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BACKGROUND: This study aimed to engineer and optimise a dysbiotic biofilm model to develop in vitro root caries for investigating microbial modulation strategies. The model involved growing complex biofilms from a saliva inoculum collected from four volunteers using two strategies. In the first strategy ("pre-treatment strategy"), bovine root slabs were used, and two natural compounds were incorporated at time 0 of the 10-day biofilm experiment, which included sucrose cycles mimicking the cariogenic environment. In the second strategy ("post-treatment strategy"), mature biofilms were grown in a modified Calgary biofilm device coated with collagen and hydroxyapatite for 7 days and then were exposed to the same natural compounds. The metatranscriptome of each biofilm was then determined and analysed. Collagenase activity was examined, and the biofilms and dentine were imaged using confocal and scanning electron microscopy (SEM). Mineral loss and lesion formation were confirmed through micro-computed tomography (µ-CT). RESULTS: The pH confirmed the cariogenic condition. In the metatranscriptome, we achieved a biofilm compositional complexity, showing a great diversity of the metabolically active microbiome in both pre- and post-treatment strategies, including reads mapped to microorganisms other than bacteria, such as archaea and viruses. Carbohydrate esterases had increased expression in the post-treated biofilms and in samples without sugar cycles, while glucosyltransferases were highly expressed in the presence of sucrose cycles. Enrichment for functions related to nitrogen compound metabolism and organic cyclic component metabolism in groups without sucrose compared to the sucrose-treated group. Pre-treatment of the roots with cranberry reduced microbial viability and gelatinase (but not collagenase) activity (p < 0.05). SEM images showed the complexity of biofilms was maintained, with a thick extracellular polysaccharides layer. CONCLUSIONS: This root caries model was optimized to produce complex cariogenic biofilms and root caries-like lesions, and could be used to test microbial modulation in vitro. Pre-treatments before biofilm development and cariogenic challenges were more effective than post-treatments. The clinical significance lies in the potential to apply the findings to develop varnish products for post-professional tooth prophylaxis, aiming at implementing a strategy for dysbiosis reversal in translational research. Video Abstract.

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Conventional views associate microbial biofilm with demineralization in root caries (RC) onset, while research on their collagenases role in the breakdown of collagen matrix has been sporadically developed, primarily in vitro. Recent discoveries, however, reveal proteolytic bacteria enrichment, specially Porphyromonas and other periodontitis-associated bacteria in subgingivally extended lesions, suggesting a potential role in RC by the catabolism of dentin organic matrix. Moreover, genes encoding proteases and bacterial collagenases, including the U32 family collagenases, were found to be overexpressed in both coronal and root dentinal caries. Despite these advancements, to prove microbial collagenolytic proteases' definitive role in RC remains a significant challenge. A more thorough investigation is warranted to explore the potential of anti-collagenolytic agents in modulating biofilm metabolic processes or inhibiting/reducing the size of RC lesions. Prospective treatments targeting collagenases and promoting biomodification through collagen fibril cross-linking show promise for RC prevention and management. However, these studies are currently in the in vitro phase, necessitating additional research to translate findings into clinical applications. This is a comprehensive state-of-the-art review aimed to explore contributing factors to the formation of RC lesions, particularly focusing on collagen degradation in root tissues by microbial collagenases.

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OBJECTIVE: The complete picture of how the human microbiome interacts with its host is still largely unknown, particularly concerning microorganisms beyond bacteria. Although existing in very low abundance and not directly linked to causing diseases, archaea have been detected in various sites of the human body, including the gastrointestinal tract, oral cavity, skin, eyes, respiratory and urinary systems. But what exactly are these microorganisms? In the early 1990 s, archaea were classified as a distinct domain of life, sharing a more recent common ancestor with eukaryotes than with bacteria. While archaea's presence and potential significance in Dentistry remain under-recognized, there are concerns that they may contribute to oral dysbiosis. However, detecting archaea in oral samples presents challenges, including difficulties in culturing, the selection of DNA extraction methods, primer design, bioinformatic analysis, and databases. DESIGN: This is a comprehensive review on the oral archaeome, presenting an in-depth in silico analysis of various primers commonly used for detecting archaea in human body sites. RESULTS: Among several primer pairs used for detecting archaea in human samples across the literature, only one specifically designed for detecting methanogenic archaea in stool samples, exhibited exceptional coverage levels for the domain and various archaea phyla. CONCLUSIONS: Our in silico analysis underscores the need for designing new primers targeting not only methanogenic archaea but also nanoarchaeal and thaumarchaeota groups to gain a comprehensive understanding of the archaeal oral community. By doing so, researchers can pave the way for further advancements in the field of oral archaeome research.

