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.

Effect of Sweeteners on Root Dentine Demineralization Using a Microcosm Biofilm Model / Efecto de los Edulcorantes en la Desmineralización de la Dentina Radicular Utilizando un Modelo de Biofilm Microcosmo

ABSTRACT: The aim of the present study was to evaluate the effect of commercial sweeteners on root dentin demineralization using a microcosm biofilm model. Bovine dentin specimens with pre-determined surface hardness were randomized into six groups according to the studied sweeteners: sucralose, stevia, saccharin, aspartame. Sucrose was used as a positive control and an untreated group as a negative control. The specimens were submitted to biofilm development from one saliva donor and the cariogenic challenge occurred on subsequent five days, twice a day. At the end, the percentage of surface hardness loss (%SHL) and biomass was determined and submitted to ANOVA followed by Tukey's test. Sucrose presented the highest rate of demineralization, however, all sweeteners tested lead to a statistically higher root demineralization compared to the negative control (p <0.05). Sucrose caused greater demineralization in root dentin, however, the sweeteners were also able to induce it under this biofilm model.

RESUMEN: El objetivo del presente estudio fue evaluar el efecto de los edulcorantes comerciales en la desmineralización de la dentina radicular utilizando un modelo de biofilm microcosmo. Se asignaron al azar muestras de dentina bovina con una dureza de la superficie predeterminada de acuerdo con los edulcorantes estudiados: sucralosa, estevia, sacarina, aspartame. La sacarosa se utilizó como control positivo y un grupo no tratado como control negativo. Las muestras se enviaron al desarrollo de biopelículas de un donante de saliva y el desafío cariogénico se produjo en los siguientes cinco días, dos veces al día. Al final, se determinó el porcentaje de pérdida de dureza de la superficie (% PDS) y biomasa y se aplicó un estudio estadístico de ANOVA seguido de la prueba de Tukey. La sacarosa presentó la mayor tasa de desmineralización; sin embargo, todos los endulzantes probados condujeron a una desmineralización de la raíz estadísticamente mayor en comparación con el control negativo (p<0,05). La sacarosa causó una mayor desmineralización en la dentina de raíz, sin embargo, los edulcorantes también fueron capaces de inducirla bajo este modelo de biofilm.

Sealing ability of MTA, CPM, and MBPc as root-end filling materials: a bacterial leakage study

ABSTRACT Objectives To evaluate the sealing ability of three root-end filling materials (white MTA, CPM, and MBPc) using an Enterococcus faecalis leakage model. Material and Methods Seventy single-root extracted human teeth were instrumented and root-ends were resected to prepare 3 mm depth cavities. Root-end preparations were filled with white MTA, CPM, and MBPc cements. Enterococcus faecalis was coronally introduced and the apical portion was immersed in BHI culture medium with phenol red indicator. The bacterial leakage was monitored every 24 h for 4 weeks. The statistical analysis was performed using the Wilcoxon-Gehan test (p<0.05). Results All cements showed bacterial leakage after 24 hours, except for the negative control group. The MBPc showed significantly less bacterial leakage compared with the MTA group (p<0.05). No significant differences were found between the CPM and the other groups. Conclusions The epoxy resin-based cement MBPc had lower bacterial leakage compared with the calcium silicate-based cements MTA and CPM.

In vivo model for microbial invasion of tooth root dentinal tubules

ABSTRACT Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF). DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0) to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine.

