Alkaline Tolerance and Biofilm Formation of Root Canal Isolates of Enterococcus faecalis: An In Vitro Study.

INTRODUCTION: The aim of this study was to compare the effects of glucose and glycerol (provided as principal fermentable supplements) on alkaline tolerance and biofilm-forming capabilities of root canal-derived strains of Enterococcus faecalis and those from other environments. METHODS: The planktonic growth kinetics and the biofilm-forming capabilities of E. faecalis isolates (identified by 16S ribosomal DNA sequencing) were compared when supplied with glucose and glycerol at pH levels of 8 and 11 in a microtiter plate. The metabolic activity of the biofilms plate that formed at a neutral pH level (supplied with either glucose or glycerol) was measured after subsequent adjustment to a pH level of 11. RESULTS: Ten isolates (7 from root canals and 3 from other sources) were examined. The lag phase and the doubling time increased under elevated alkalinity irrespective of either the fermentable supplement (glucose or glycerol) or the origin of the isolate. Biofilm formation and metabolic activity varied among strains, but neither was related to the source of isolation. In general, biofilm formation was enhanced when grown in glucose compared with glycerol and at a pH of 8 compared with a pH of 11 (irrespective of the fermentable supplement). The provision of glycerol did not increase either the planktonic growth rate or biofilm development compared with glucose but significantly increased the metabolic activity of biofilms, especially at a pH of 11 compared with a pH of 8. CONCLUSIONS: In the nutrient-deprived environment of a necrotic or root-filled root canal, glycerol may be an alternative energy source that can promote increased metabolic activity of E. faecalis under alkaline treatment conditions.

Identificación de microorganismos por reacción en cadena de la polimerasa en necrosis pulpar y periodontitis apical / Identification of microorganisms by polymerase chain reaction in pulp necrosis and apical periodontitis

Objetivos: identificar, mediante la técnica de reacción en cadena de la polimerasa, diez especies bacterianas procedentes de conductos radiculares necróticos, así como analizar su asociación con signos y síntomas de la periodontitis apical (dolor, exudado y movilidad), dentro de un área geográfica específica, Santiago de Compostela (noroeste de España). Métodos: se realizó un estudio descriptivo, en un plazo de recogida de muestras de un año. Se extrajeron 43 muestras de los conductos radiculares necróticos, a razón de una muestra por pacientes, quienes fueron examinados previamente para determinar la presencia de periodontitis apical radiográfica, de la cual fueron descritos sus signos y síntomas. Se utilizaron puntas de papel absorbentes estériles y se procedió a extraer de dichas muestras el ADN por medio de la técnica del fenol-cloroformo. El producto se amplificó por medio de la reacción en cadena de la polimerasa, usando cebadores específicos para diez microorganismos. El resultado se visualizó por medio de electroforesis, utilizando un transiluminador UV. Los resultados se analizaron estadísticamente, para así establecer qué microorganismos estaban presentes de forma individual y cuáles se presentaron formando asociaciones como el llamado red complex, en relación con los signos y síntomas presentes en la periodontitis apical. Resultados: Fusobacterium nucleatum fue el microorganismo con presencia de forma individual más frecuente (83,72 por ciento de las muestras). En cuanto al estudio de la asociación de los microorganismos con los signos y síntomas, Porphyromonas endodontalis presentó una asociación significativa con respecto al exudado (p< 0,05) y movilidad (p< 0,05). En cambio, Enterococcus spp. y Treponema denticola (p< 0,01) presentaron ambos una asociación significativa con respecto al dolor. En cuanto a la incidencia del red complex fue de 6 casos en total. Conclusiones: Fusobacterium nucleatum y Streptococcus spp. son los microorganismos identificados con mayor porcentaje en comparación a los restantes estudiados. Porphyromonas endodontalis es el microorganismo con mayor asociación estadística con respecto a los signos y síntomas de la periodontitis apical(AU)

Objectives: identify by polymerase chain reaction technique ten bacterial species obtained from necrotic root canals, and analyze their association with signs and symptoms of apical periodontitis (pain, exudate and mobility) in a specific geographic area: Santiago de Compostela (northwestern Spain). Methods: a descriptive study was conducted based on a one-year sample collection period. Forty-three samples were taken from necrotic root canals, at a rate of one sample per patient, who had been previously examined for radiographic apical periodontitis, of which the signs and symptoms were described. Sterile absorbent paper points were used to extract the DNA samples, applying the phenol-chloroform technique. The product was amplified by polymerase chain reaction, using specific primers for ten microorganisms. The result was visualized by electrophoresis using a UV transilluminator. Outcomes were analyzed statistically to determine which microorganisms were present individually and which formed associations such as the so-called red complex, according to the signs and symptoms present in the apical periodontitis. Results: Fusobacterium nucleatum was the most common individual microorganism (83.72 percent of the samples). As to the association of microorganisms with signs and symptoms, Porphyromonas endodontalis was found to be significantly associated with exudate (p< 0.05) and mobility (p< 0.05), while both Enterococcus spp. and Treponema denticola (p< 0.01) had a significant association with pain. Incidence of the red complex was 6 cases in all. Conclusions: Fusobacterium nucleatum and Streptococcus spp. were the microorganisms identified as displaying the highest percentages. Porphyromonas endodontalis showed the greatest statistical association with signs and symptoms of apical periodontitis(AU)