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Abstract Conventional views associate microbial biofilm with demineralization in root caries (RC) onset, while research on their collagenases role in the breakdown of collagen matrix has been sporadically developed, primarily in vitro. Recent discoveries, however, reveal proteolytic bacteria enrichment, specially Porphyromonas and other periodontitis-associated bacteria in subgingivally extended lesions, suggesting a potential role in RC by the catabolism of dentin organic matrix. Moreover, genes encoding proteases and bacterial collagenases, including the U32 family collagenases, were found to be overexpressed in both coronal and root dentinal caries. Despite these advancements, to prove microbial collagenolytic proteases' definitive role in RC remains a significant challenge. A more thorough investigation is warranted to explore the potential of anti-collagenolytic agents in modulating biofilm metabolic processes or inhibiting/reducing the size of RC lesions. Prospective treatments targeting collagenases and promoting biomodification through collagen fibril cross-linking show promise for RC prevention and management. However, these studies are currently in the in vitro phase, necessitating additional research to translate findings into clinical applications. This is a comprehensive state-of-the-art review aimed to explore contributing factors to the formation of RC lesions, particularly focusing on collagen degradation in root tissues by microbial collagenases.

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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.

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Background: Obesity is a prevalent chronic condition affecting children and adults worldwide, and it seems to influence the timing of tooth eruption. The aim of this study was to assess the as-sociation between weight status at age 12 and the eruption of permanent teeth at ages 12 and 14-15 among schoolchildren from southern Brazil. Material and Methods: A cross-sectional survey was conducted in Porto Alegre, southern Brazil, and included a representative sample of 1,528 12-year-old schoolchildren. After 2.5 years, 801 individuals were reexamined. Baseline data collection included a questionnaire, the record-ing of anthropometric measures (height and weight), and clinical examination to register the number of erupted permanent teeth. At follow-up, the eruption stage of second perma-nent molars was recorded. Statistical analysis used Poisson regression. Results: Overweight and obese individuals were 32% and 88% more likely to have com-plete permanent dentition at age 12, respectively (overweight, PR=1.32, 95%CI=1.13-1.55; obese, PR=1.88, 95%CI=1.75-2.02). Obese 12-year-olds were more likely to present erupt-ed #17, #27, #37, and #47 at age 12 and to present completely erupted second molars at age 14-15 than normal weight ones. Conclusions: This population-based study found a significant association between over-weight/obesity at age 12 and early tooth eruption at ages 12 and 14-15 among schoolchil-dren from southern Brazil. Key words:Tooth eruption, Obesity, Permanent teeth, Epidemiology.

