Qualitative and quantitative molecular analysis of bacteria in root canals of primary teeth with pulp necrosis

Abstract Information about bacterial diversity, such as the number of each species in the root canals of primary teeth, contributes to improving our effective management of infections of endodontic origin in primary teeth. This study made a qualitative and quantitative assessment of the bacteria in the root canals of primary teeth with necrotic pulp, using the fluorescence in situ hybridization (FISH) technique. Thirty-one primary teeth with pulp necrosis from 31 children were evaluated using the FISH technique, to detect the presence and density of Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Streptococcus, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythia and Treponema denticola. Descriptive measures explained the data related to density, and Student's t-test assessed the differences among the densities of each bacterium, according to signs and symptoms. The bacterial density was paired and correlated. All bacteria tested were detected and identified in all the samples. The average number of bacterial individuals from each species ranged from 1.9 x 108 cells/mL (S. mutans) to 3.1 x 108 cells/mL (F. nucleatum) (p > 0.05). The sum of the mean counts of each bacterium represented almost 80% of the entire microbial community. Patients with pain had significantly more T. denticola, and those with edema showed a greater density of Streptococcus and P. nigrescens (p < 0.05). This study revealed that all 12 bacteria evaluated were found in all primary teeth with pulp necrosis. There was no predominance among the species studied; all species had a similar number of individuals.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries

Abstract A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite

Abstract Objective This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy

Abstract Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. Objectives The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. Material and Methods Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. Results Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). Conclusion The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA.

Successful and failed mini-implants: microbiological evaluation and quantification of bacterial endotoxin

Abstract Objectives Using two groups of mini-implants (successful and failed) the objectives of this in vivo study were: to evaluate the microbial contamination by the checkerboard DNA-DNA hybridization technique and to quantify the bacterial endotoxin by the limulus amebocyte lysate assay. Material and Methods The 15 successful and 10 failed mini-implants (1.6 mm diameter × 7.0 or 9.0 mm long), placed in the maxilla and/or mandible, were obtained from 15 patients undergoing orthodontic treatment. Data were analyzed statistically by the Wilcoxon rank-sum test using the SAS software (a=0.05). Results All 40 microbial species were detected in both groups of mini-implants, with different frequencies. No differences were observed between the groups with respect to microbial complexes (blue, purple, yellow, green, orange, red and other species) and endotoxin quantification (p>0.05). Conclusion Neither microbial contamination nor endotoxin quantification was determinant for the early loss of stability of the mini-implants.

Dental caries and bacterial load in saliva and dental biofilm of type 1 diabetics on continuous subcutaneous insulin infusion

Abstract Objectives Since most of the studies evaluates diabetics on multiple daily injections therapy and continuous subcutaneous insulin infusion may help gain better metabolic control and prevent complications, the objective of this study was to evaluate the prevalence of dental caries, the unstimulated salivary flow rate and the total bacteria load, Streptococcus spp. levels and Lactobacillus spp. levels in saliva and supragingival dental biofilm of type 1 diabetics on insulin pump. Material and Methods Sixty patients with type 1 diabetes on insulin pump and 60 nondiabetic individuals were included. The dental caries evaluation was performed using ICDAS and the oral hygiene was assessed according to Greene and Vermillion Simplified Oral Hygiene Index. Unstimulated saliva and supragingival dental biofilm were collected. Total bacteria, Streptococcus spp. and Lactobacillus spp. was quantified by qPCR. Results Patients with type 1 diabetes had a higher prevalence of dental caries and filled and missing teeth when compared with the control group. These patients were associated with more risk factors for the development of dental caries, namely a lower unstimulated salivary flow rate and a higher bacterial load in saliva and dental biofilm. Conclusion Some risk factors related to dental caries were associated with type 1 diabetics. An early diagnosis combined with the evaluation of the risk profile of the diabetic patient is imperative, allowing the dental caries to be analyzed through a perspective of prevention and the patient to be integrated into an individualized oral health program.

The effects of different restorative materials on periodontopathogens in combined restorative-periodontal treatment

Abstract Objective The aim of the study was to evaluate the association between subgingival restorations and the target periodontopathogenic bacteria (Pg, Td and Pi) in subgingival biofilm during one year after combined restorative-periodontal treatment. Material and Methods Seventeen systemically healthy subjects, who were positive for the presence of three cervical lesions associated with gingival recessions in three different adjacent teeth, were included in the study. A total of 51 combined defects were treated with connective tissue graft plus a nanofilled composite resin (NCR+CTG), a resin-modified glass ionemer cement (RMGI+CTG) and a fluoride-releasing resin material with pre-reacted glass (PRG), called giomer (Giomer+CTG). Periodontal clinical measurements and subgingival plaque samples were obtained from all combined defects at baseline and at 6 and 12 months after the surgery. The number of bacteria were evaluated by the real-time polymerase chain reaction (qPCR) method. Results No statistically significant difference in the amount of DNA copies of Pg, Td and Pi was observed in any of the groups at any time points (p>0.05). In addition, there was no statistically significant difference in the amount of DNA copies of the bacteria at baseline and at 6 and 12 months postoperatively, regardless of treatment group (p>0.05). Conclusion This study suggests that subgingivally placed NCR, RMGI and giomer restorations can show similar effects on periodontopathogenic bacteria in the treatment of gingival recessions that are associated with noncarious cervical lesions (NCCLs).