Identificación de microorganismos por reacción en cadena de la polimerasa en necrosis pulpar y periodontitis apical / Identification of microorganisms by polymerase chain reaction in pulp necrosis and apical periodontitis

Objetivos: identificar, mediante la técnica de reacción en cadena de la polimerasa, diez especies bacterianas procedentes de conductos radiculares necróticos, así como analizar su asociación con signos y síntomas de la periodontitis apical (dolor, exudado y movilidad), dentro de un área geográfica específica, Santiago de Compostela (noroeste de España). Métodos: se realizó un estudio descriptivo, en un plazo de recogida de muestras de un año. Se extrajeron 43 muestras de los conductos radiculares necróticos, a razón de una muestra por pacientes, quienes fueron examinados previamente para determinar la presencia de periodontitis apical radiográfica, de la cual fueron descritos sus signos y síntomas. Se utilizaron puntas de papel absorbentes estériles y se procedió a extraer de dichas muestras el ADN por medio de la técnica del fenol-cloroformo. El producto se amplificó por medio de la reacción en cadena de la polimerasa, usando cebadores específicos para diez microorganismos. El resultado se visualizó por medio de electroforesis, utilizando un transiluminador UV. Los resultados se analizaron estadísticamente, para así establecer qué microorganismos estaban presentes de forma individual y cuáles se presentaron formando asociaciones como el llamado red complex, en relación con los signos y síntomas presentes en la periodontitis apical. Resultados: Fusobacterium nucleatum fue el microorganismo con presencia de forma individual más frecuente (83,72 por ciento de las muestras). En cuanto al estudio de la asociación de los microorganismos con los signos y síntomas, Porphyromonas endodontalis presentó una asociación significativa con respecto al exudado (p< 0,05) y movilidad (p< 0,05). En cambio, Enterococcus spp. y Treponema denticola (p< 0,01) presentaron ambos una asociación significativa con respecto al dolor. En cuanto a la incidencia del red complex fue de 6 casos en total. Conclusiones: Fusobacterium nucleatum y Streptococcus spp. son los microorganismos identificados con mayor porcentaje en comparación a los restantes estudiados. Porphyromonas endodontalis es el microorganismo con mayor asociación estadística con respecto a los signos y síntomas de la periodontitis apical(AU)

Objectives: identify by polymerase chain reaction technique ten bacterial species obtained from necrotic root canals, and analyze their association with signs and symptoms of apical periodontitis (pain, exudate and mobility) in a specific geographic area: Santiago de Compostela (northwestern Spain). Methods: a descriptive study was conducted based on a one-year sample collection period. Forty-three samples were taken from necrotic root canals, at a rate of one sample per patient, who had been previously examined for radiographic apical periodontitis, of which the signs and symptoms were described. Sterile absorbent paper points were used to extract the DNA samples, applying the phenol-chloroform technique. The product was amplified by polymerase chain reaction, using specific primers for ten microorganisms. The result was visualized by electrophoresis using a UV transilluminator. Outcomes were analyzed statistically to determine which microorganisms were present individually and which formed associations such as the so-called red complex, according to the signs and symptoms present in the apical periodontitis. Results: Fusobacterium nucleatum was the most common individual microorganism (83.72 percent of the samples). As to the association of microorganisms with signs and symptoms, Porphyromonas endodontalis was found to be significantly associated with exudate (p< 0.05) and mobility (p< 0.05), while both Enterococcus spp. and Treponema denticola (p< 0.01) had a significant association with pain. Incidence of the red complex was 6 cases in all. Conclusions: Fusobacterium nucleatum and Streptococcus spp. were the microorganisms identified as displaying the highest percentages. Porphyromonas endodontalis showed the greatest statistical association with signs and symptoms of apical periodontitis(AU)

Endodontic Microbiology and Pathobiology: Current State of Knowledge.

Newer research tools and basic science knowledge base have allowed the exploration of endodontic diseases in the pulp and periapical tissues in novel ways. The use of next generation sequencing, bioinformatics analyses, genome-wide association studies, to name just a few of these innovations, has allowed the identification of hundreds of microorganisms and of host response factors. This review addresses recent advances in endodontic microbiology and the host response and discusses the potential for future innovations in this area.

Antimicrobial peptide-based treatment for endodontic infections--biotechnological innovation in endodontics.