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Introduction and aim: The presence of host collagenases in the degradation of the protein matrix at later stages of carious dentin lesions development, as well as the potential involvement of bacterial collagenases, have been suggested but lack conclusive evidence. This study aims to conduct a systematic review to comprehensively assess the profile of host and bacterial-derived collagenolytic proteases in both root and coronal dentin carious lesions. Methods: The search was performed in eight databases and the grey literature. Studies evaluating ex vivo dentin, extracted teeth, or biofilms from natural caries lesions were included. The methodological quality of studies was assessed using the Joanna Briggs Institute tool. Synthesis of the results and the certainty of evidence were performed following the Synthesis without Meta-analysis (SWiM) checklist and GRADE approach for narrative synthesis, respectively. Results: From 935 recovered articles, 18 were included. Although the evidence was very uncertain, it was possible to suggest that 1) MMP-2, MMP-9, MMP-13, and CT-B may be increased in carious dentin when compared to sound dentin; 2) there is no difference in MMP-2 presence, while MMP-13 may be increased in root when compared to coronal carious dentin; 3) there is no difference of MMP-2 and MMP-9 expression/activity before and after cavity sealing; 4) MMP-8 may be increased in the dentin before cavity sealing compared to dentin after cavity sealing; 5) there is no difference of MMP-20 in irradiated vs. non-irradiated carious dentin. MMP-20 probably reduces in carious outer dentin when compared to carious inner dentin (moderate certainty). Genes encoding bacterial collagenolytic proteases and protein-degrading bacteria were detected in coronal and root carious lesions. Conclusion: Trends in the direction of the effect were observed for some collagenolytic proteases in carious dentin, which may represent a potential target for the development of new treatments. (Protocol register-PROSPERO: CRD42020213141).

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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.

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Lactobacillus spp. are acidogenic and aciduric bacteria and are among the main cariogenic microorganisms associated with the carious process. OBJECTIVE: This study aimed to identify genes involved in the acid-tolerance of Lactobacillus spp. and potential functions attributed to these genes within the metatranscriptome of sound root surfaces and carious root surfaces. DESIGN: Genomic libraries were built from mRNA isolated from the biofilm samples (10 from sound root and 9 from carious root using Illumina HiSeq 2500). Reads generated by RNA-seq were mapped against 162 oral microbial genomes and genes potentially related to acid tolerance were manually extracted from the Lactobacillus spp. genomes using L. paracasei ATCC 344 as reference genome. The R package DESeq2 was used to calculate the level of differential gene expression between those two clinical conditions. RESULTS: Fifteen Lactobacillus spp. genomes were identified and a total of 653 acid tolerance genes were overexpressed in carious root surfaces. Multiple functions, as translation, ribosomal structure and biogenesis, transport of nucleotides and amino acids, are involved in Lactobacillus spp. acid tolerance. Species-specific functions also seem to be related to the fitness of Lactobacillus spp. in acidified environments such as that of the cariogenic biofilm associated with carious root lesions. CONCLUSIONS: The response of Lactobacillus spp. to an acidic environment is complex and multifaceted. This finding suggests several possible avenues for further research into the adaptive mechanisms of these bacteria.

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INTRODUCTION: The controversial issue of whether the Archaea domain plays a role in endodontic infections is the focus of this systematic review with meta-analysis. The aim is to emphasize the significance of minority microbial domains in oral dysbiosis by evaluating the prevalence of archaea in root canals and its association with clinical parameters such as symptomatology and type of endodontic infection. METHODS: The search strategy involved researching 6 databases and the gray literature. Publications were accepted in any year or language that identified archaea in samples from endodontic canals. A 2-step selection process narrowed the final choice to 16 articles. The methodological quality of the studies was evaluated using tools from the Joanna Briggs Institute, and the certainty of evidence was assessed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach. RESULTS: The results showed that archaea were present in 20% (95% [confidence interval] CI = 8%-32%) of individuals with endodontic samples analyzed. The samples were about twice as likely to be archaeal-positive if collected from individuals with primary vs. persistent/secondary infection (odds ratio = 2.33; 95% CI = 1.31-4.14; I2 = 0%), or individuals with self-reported vs. symptom-free infections (odds ratio = 2.67; 95% CI = 1.47-4.85; I2 = 0%). Methanogenic archaea were reported in 66% of the included studies. Representative members of phyla Thaumarchaeota and Crenarchaeota were also identified. CONCLUSIONS: Archaea are present in about one-fifth of the infected root canals. Recognized biases in experimental approaches for researching archaea must be addressed to understand the prevalence and roles of archaea in endodontic infections, and to determine whether the decontamination process should include the elimination or neutralization of archaea from root canals (International Prospective Register of Systematic Reviews protocol = CRD42021264308).