Diversidade bacteriana nas infecções endodônticas primárias e secundárias/persistentes através da técnica de Checkerboard DNA-DNA Hybridization / Bacterial diversity in primary and secondary/persistent endodontic infections by Checkerboard DNA-DNA Hybridization Technique

Introdução: os métodos moleculares de diagnóstico apresentam como uma das principais vantagens a detecção de microrganismos por meio do DNA bacteriano, levando a uma caracterização microbiana mais acurada. Objetivo: o presente estudo visou investigar a diversidade bacteriana presente nas infecções endodônticas primárias e secundárias/persistentes, comparando o perfil das comunidades microbianas existentes antes e após a terapia endodôntica. Métodos: as amostras microbiológicas foram coletadas antes (T1) e após terapia endodôntica (T2), utilizando cone de papel estéril/apirogênico em dentes com infecções endodônticas primárias (n = 10) e secundárias / persistentes (n = 10). A presença e os níveis de 40 espécies bacterianas nas infecções endodônticas foram investigados por meio da técnica de Checkerboard DNA-DNA Hybridization. Resultados: nas infecções endodônticas primárias em T1, as espécies encontradas em maiores níveis foram P. micra, F. nucleatum sp. nucleatum, S. constellatus, P. gingivalis, G. morbillorum, P. endodontalis, T. denticola, P. acnes, S. gordonii, S. mitis, V. parvula e C. rectus. Em T2, as bactérias mais encontradas foram P. micra, S. oralis e P. acnes. Nas infecções endodônticas secundárias em T1, as espécies mais frequentemente encontradas foram P. acnes, P. micra, S. constellatus, G. morbillorum, C. rectus, A. naeslundii, S. mitis e S. oralis. Em T2, as espécies mais encontradas foram Enterococcus faecalis e Propionibacterium acnes. Conclusão: o presente estudo confirmou comunidades microbianas distintas em infecções endodônticas primárias e secundárias. Além disso, os procedimentos clínicos endodônticos mostraram-se eficazes na redução significativa da prevalência, dos níveis de detecção e na diversidade bacteriana.

Bacteriological analysis of necrotic pulp and fistulae in primary teeth

Objectives: Primary teeth work as guides for the eruption of permanent dentition, contribute for the development of the jaws, chewing process, preparing food for digestion, and nutrient assimilation. Treatment of pulp necrosis in primary teeth is complex due to anatomical and physiological characteristics and high number of bacterial species present in endodontic infections. The bacterial presence alone or in association in necrotic pulp and fistula samples from primary teeth of boys and girls was evaluated. Material and Methods: Necrotic pulp (103) and fistula (7) samples from deciduous teeth with deep caries of 110 children were evaluated. Bacterial morphotypes and species from all clinical samples were determined. Results: A predominance of gram-positive cocci (81.8%) and gram-negative coccobacilli (49.1%) was observed. In 88 out of 103 pulp samples, a high prevalence of Enterococcus spp. (50%), Porphyromonas gingivalis (49%), Fusobacterium nucleatum (25%) and Prevotella nigrescens (11.4%) was observed. Porphyromonas gingivalis was detected in three out of seven fistula samples, Enterococcus spp. in two out of seven samples, and F. nucleatum, P. nigrescens and D. pneumosintes in one out of seven samples. Conclusions: Our results show that Enterococcus spp. and P. gingivalis were prevalent in necrotic pulp from deciduous teeth in boys from 2 to 5 years old, and that care of the oral cavity of children up to five years of age is important. .

Preliminary results of the use an ultrasonic equipment in the treatment of moderate periodontitis: clinical and microbiological real time polimerase chain reacion-based (PCR) study

OBJETIVE: This preliminary study aimed to evaluate clinically and microbiologically the treatment of patients with light to moderate periodontal disease by using an ultrasonic device. MATERIAL AND METHODS: Sixteen adults with moderate periodontitis were studied. A Real-time PCR protocol was optimised using specific primers for the three microorganisms. Clinical and microbiological testing was performed prior and following treatment. RESULTS: Despite clinical improvement, bacteria quantification didn’t change. All the patients were positive for P. gingivalis and P. intermedia and nine for A. actinomycetemcomitans. CONCLUSION: VectorTM-system treatment showed clinical improvement. A Real-time PCR protocol is now available for periodontal diagnostics and monitorization. However, further studies with larger populations are indicated.

OBJETIVOS: Este estudo preliminar teve a finalidade de avaliar clinicamente microbiologicamente o tratamento de pacientes portadores de periodontite leve a moderada, utilizando-se um novo aparelho piezo-elétrico disponível no mercado (VectorTM system). MATERIAL E MÉTODO: Dezesseis pacientes adultos, portadores de periodontite leve a moderada, foram submetidos ao tratamento. Utilizou-se um protocolo de PCR em tempo real utilizando primers específicos para três microrganismos. Realizaram-se avaliação clínica e um estudo microbiológico simultâneo antes e após os tratamentos. RESULTADOS: A avaliação clínica foi positiva, embora não tendo ocorrido alterações na quantidade de bactérias. Todos os pacientes apresentaram amplificação específica para P. intermedia e P. gingivalis e nove pacientes para A. Actinomycetemcomitans. CONCLUSÃO: Considerando-se os resultados deste estudo preliminar, tratamento com o aparelho testado mostrou eficácia clínica. O protocolo utilizado neste estudo está agora disponível para diagnóstico periodontal e monitoração de resultados de tratamento. Entretanto, indica-se estudos clínicos e microbiológicos mais aprofundados, com amostras maiores, para conclusões mais consistentes.

Hidróxido de cálcio em endondontia: suposiçöes x comprovaçäo científica / The calcium hydroxide in endodontics: supposition x cientifc proof

Várias propriedades biológicas têm sido atribuídas ao hidróxido de cálcio, tais como: açäo antiinflamatória, açäo antimicrobiana, solvente de matéria orgânica, inibiçäo da reabsorçäo e induçäo de reparo mineralizado. Neste trabalho, estas propriedades säo discutidas baseado em achados científicos