Clarissa Cavalcanti Fatturi Parolo

Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However π, nucleotide diversity per site, was low for all loci being in the range 0.019–0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3–1.15] compared to 8.3 [95% confidence interval 5.0–14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

Comparative analysis of the effect of two chlorhexidine mouthrinses on plaque accumulation and gingival bleeding

The aim of the present study was to evaluate the effect of two chlorhexidine rinsing solutions (0.12% and 0.2%) on plaque and gingival bleeding. Ten dental students participated in this double-blind, cross-over study, rinsing twice a day, for one minute, with each one of the tested solutions for fourteen days. A wash-out period of one week between treatments was observed. In order to assess gingival bleeding, the van der Weijden et al.(1) (1994) index was used. The plaque indexes used were those of Quigley, Hein(2) (1962) and Silness, Löe(3) (1964). In the pre-experimental period, subjects received oral hygiene instructions and dental prophylaxis. The results revealed no significant differences between both concentrations in relation to plaque and gingival bleeding. Mean values (+/- standard deviation) of the Quigley & Hein index were 0.25 +/- 0.16 for the 0.12% solution and 0.23 +/- 0.26 for the 0.2% solution (p = 0.4838). Mean values (+/- standard deviation) of the Silness-Löe index were 0.12 +/- 0.10 for the 0.12% solution and 0.11 +/- 0.11 for the 0.2% solution (p = 0.7592). The bleeding index mean values at the end of the study were not different for both concentrations with mean values (+/- standard deviation) of 14.93% +/- 6.68% and 13.95 +/- 9.24% for the 0.12% and 0.2% solutions, respectively. Although an increase in gingival bleeding was observed, both concentrations were able to control dental plaque.

Microbial Contamination of Noncavitated Caries Lesions: A Scanning Electron Microscopic Study

Contamination by microorganisms of active and inactive noncavitated lesions was analyzed by scanning electron microscopy. The sample comprised 10 active and 14 inactive smooth surface lesions and 5 sound tooth surfaces. The active lesions were obtained through an in situ model. The inactive lesions and the sound surfaces were obtained from extracted teeth. The samples were split and the two resulting halves were analyzed. The lesion areas were measured in order to compare bacterial contamination in active and inactive lesions. Microorganisms were detected within the enamel in all lesions studied (active and inactive). Sound teeth did not harbor bacteria with the exception of one half tooth where one rod was observed in the enamel. Great variation was observed in bacterial contamination in both active and inactive lesions. Microorganisms penetrated rather deeply into some active as well as into inactive lesions, reaching the dentin in 5 inactive lesions. Bacteria were present in the tubular and intertubular dentin. No difference was observed in the number of microorganisms per square millimeter in the active and inactive lesions (p > 0.05). Cocci and rods comprised 99% of the organisms, while filamentous and spiral bacteria were seldom present. Yeast-like microorganisms were found in inactive lesions. The presence of microorganisms in inactive as well as active noncavitated lesions shows that bacteria inside dental tissue (enamel and dentin) do not impede the arrestment of the caries process.

Evaluation of the Effect of Enterococcus faecalis Biofilm on the 2% Chlorhexidine Substantivity: An In Vitro Study

INTRODUCTION The aim of this study was to correlate the bacterial viability and the presence of 2% chlorhexidine (CHX) solution on dentin by means of confocal laser scanning microscopy and high-performance liquid chromatography for 48 hours, 7 days, and 30 days. METHODS One hundred twenty-three extracted human teeth were used. Samples were divided into 4 groups according to the solution (CHX or saline) and the presence of Enterococus faecalis biofilm. Samples were kept in contact with 5 mL of the solution for 5 minutes. Each group was divided into 3 subgroups according to the evaluation period (n = 10). Statistical analysis was performed by using the Kruskal-Wallis test, the Mann-Whitney U test (P < .05), and the Spearman rank correlation coefficient (P < .01). RESULTS There was a negative correlation between the percentage of live cells and the amount of remaining CHX (P = .000). CHX significantly reduced the percentage of viable cells compared with saline after 48 hours (P = .007). Differences were maintained in the 7-day evaluation period (P = .001). After 30 days, the CHX group presented an increase of viable cells, thereby becoming similar to saline (P = .623). Simultaneously, the remaining CHX was significantly reduced in the 30-day specimens (P = .000). CONCLUSIONS The results of this study indicate that 2% CHX solution was detected for 48 hours and 7 days with a low percentage of viable cells. The presence of microorganisms on human dentin did not affect 2% CHX maintenance.