Influence of Temperature on the Antibacterial Activity of Sodium Hypochlorite

Abstract The aim of this study was to compare the antimicrobial activity of 5.25% NaOCl, Hypoclean and Chlor-Xtra at 20 °C and 45 °C in bovine root dentin. One-hundred-and-seventy dentin tubes prepared from bovine maxillary incisors were infected for 21 days with Enterococcus faecalis. The specimens were divided into the following groups: 1. 5.25% NaOCl 20 °C; 2. Hypoclean 20 °C; 3. Chlor-Xtra 20 °C; 4. 5.25% % NaOCl 45 °C; 5. Hypoclean 45 °C; 6. Chlor-Xtra 45 °C; 7. positive control; 8. negative control. Dentin chips were collected with round burs into Brain Heart Infusion (BHI) broth. After culturing, the number of colony-forming units (CFU) was counted. Statistical analyses were performed using descriptive statistics (mean, standard deviation, median), Shapiro-Wilk test, ANOVA and Tukey test. Significance level was set at p<0.05. In all experimental groups, CFU was minimum after treatment (day 0) and the obtained results were significantly different from each other at any period (p<0.05). After treatment, the Hypoclean and Chlor-Xtra showed the lowest numbers of CFU at 20 °C and 45 °C, whereas 5.25% NaOCl showed the highest number of CFU at both temperatures. In each group, the number of CFUs increased significantly with time (p<0.05). The antibacterial activity of Hypoclean and Chlor-Xtra at 45 °C were significantly greater than other tested solutions.

Resumo O objetivo deste estudo foi comparar a atividade antimicrobiana do hipoclorito de sódio a 5,25%, Hypoclean e Cloro-Xtra a 20 °C e 45 °C em dentina radicular bovina. Um total de 170 tubos de dentina foram preparados a partir de incisivos superiores bovinos infectados por 21 dias com Enterococcus faecalis. Os espécimes foram divididos nos seguintes grupos: 1. NaOCl - 5,25% a 20 °C; 2. Hypoclean 20 °C; 3. Cloro-Xtra 20 °C; 4. NaOCl - 5,25% a 45 °C; 5. Hypoclean 45 °C; 6. Cloro-Xtra 45 °C; 7. Controle positivo; 8. Controle negativo. Raspas de dentina foram coletadas com brocas esféricas e cultivadas em infusão cérebro coração (brain heart infusion - BHI). Após a cultura, o número de unidades formadoras de colônias (UFC) foi contado. As análises estatísticas foram realizadas utilizando estatística descritiva (média, desvio padrão, mediana), teste de Shapiro-Wilk, ANOVA e teste de Tukey. O nível de significância foi estabelecido p<0,05. Em todos os grupos experimentais, o número de UFC foi mínimo após o tratamento inicial. Os resultados obtidos foram significativamente diferentes nos períodos de tempos experimentais (p<0,05). O Hypoclean e o Cloro-Xtra mostraram o menor número de UFC a 20 °C e a 45 °C, enquanto que o NaOCl - 5,25% apresentou maior número de UFC em ambas as temperaturas. Em cada grupo, o número de CFUs foi significativamente aumentado por período de tempo (p<0,05). As atividades antibacterianas do Hypoclean e Chlor-Xtra a 45 °C foram significativamente maiores do que nas outras soluções testadas.

In vitro antimicrobial activity of different gutta-percha points and calcium hydroxide pastes

The aim of this study was to evaluate the antimicrobial activity of different trademarks and compositions of gutta-percha points and calcium hydroxide pastes used in endodontic therapy. The evaluated material consisted of gutta-percha points containing calcium hydroxide (RoekoTM), gutta-percha points containing chlorhexidine (RoekoTM), two convencional gutta-percha points (Endo PointsTM and RoekoTM) and two calcium hydroxide pastes (CalenTM and Calen/PMCC TM). Antimicrobial tests included five species of microorganisms: Escherichia coli (ATCC10538), Staphylococcus epidermidis (ATCC12228), Staphylococcus aureus (ATCC6538), Pseudomonas aeruginosa (ATCC27853), and Micrococcus luteus (ATCC9341). The Agar difusion method was employed. The plates were kept at room temperature for 2 h for prediffusion and then incubated at 37°C for 24 h. The triphenyltetrazolium chloride gel was added for optimization and the zones of inhibition were measured. Statistical evaluation was carried out using analysis of variance and Tukey Test. The obtained results showed that all microbial species used in the study were inhibited by the gutta-percha points containing chlorhexidine and by the calcium hydroxide pastes (CalenTM and Calen/PMCC TM), with similar results (p > 0.05). No antimicrobial activity was observed for the other groups. It was concluded that the gutta-percha points containing chlorhexidine presented antimicrobial activity, whereas the gutta-percha points containing calcium hydroxide did not.

