in reducing the total anaerobic microorganisms associated with carious dentine.

Fatores de risco da doença cárie de acometimento precoce / Risk factors of early childhood caries

Neste artigo os autores fazem uma revisão da literatura sobre cárie de acometimento precoce em crianças (ECC) e sugerem que essa modalidade de acometimento de doença cárie, se tornou um problema de saúde pública, devido sua alta prevalência e rápida progressão, podendo levar a destruição dos elementos dentários. Os autores discutem os critérios de diagnóstico da cárie de acometimento precoce, revelando suas causas e determinantes de risco sociais e econômicos como fatores de risco da doença. A doença cárie de acometimento precoce é um problema crescente em muitos países e a complexidade de seus determinantes e fatores de risco, faz com que a evidencia científica no que tange modelos explicativos confiáveis ainda é insuficiente, necessitando de mais pesquisas para esclarecer seu mecanismo e prevenir sua ocorrência.