Response of E. faecalis biofilms to different associations of auxiliary substances during root canal preparation: A confocal laser microscopy analysis

Objective: This in vitro study evaluated the effect of different endodontic auxiliary chemical substances over Enterococcus faecalis (Ef) biofilm through confocal laser scanning microscopy (CLSM). Methods: Forty‐five bovine incisors were infected with Ef for 21 days. Teeth were divided into five groups: group 1: 2.5% NaOCl + EDTA, group 2: 2% CHX gel + EDTA, group 3: 2% CHX liquid + EDTA, group 4: 2.5% NaOCl + 2% CHX gel + EDTA, group 5: 2.5% NaOCl + 2% CHX liquid + EDTA and a negative and a positive control group (NCG; PCG). The samples were stained with SYTO9 and propidium iodide and analyzed by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Scores were standardized according to the total bacterial load (TBL)—1: ≤25%, 2: >25 ≤50%, 3: >50 ≤75%, 4: >75% and debris—1: absence of debris; 2: presence of debris. Statistical analysis was carried out through the Kruskal–Wallis and the Fischer exact tests (P = 0.05). Results: No statistical differences were observed to CFU, debris and bacterial viability. Conclusion: None of the tested substances could completely eliminate Ef from the root canal space. Microsc. Res. Tech. 76:658–662, 2013.

Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms

Aim:  To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm.

Acid susceptibility of arrested enamel lesions: in situ study.

Arrested lesions are more resistant to a new cariogenic challenge, but the degree of surface rehardening needed to achieve this is unknown. The aim of this in situ study was to analyze the acid susceptibilityof newly formed and arrested enamel lesions with known arrestment period and surface microhardness. Six individuals wore an oral appliance with human enamel blocks for 3 periods: (1) 21 days of demineralization due to plaque accumulation and cariogenic challenge, 4 blocks/person (nonfluoride dentifrice); (2) 75 days of arrestment, brushing with fluoride dentifrice, 2 blocks/person; (3) 21 days of demineralization, 5 blocks/person: 1 sound block, 2 demineralized blocks and 2 demineralized and arrested blocks (nonfluoride dentifrice). After period 1, all blocks showed a dull whitish surface characteristic of active, noncavitated lesions. After arrestment, the surfaces assumed a shiny and smooth aspect. The Knoop hardness number (KHN, mean ± SD) of the sound blocks was 307.6 ± 15.0. After period 1, microhardness decreased significantly to 162.6 ± 33.5 KHN (p < 0.001). The microhardness of subsequently arrested lesions (279.8 ± 23.1 KHN) was significantly greater than after demineralization, but lower than that of sound enamel. Arrested enamel did not show a decrease in microhardness when subjected to a new cariogenic challenge and after the same cariogenic challenge showed similar microhardness to sound enamel. The results showed that, although noncavitated lesions probably take years to reach microhardness levels like sound enamel, this does not imply that special care, in addition to the ones normally given to sound tooth surfaces, is necessary.