The presence/persistence of microorganisms in the pulp and periapical area corresponds to the maintenance of an exacerbated immune response that leads to the start of periradicular bone resorption and its perpetuation. In endodontic treatment, the available intracanal medications do not have all the desirable properties in the context of endodontic infection and apical periodontitis; they need to include not only strong antimicrobial performance but also an immunomodulatory and reparative activity, without host damage. In addition, there are various levels of resistance to root canal medications. Thus, antimicrobial agents that effectively eliminate resistant species in root canals could potentially improve endodontic treatment. In the emergence of new therapies, an increasing number of studies on antimicrobial peptides (AMPs) have been seen over the past few years. AMPs are defense biomolecules produced in response to infection, and they have a wide spectrum of action against many oral microorganisms. There are some studies that correlate peptides and oral infections, including oral peptides, neuropeptides, and bacterial, fish, bovine and synthetic peptides. So far, there are around 120 published studies correlating endodontic microbiota with AMPs but, according to our knowledge, there are no registered patents in the American patent database. There are a considerable number of AMPs that exhibit excellent antimicrobial activity against endodontic microbiota at a small inhibitory concentration and modulate an exacerbated immune response, down-regulating bone resorption. All these reasons indicate the antimicrobial peptide-based endodontic treatment as an emerging and promising option.

Microbial invasion of dentinal tubules: a literature review and a new perspective.

Various features of endodontic microbiology have been investigated using various methods. The aim of the present study was to review the existing literature on endodontic microbiology in dentinal tubules, and to present the features of two cases with endodontic pathology. An electronic search was performed with a search string created ad hoc. Ex vivo and in vitro studies were included, recording the method of detection and characteristics of analyzed teeth. Twenty studies fulfilled the inclusion criteria. Seven of them were in vitro laboratory studies on teeth inoculated after extraction, while 13 were ex vivo studies on extracted, infected teeth. Endodontic bacteria were detected in dentinal tubules, both as single units and as biofilm aggregates. Two similar in vitro cases presented here corroborate the latter findings. A number of techniques have been utilized to observe bacteria in the dentinal tubule ecosystem. Dentinal tubules are favorable niches for microbial survival, either in the form of monomicrobial or polymicrobial communities.

Effectiveness of two intracanal dressings in adult Portuguese patients: a qPCR and anaerobic culture assessment.

AIM: To quantify bacterial equivalents before and after chemomechanical preparation using 3% sodium hypochlorite (NaOCl) and intracanal dressing with calcium hydroxide paste (Ca(OH)2 ) or 2% Chlorhexidine digluconate gel (CHX) in necrotic pulps associated or not with apical periodontitis and to further compare this quantification with counts of anaerobic microorganisms. METHODOLOGY: Prospective clinical trial in 69 single-rooted adult teeth (strict inclusion criteria); CHX group: 34; Ca(OH)2 group: 35. Bacteria samples were taken at baseline (S1), after chemomechanical preparation (S2) and after 14 days of intracanal dressing (S3). Bacterial equivalents were assessed by broad-range real-time polymerase chain reaction (qPCR), and live viable bacteria measured with conventional anaerobic culture (CFU/mL). Descriptive/inferential analysis was performed with spss vs. 20.0 (α = 0.05) using the Kruskal-Wallis, Mann-Whitney and chi-squared tests and Spearman's correlation coefficients. RESULTS: Both groups showed a significant decrease between S1 and S2 (Mann-Whitney U-test; P < 0.001) both in qPCR and in culture. In the Ca(OH)2 -group, no variation was observed between S2 and S3 by qPCR and culture. In contrast, the CHX group showed a significant increase from S2 to S3 by both techniques. The two groups were only significantly different in S3 (Mann-Whitney U-test; P ≤ 0.001), with a worse performance in the CHX group. Again, these results were congruent by both approaches. Data from both approaches correlate reasonably (rS < 0.5). CONCLUSIONS: Infected root canals contained a high bacterial load, and the chemomechanical root canal preparation reduced bacterial equivalents by 99.1% and anaerobic counts by 98.5%. Intracanal dressings were not efficient at reducing bacterial load, but the 14-day intracanal dressing with Ca(OH)2 performed significantly better than CHX, particularly in cases with apical periodontitis.

Enterococcus faecalis and Candida albicans in the dental root canal and periapical infections.

OBJECTIVE: The aim of the present study was to examine the prevalence of Enterococcus faecalis and Candida albicans in endodontic infections. METHODS: Samples for microbiological examination were collected from 32 patients with deep dental caries, infected dental root canal, or periapical infection. RESULTS: Cultivation of the dental samples yielded four strains of Enterococcus faecalis (12.5 %), and three strains of Candida albicans (9.4 %). All Enterococcus faecalis isolates were susceptible to ampicillin, one isolate was resistant to tetracycline, two to erythromycin and azithromycin (additional 2 had intermediate susceptibility), and one strain had intermediate susceptibility to ciprofloxacin and moxifloxacin. CONCLUSION: We conclude that Enterococcus faecalis and Candida albicans can participate in the dental root canal and periapical infections, and the use of effective irrigant solutions and intracanal medicaments active against these microbes is important in order to prevent endodontic therapy failures. Unexpected was the isolation of C. albicans from a nine-year-old child with periodontitis apicalis. This finding must draw attention to the possibility that even at such a young age, this microorganism could be a potential etiological agent in endodontic infections (Tab. 2, Ref. 34). Text in PDF www.elis.sk.