Biofilm dysbiosis and caries activity: a surface or an individual issue?

OBJECTIVE: This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. METHODOLOGY: A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). RESULTS: Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. CONCLUSION: Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

Hub genes and pathways related to caries-free dental biofilm: clinical metatranscriptomic study.

OBJECTIVE: This study aimed to evaluate the microbial functional profile of biofilms related to caries-free (CF, n = 6) and caries-arrested (CI, n = 3) compared to caries-active (CA, n = 5) individuals. MATERIALS AND METHODS: A metatranscriptomic was performed in supragingival biofilm from different clinical conditions related to caries or health. Total RNA was extracted and cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data (SortMeRNA) were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2) and weighted gene co-expression network analysis (WCGNA) to explore and identify gene modules related to these clinical conditions. RESULTS: A total of 5303 genes were found in the metatranscriptomic analysis. A co-expression network identified the most relevant modules strongly related to specific caries status. Correlation coefficients were calculated between the eigengene modules and the clinical conditions (CA, CI, and CF) discriminating multiple modules. CA and CI showed weak correlation coefficient strength across the modules, while the CF condition presented a very strong positive correlation coefficient (r = 0.9, p value = 4 × 10-9). Pearson's test was applied to further analyze the module membership and gene significance in CF conditions, and the most relevant were HSPA1s-K03283, Epr- K13277, and SLC1A-K05613. Gene Ontology (GO) shows important bioprocesses, such as two-component system, fructose and mannose metabolism, pentose and glucuronate interconversions, and flagellar assembly (p-adjust < 0.05). The ability to use different carbohydrates, integrate multiple signals, swarm, and bacteriocin production are significant metabolic advantages in the oral environment related to CF. CONCLUSIONS: A distinct functional health profile could be found in CF, where co-occurring genes can act in different pathways at the same time. Genes HSPA1s, Epr, and SLC1A may be appointed as potential biomarkers for caries-free biofilms. CLINICAL RELEVANCE: Potential biomarkers for caries-free biofilms could contribute to the knowledge of caries prevention and control.

Biofilm dysbiosis and caries activity: a surface or an individual issue?

Abstract Objective This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. Methodology A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). Results Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. Conclusion Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

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.

Characterization of Lactobacilli isolated from carious dentin after selective caries removal and cavity sealing.

OBJECTIVE: To characterize lactobacilli isolated from residual carious dentin after selective caries removal (SCR), by observing the changes detected in their prevalence, diversity, and cariogenic potential after starvation stress caused by cavity sealing (CS). DESIGN: Lactobacilli were cultured from carious dentin lesions (n = 16 patients) treated in a clinical trial, three months before and after CS. Presumptive lactobacilli were selected, isolated, and analyzed by Gram staining. Housekeeping gene sequences were used to identify the species (groEL, rpoA, pheS, and 16S rRNA). RESULTS: N = 86 Lactobacillus spp. (n = 41 before and n = 45 after sealing) were genotyped by AP-PCR and analyzed for their cariogenic potential (acid production and acid tolerance). The proportion of lactobacilli to the total anaerobic counts was high, and a significant decrease was observed after sealing (median before sealing = 78.9; 25th-75th = 60.25-97.35; median after sealing = 0.00; 25th-75th = 0.00-77.08; p = 0.001). L. paracasei was the most prevalent species of lactobacilli in carious dentin (p = 0.02). L. rhamnosus prevalence increased to a proportion similar to L. paracasei after CS (p = 0.001). A total of 28 and 14 different genotypes were found before and after CS, respectively. There was no difference between the L. paracasei and the L. rhamnosus isolated from carious dentin, neither regarding acid production nor acid tolerance. CONCLUSIONS: Although there was a significant reduction in lactobacilli in the residual carious dentin after SCR, some strains were capable of surviving after three months of CS. However, the sealed available nutrients are low and not sufficient for caries progression. Also, we believe that a longer follow up period may eliminate all the residual lactobacilli. L. paracasei prevailed in carious dentin in a proportion similar to L. rhamnosus in the sealed dentin. Characterization of lactobacilli after SCR and sealing may help the understanding the importance of genotyping of lactobacilli in carious microbiota.

The role of Candida albicans in root caries biofilms: an RNA-seq analysis.

Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.

The role of Candida albicans in root caries biofilms: an RNA-seq analysis

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.

Fechamento de diastema com resina composta direta: relato de caso clínico / Diastema closure with direct composite restorations: a case report

Com a evolução dos materiais restauradores adesivos diretos e das técnicas restauradoras minimamente invasivas, é possível realizar o fechamento de diastemas inter incisivos superiores de maneira efetiva e estética em uma única sessão. O presente trabalho consiste em um relato de caso clínico que aborda o clareamento dental e o fechamento de diastema entre os dentes 11 e 21 de um paciente jovem. Previamente ao procedimento restaurador foi realizado clareamento de consultório em todos os dentes com peróxido de hidrogênio 35%. Então, o planejamento restaurador foi auxiliado por modelos de estudo associado ao enceramento diagnóstico.A técnica restauradora de eleição foi a resina composta direta com o auxílio de uma matriz/guia de silicona para a confecção das faces palatinas das restaurações. Após o acabamento e polimento, obteve-se a reanatomização dos incisivos centrais com a eliminação do diastema. O tratamento realizado baseado no clareamento dental seguido por restaurações diretas de resina composta foi eficaz na solução estética do sorriso, atingindo a expectativa do paciente e dentro dos princípios de máxima preservação dos tecidos dentais.

After the evolution of restorative adhesive materials and minimal invasive restorations, it ispossible to close a maxilar interincisal diastema in an effective and aesthetical way, performingit in only one session. This clinical case report approaches a teeth bleaching and diastema closurewith composites between teeth 11 and 21, of a young male patient. The elective restorative technique was direct composite restoration with a silicon putty matrix to make the shape of thelingual surfaces of the restorations. Before the restoration procedure, it was performed “in office”bleaching technique, in addition to the case planning with the assistance of a wax-up. The tooth whitening therapy followed by direct composite restorations was effective, contributing to clinical success, esthetic and patient´s satisfaction.