Functionally Active Microbiome in Supragingival Biofilms in Health and Caries.

The oral microbiome is unique at inter and intra-individual levels at various sites due to physical and biological factors. This study aimed to compare the bacterial composition of supragingival biofilms collected from enamel sites with different caries activity, from active and inactive-caries subjects, and from caries-free (CF) subjects. Twenty-two individuals (aged between 13 and 76 years old; med = 23.5 years old) were allocated into 3 groups: caries-active (CA) (n = 10), caries-inactive (CI) (n = 6), and CF (n = 6). From the CA group, 3 sites were sampled: CA (active non-cavitated lesion), CI (inactive non-cavitated lesion), and sound enamel surface (S). From the subjects of the CI group, biofilm from a CI lesion was collected (INCL), while for the CF subjects, a pool of biofilm from sound enamel surfaces was sampled. The total RNA was extracted, and cDNA libraries were prepared and paired-end sequenced (Illumina HiSeq 3,000). Final dental biofilm samples analysed from CA was 16 (ANCL-CA = 6, INCL-CA = 4, S-CA = 6); from CI, 3 (INCL-CI = 3); and from CF, 6 (S-CF = 6) (some samples were lost by insufficient genetic material). Read sequences were processed and analysed using the Metagenomics RAST server. High-quality sequences (3,542,190) were clustered into operational taxonomic units (97% identity; SILVA SSU), representing 915 genera belonging to 29 phyla (higher abundant: Actinobacteria, Firmicutes, Bacteroidetes, and Fusobacteria). The presence of a core microbiome was observed (123 shared genera). The alpha diversity analysis showed less bacterial diversity in disease (S-CA) compared to health (S-CF). The dominant genera included Actinomyces, Corynebacterium, Capnocytophaga, Leptotrichia, Veillonella, Prevotella, Streptococcus, Eubacterium, and Neisseria. Veillonella and Leptotrichia were related with disease and Prevotella with health. Corynebacterium, Capnocytophaga, and Actinomyces clustered together presenting high abundance in health and disease. The Metric Multidimensional Scaling Ordination analysis shows that sites from active subjects (ANCL-CA, INCL-CA, and S-CA) are closer to each other than either INCL-CI subjects or S-CF subjects. In conclusion, supragingival bacterial communities presented intra-individual similarities, but inter-individual diversity and difference in bacterial composition reveal that the subject's caries activity status matters more than sites.

Effect of human, dentin, albumin and lipopolysaccharide on the antibacteerial activity of endodontic activity of endodontic irrigants.

CONTEXT: Human dentin powder (HD), bovine serum albumin (BSA) and endotoxin (LPS) may affect the antimicrobial activity of irrigating solutions. AIM: To evaluate the inhibitory effect of HD powder, BSA, and LPS on the antibacterial activity of 0.5%, 1%, 2.5%, and 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) gel, BioPure mixture of tetracycline, citric acid, and detergent (MTAD), and QMix. METHODS: The direct contact test against Enterococcus faecalis (ATCC 29212) for 2-min, 30-min and 6-h was used. Sterile pyrogen-free water was the negative control. After experimental periods, a neutralizing agent was used. Colony-forming units were determined by 10-fold serial dilutions and culture on agar plates. Data were analyzed by Kruskal-Wallis and Dunn's test (α = 5%). RESULTS: In the absence of inhibitors, all irrigants eliminated E. faecalis. In contact with HD, all solutions eliminated E. faecalis within 2-min, with the exception of MTAD. In the presence of BSA, only 5% NaOCl killed E. faecalis within 2-min. LPS did not affect the antibacterial effect of any irrigant. At 30-min and at 6-h, all substances eliminated E. faecalis. CONCLUSIONS: In the presence of albumin, irrigants needed >2-min to eliminate E. faecalis, except for 5% NaOCl. The same was observed in the presence of dentin when E. faecalis was exposed to MTAD.

Occlusal Caries: Biological Approach for Its Diagnosis and Management.