O objetivo deste estudo foi a avaliação da atividade antimicrobiana de diferentes marcas e composições de cones de guta-percha e pastas à base de hidróxido de cálcio utilizados em endodontia. Os materiais avaliados foram: cones de guta-percha contendo hidróxido de cálcio (RoekoTM), cones de guta-percha contendo clorexidina (RoekoTM), duas marcas de cones de guta-percha (Endo PointsTM e RoekoTM) e duas pastas à base de hidróxido de cálcio (CalenTM e Calen/PMCC TM). Os testes antimicrobianos incluíram 5 espécies de microrganismos: Escherichia coli (ATCC10538), Staphylococcus epidermidis (ATCC12228), Staphylococcus aureus (ATCC6538), Pseudomonas aeruginosa (ATCC27853) e Micrococcus luteus (ATCC9341). O método empregado foi o de difusão em Agar. As placas foram mantidas em temperatura ambiente por 2 horas para pré-difusão e então incubadas a 37°C por 24 horas. O gel de cloreto de trifeniltetrazólio foi acrescentado para otimização e as zonas de inibição foram medidas. A análise estatística foi realizada pela análise de variância e pelo teste de Tukey. Os resultados obtidos demonstraram que todas as espécies microbianas usadas foram inibidas pelos cones de guta-percha com clorexidina e pelas pastas à base de hidróxido de cálcio (CalenTM e Calen/PMCC TM), com resultados similares entre os materiais (p > 0.05). Nenhuma atividade antimicrobiana foi observada para os demais grupos. Concluiu-se que os cones de guta-percha com clorexidina apresentaram atividade antimicrobiana, enquanto os cones com hidróxido de cálcio não demonstraram esta propriedade.

O terço apical da raiz: características morfológicas, microbiota e considerações terapêuticas / Apical third of the root: morphological characteristics, microbiota and therapeutic implications

Devido à localização estratégica do terço apical do canal radicular em relação aos tecidos perirradiculares, todos os esforços terapêuticos deveriam ser direcionados à realização de limpeza, modelagem desinfecção e selamento adequados dessa região visando o sucesso do tratamento endodôntico. O objetivo deste estudo é revisar a morfologia e a microbiologia do terço apical da raiz, discutindo as implicações clínicas desse conhecimento.

Presencia de Organismos Micologicos en las Lesiones Periapicales / Presence of Fungus in Periapical Lesions

Las lesiones pulpares y periapicales en general parecen poseer como agentes etiologicos la combinacion de las bacterias de las lesiones por caries, ademas de los antigenos bacterianos y los productos metabolicos. Pero igualmente la pulpa puede verse atacada por agentes mecanicos, quimicos o termicos. En cavidad oral encontramos organismos bacteriologicos y micologicos como flora normal de la misma, pero se ha observado que las bacterias pueden encontrarse como organismos presentes en las lesiones,- para nuestro estudio en lo concerniente a lesiones periapicales- al existir una alteracion del equilibrio existente; mas hasta el momento no se ha estudiado la presencia de organismos micologicos en dicahs lesiones. El analisis de los doce casos elegidos, se realizo previa eliminacion quirurgica de las lesiones, teniendo en cuenta los diferentes medios de asepcia necesarios para que las muestras no se contaminaran con el medio ambientre. Luego de obtener los resultados correspondientes al laboratorio bacteriologico e histopatologico se pudo determinar si las lesiones periapicales, absceso periapical, granuloma y quiste periapical- poseen organismos micologicos ademas de agentes bacteriologicos..