PCR identification of endodontic pathogens and DNA quantification in samples from teeth with posttreatment apical periodontitis

Abstract

The aim of this clinical study was to quantify the concentration of DNA and to detect selected bacterial species from samples of infected root-fi lled teeth with post-treatment apical periodontitis after removal of gutta-percha (S1), after chemo-mechanical preparation at the fi rst appointment (S2), 5 days after the canal was fi lled with sterile physiological solution (S3), after reinstrumentation at the second appointment (S4), and 14 days after an intracanal dressing was placed at the third appointment (S5). Methods: Fifteen root-fi lled teeth were selected. Removal of gutta-percha was performed using the crown-down technique. Chemo-mechanical preparation was performed with hand fi les associated with 2% chlorhexidine gel. An intracanal dressing based on Ca(OH) 2 was used. DNA was extracted from the samples and 14 endodontic 16S rDNA species-specifi c primers were tested. The concentration of DNA was quantifi ed using a NanoDropTM 2000 spectrophotometer. Results: Bacteria were present in all cases at all sampling times, as revealed by a universal primer. DNA was isolated from all samples, with an average concentration of 4.24 ± 2.9 ng/µL (S1), 3.39 ± 1.54 ng/µL (S2), 4.0 ± 1.94 ng/µL (S3), 2.66 ± 0.98 ng/µL (S4) and 3.97 ± 2.32 ng/µL (S5). Parvimonas micra and Enterococcus faecalis (S1), P. micra (S2), Porphyromonas endodontalis and E. faecalis (S3), E. faecalis and Prevotella nigrescens (S4/S5) were the species most frequently deteced. DNA concentration reductions were detected between S3 and S4 (p = 0.0256), whereas an increase was found between S4 and S5. Conclusion: A wide variety of bacterial species was detected in root-fi lled teeth with post-treatment apical periodontitis. Moreover, the use of an intracanal dressing was unable to further reduce the concentration of bacterial DNA.descrIPtors | Endodontics; Treatment Failure; Periapical Periodontitis; Bacteria; Polymerase Chain Reaction; DNA; Chlorhexidine.resuMo | Identifi cação de patógenos endodônticos por PCR e quantifi cação de DNA em amostras extraídas de dentes com periodontite apical pós-tratamento • Objetivo: O objetivo deste estudo clínico foi quantifi car a concentração de DNA e detectar algumas espécies bacterianas de amostras de dentes tratados endodonticamente com periodontite apical após a remoção da guta-percha (S1), após o preparo químico-mecânico na primeira sessão (S2), 5 dias após o preenchimento do canal com solução fi siológica estéril (S3), após reinstrumentação na segunda sessão (S4), e 14 dias após a inserção da medicação intracanal na terceira sessão (S5). Métodos: Quinze dentes tratados endodonticamente foram selecionados. A remoção da guta-percha foi realizada por meio da técnica coroa-ápice. Utilizaram-se limas manuais associadas à clorexidina gel a 2% durante o preparo químico-mecânico. A medicação intracanal selecionada foi à base de hidróxido de cálcio. DNA foi isolado das amostras e foram investigadas 14 espécies bacterianas (primer espécie-específi co16S rDNA). A concentração de DNA foi quantifi cada utilizando o espect...

A novel experimental approach to investigate the effect of different agitation methods using sodium hypochlorite as an irrigant on the rate of bacterial biofilm removal from the wall of a simulated root canal model

“…Furthermore, E. faecalis is able to develop a biofilm under different growth conditions, including aerobic, anaerobic, nutrient-rich and nutrient-deprived environments [50]. Moreover, this species has been used to evaluate the efficacy of irrigation solutions [48,51]. However, single species biofilm may be considered a limitation of the present study and future investigations using multi-species biofilms, including Gram-negative species may be valuable to be explored in the future.…”

“…The Gram-positive facultative E. faecalis type strain was selected to generate the biofilms as these species have been associated with secondary root canal infections [48]. It has been reported that E. faecalis exhibits an inherent resistance to antimicrobial agents, as well as possessing the capacity to adapt to changes in environmental conditions [49].…”

Investigation to test potential stereolithography materials for development of anin vitroroot canal model

“…The Gram‐positive facultative E. faecalis type strain was selected to generate the biofilms because these species have been associated with secondary root canal infections (Endo et al, ). It has been reported that E. faecalis has a hydrophilic and negatively charged cell wall (George & Kishen, ) and exhibits an inherent resistance to antimicrobial agents, as well as possessing the capacity to adapt to changes in environmental conditions (Laplace et al, ).…”

“…The Gram-positive facultative E. faecalis type strain was selected to generate the biofilms because these species have been associated with secondary root canal infections (Endo et al, 2014). It has been reported that E. faecalis has a hydrophilic and negatively charged cell wall (George & Kishen, 2007) and exhibits an inherent resistance to FIG URE 1 SEM images (x2000, x10000 magnification) illustrate that the E. faecalis biofilm grown onto the surface of the (a) dentine, (b) Photopolymer, or (c) Accura sample surface after ten-day incubation.…”

TÉCNICAS DE BIOLOGÍA MOLECULAR PARA LA INVESTIGACIÓN EN ODONTOLOGÍA Y BIOLOGÍA ORAL (1a PARTE)

“…10 De igual manera, su uso para estudiar patógenos endodónticos también se ha comprobado. 11 Finalmente, la posibilidad de detectar mutaciones en genes humanos (o marcadores moleculares) relacionados con la manifestación de cáncer bucal es otra posibilidad que, ha quedado firmemente establecida (Figura 5). 12…”