The management of occlusal caries still remains a major challenge for researchers as well as for general practitioners. The present paper reviews and discusses the most up-to-date knowledge and evidence of the biological principles guiding diagnosis, risk assessment, and management of the caries process on occlusal surfaces. In addition, it considers the whole spectrum of the caries process on occlusal surfaces, ranging from the molecular ecology of occlusal biofilms to the management of deep occlusal caries lesions. Studies using molecular methods with focus on biofilms in relation to occlusal caries should explore the relationship between the function and the structural composition of these biofilms to understand the role of occlusal biofilms in caries development. State-of-the-art measures to evaluate risk for occlusal caries lesion activity, caries incidence, and progression should include the assessment of the occlusal biofilm and the stage of tooth eruption. Careful clinical examination of non-cavitated lesions, including assessment of the lesion activity status, remains the major tool to determine the immediate treatment need and to follow on the non-operative treatment outcome. Even medium occlusal caries lesions in the permanent dentition may be treated by non-invasive fissure sealing. By extending the criteria for non-invasive treatments, traditional restoration of occlusal surfaces can be postponed or even avoided, and the dental health in children and adolescents can be improved. Selective removal (incomplete) to soft dentin in deep carious lesions has greater success rates than stepwise excavation. Selective (complete) removal to firm dentin has a lower success rate due to increased pulp exposure.

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.

Saliva, supragingival biofilm and root canals can harbor gene associated with resistance to lactamic agents.

This study aimed to determine the presence of Prevotella strains and genes associated with resistance to lactamics in different oral niches from patients with/without primary endodontic infections. Saliva (S) and supragingival biofilm (SB) were collected from three patient groups: Group I - no endodontic infection (n = 15); Group II - acute endodontic infection (n = 12); and Group III - chronic endodontic infection (n = 15). Root canal (RC) samples were collected from Groups II and III. The presence of P. intermedia, P nigrescens, P. tannerae and cfxA/cfxA2 gene was assessed by PCR. The cfxA/cfxA2 gene was not detected in all environments within the same patient. The cfxA/cfxA2 gene was present in 23.81% of S samples, 28.57% of SB samples, and 7.41% of RC samples. Prevotella species were detected in 53.97%, 47.62% and 34.56% of the S, SB, and RC samples, respectively. P. intermedia had a high frequency in saliva samples from Group 3. Saliva samples from Group 1 had higher detection rates of P. nigrescens than did Groups 2 and 3. Patients without endodontic disease had high frequencies of P. nigrescens in the SB samples. The presence or absence of spontaneous symptoms was not related to the detection rates for resistance genes in the RC samples. Saliva, supragingival biofilm and root canals can harbor resistant bacteria. The presence of symptomatology did not increase the presence of the cfxA/cfxA2 gene in the supragingival biofilm and inside root canals.

Saliva, supragingival biofilm and root canals can harbor gene associated with resistance to lactamic agents

This study aimed to determine the presence of Prevotella strains and genes associated with resistance to lactamics in different oral niches from patients with/without primary endodontic infections. Saliva (S) and supragingival biofilm (SB) were collected from three patient groups: Group I – no endodontic infection (n = 15); Group II – acute endodontic infection (n = 12); and Group III – chronic endodontic infection (n = 15). Root canal (RC) samples were collected from Groups II and III. The presence of P. intermedia, P nigrescens, P. tannerae and cfxA/cfxA2 gene was assessed by PCR. The cfxA/cfxA2 gene was not detected in all environments within the same patient. The cfxA/cfxA2 gene was present in 23.81% of S samples, 28.57% of SB samples, and 7.41% of RC samples. Prevotella species were detected in 53.97%, 47.62% and 34.56% of the S, SB, and RC samples, respectively. P. intermedia had a high frequency in saliva samples from Group 3. Saliva samples from Group 1 had higher detection rates of P. nigrescens than did Groups 2 and 3. Patients without endodontic disease had high frequencies of P. nigrescens in the SB samples. The presence or absence of spontaneous symptoms was not related to the detection rates for resistance genes in the RC samples. Saliva, supragingival biofilm and root canals can harbor resistant bacteria. The presence of symptomatology did not increase the presence of the cfxA/cfxA2 gene in the supragingival biofilm and inside root canals.

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.

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 pi, 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.