INFLUENCE OF ADDITION OF 2-(3-(2H-BENZOTRIAZOL-2-YL)- 4-HYDROXYPHENYL)ETHYL METHACRYLATE TO AN EXPERIMENTAL ADHESIVE SYSTEM

The aim of this study was to evaluate the addition of 2-[3-(2HBenzotriazol- 2-yl)-4-hydroxyphenyl]ethyl methacrylate (BTAM) to an experimental adhesive resin. An experimental base adhesive resin was formulated with BisGMA, TEGDMA and HEMA, to which BTAM was added at 1, 2.5 and 5%, in weight. One group with no addition was used as control. The experimental adhesives were evaluated for antibacterial potential (against Streptococcus mutans), degree of conversion with FTIR, softening in solvent and microRaman interface analyses. Data were analyzed by Kruskal-Wallis, paired t test and ANOVA and Tukey, considering a 5% level of significance. The results showed antibacterial activity of 5% BTAM against S. mutans (p<0.05), however, no difference was found among BTAM groups (p> 0.05). The results of degree of conversion and softening of solvent showed no statistical difference between BTAM and control groups (p>0.05). The addition of 5% BTAM showed higher antibacterial activity than the negative control, and copolymerization with comonomer blend of adhesive resin and BTAM was detected at the dentin/ adhesive interface.

Actinomyces spp. gene expression in root caries lesions

Background The studies of the distribution of Actinomyces spp. on carious and non-carious root surfaces have not been able to confirm the association of these bacteria with root caries, although they were extensively implicated as a prime suspect in root caries. Objective The aim of this study was to observe the gene expression of Actinomyces spp. in the microbiota of root surfaces with and without caries. Design The oral biofilms from exposed sound root surface (SRS; n=10) and active root caries (RC; n=30) samples were collected. The total bacterial RNA was extracted, and the mRNA was isolated. Samples with low RNA concentration were pooled, yielding a final sample size of SRS=10 and RC=9. Complementary DNA (cDNA) libraries were prepared and sequenced on an Illumina® HiSeq 2500 system. Sequence reads were mapped to eight Actinomyces genomes. Count data were normalized using DESeq2 to analyse differential gene expression applying the Benjamini-Hochberg correction (false discovery rate [FDR]<0.001). Results Actinomyces spp. had similar numbers of reads (Mann-Whitney U-test; p>0.05), except for Actinomyces OT178 (p=0.001) and Actinomyces gerencseriae (p=0.004), which had higher read counts in the SRS. Genes that code for stress proteins (clp, dnaK, and groEL), enzymes of glycolysis pathways (including enolase and phosphoenolpyruvate carboxykinase), adhesion (Type-2 fimbrial and collagen-binding protein), and cell growth (EF-Tu) were highly – but not differentially (p>0.001) – expressed in both groups. Genes with the most significant upregulation in RC were those coding for hypothetical proteins and uracil DNA glycosylase (p=2.61E-17). The gene with the most significant upregulation in SRS was a peptide ABC transporter substrate-binding protein (log2FC=−6.00, FDR=2.37E-05). Conclusion There were similar levels of Actinomyces gene expression in both sound and carious root biofilms. These bacteria can be commensal in root surface sites but may be cariogenic due to survival mechanisms that allow them to exist in acid environments and to metabolize sugars, saving energy.

Genotypic diversity and virulence traits of Streptococcus mutans isolated from carious dentin after partial caries removal and sealing

The aim of this study was to compare the genotypic diversity and virulence traits of Streptococcus mutans isolated from carious dentin before and after partial dentin caries removal (PDR) and sealing. Carious dentin samples were obtained three months before and after the PDR and cavity sealing. Up to seven isolates of each morphological type of S. mutans were selected and strain identity was confirmed using gtfB primer. Genotyping was performed by arbitrary primer-PCR (AP-PCR). Acidogenesis and acidurance of the genotypes were evaluated as virulence traits. A paired t-test and a Wilcoxon test were used to compare the virulence of genotypes. A total of 48 representative S. mutans isolates were genotyped (31 before and 17 after the sealing). At least one of the genotypes found before the sealing was also found on dentin after the sealing. The number of genotypes found before the sealing ranged from 2 to 3 and after the sealing from 1 to 2 genotypes. No difference was observed in the acidogenesis and acidurance between genotypes isolated before and after the sealing. In conclusion, genotypic diversity of S. mutans decreased after the PDR and sealing, but the virulence traits of S. mutans remained unchangeable.