Antimicrobial Activity of Phytic Acid: An Emerging Agent in Endodontics.

Background: Phytic acid (IP6) is a promising and emerging agent, and because of its unique structure and distinctive properties, it lends itself to several applications in dentistry. Recently, IP6 was proposed as a potential chelating agent in endodontics. However, there is limited knowledge regarding its antimicrobial and antibiofilm effectiveness. The aims of this study, were therefore to evaluate the antimicrobial and antibiofilm activities of IP6 against a range of microbial species and compare these with ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite (NaOCl). The contact time required for IP6 to exert its bactericidal effect on Enterococcus faecalis was also determined. Methods: The inhibitory and biocidal activities of IP6, EDTA and NaOCl were assessed using a broth microdilution assay against 11 clinical and reference strains of bacteria and a reference strain of Candida albicans. The contact time required for various IP6 concentrations to eliminate planktonic cultures of E. faecalis was determined using a membrane filtration method according to BS-EN-1040:2005. IP6 bactericidal activity was also evaluated using fluorescent microscopy, and the antibiofilm activity of the test agents was also determined. Results: IP6 was biocidal against all tested microorganisms. At concentrations of 0.5%, 1% and 2%, IP6 required 5 min to exert a bactericidal effect on E. faecalis, while 5% IP6 was bactericidal after 30 s. IP6 also eradicated biofilms of the tested microorganisms. In conclusion, IP6 had notable antimicrobial effects on planktonic and biofilm cultures and exhibited rapid bactericidal effects on E. faecalis. This research highlighted, for the first time the antimicrobial and antibiofilm properties of IP6, which could be exploited, not only in dental applications, but also other fields where novel strategies to counter antimicrobial resistance are required.

The effect of sodium hypochlorite concentration and irrigation needle extension on biofilm removal from a simulated root canal model.

To investigate the effect of sodium hypochlorite concentration and needle extension on removal of Enterococcus faecalis biofilm, sixty root canal models were 3D printed. Biofilms were grown on the apical 3 mm of the canal for 10 days. Irrigation for 60s with 9 mL of either 5.25% or 2.5% NaOCl or water was performed using a needle inserted either 3 or 2 mm from the canal terminus and imaged using fluorescence microscopy and residual biofilm percentages were calculated using imaging software. The data were analysed using analysis of covariance and two-sample t-tests. A significance level of 0.05 was used throughout. Residual biofilm was less using 5.25% than with 2.5% NaOCl. Statistically significant biofilm removal was evident with the needle placed closer to the canal terminus. A greater reduction of available chlorine and pH was noted as the concentration increased. One-minute irrigation was not sufficient for complete biofilm removal.

Confocal laser scanning, scanning electron, and transmission electron microscopy investigation of Enterococcus faecalis biofilm degradation using passive and active sodium hypochlorite irrigation within a simulated root canal model.

Root canal irrigation is an important adjunct to control microbial infection. The aim of this study was to investigate the effect of 2.5% (wt/vol) sodium hypochlorite (NaOCl) agitation on the removal, killing, and degradation of Enterococcus faecalis biofilm. A total of 45 root canal models were manufactured using 3D printing with each model comprising an 18 mm length simulated root canal of apical size 30 and taper 0.06. E. faecalis biofilms were grown on the apical 3 mm of the models for 10 days. A total of 60 s of 9 ml of 2.5% NaOCl irrigation using syringe and needle was performed, the irrigant was either left stagnant in the canal or agitated using manual (Gutta-percha), sonic, and ultrasonic methods for 30 s. Following irrigation, the residual biofilms were observed using confocal laser scanning, scanning electron, and transmission electron microscopy. The data were analyzed using one-way ANOVA with Dunnett post hoc tests at a level of significance p ≤ .05. Consequence of root canal irrigation indicate that the reduction in the amount of biofilm achieved with the active irrigation groups (manual, sonic, and ultrasonic) was significantly greater when compared with the passive and untreated groups (p < .05). Collectively, finding indicate that passive irrigation exhibited more residual biofilm on the model surface than irrigant agitated by manual or automated (sonic, ultrasonic) methods. Total biofilm degradation and nonviable cells were associated with the ultrasonic group.

Investigation to test potential stereolithography materials for development of an in vitro root canal model.

The aims were to compare the physico-chemical properties (zeta-potential, wettability, surface free energy) of stereolithography materials (STL) (Photopolymer, Accura) to dentine and to evaluate the potential of each material to develop Enterococcus faecalis biofilm on their respective surfaces. Eighteen samples of each test material (Photopolymer, Accura, dentine) were employed (total n = 54) and sectioned to 1 mm squares (5 mm x 5 mm) (n = 15) or ground into a powder to measure zeta-potential (n = 3). The zeta-potential of the powder was measured using the Nano-Zetasizer technique. The contact angle (wettability, surface free energy tests) were measured on nine samples using goniometer. The biofilm attachment onto the substrate was assessed on the samples of each material using microscope and image processing software. The data were compared using one-way ANOVA with Dunnett post-hoc tests at a level of significance P ≤ 0.05. Both STL materials showed similar physico-chemical properties to dentine. The materials and dentine had negative charge (Accura: -23.7 mv, Photopolymer: -18.8 mv, dentine: -9.11 mv). The wettability test showed that all test materials were hydrophilic with a contact angle of 47.5°, 39.8°, 36.1° for Accura, Photopolymer and dentine respectively, and a surface free energy of 46.6, 57.7, 59.6 mN/m for Accura, Photopolymer and dentine, respectively. The materials and dentine proved suitable for attachment and growth of E. faecalis biofilm with no statistical differences (P > 0.05). Stereolithography materials show similar physico-chemical properties and growth of E. faecalis biofilm to dentine. Therefore, they may be an alternative to tests requiring dentine.

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.

OBJECTIVE: Root canal irrigation is an important adjunct to control microbial infection. This study aimed primarily to develop a transparent root canal model to study in situ Enterococcus faecalis biofilm removal rate and remaining attached biofilm using passive or active irrigation solution for 90s. The change in available chlorine and pH of the outflow irrigant were assessed. METHODS: A total of forty root canal models (n=10 per group) were manufactured using 3D printing. Each model consisted of two longitudinal halves of an 18mm length simulated root canal with size 30 and taper 0.06. E. faecalis biofilms were grown on the apical 3mm of the models for 10days in Brain Heart Infusion broth. Biofilms were stained using crystal violet for visualization. The model halves were reassembled, attached to an apparatus and observed under a fluorescence microscope. Following 60s of 9mL of 2.5% NaOCl irrigation using syringe and needle, the irrigant was either left stagnant in the canal or activated using gutta-percha, sonic and ultrasonic methods for 30s. Images were then captured every second using an external camera. The residual biofilm percentages were measured using image analysis software. The data were analyzed using Kruskal-Wallis test and generalized linear mixed model. RESULTS: The highest level of biofilm removal was with ultrasonic agitation (90.13%) followed by sonic (88.72%), gutta-percha (80.59%), and passive irrigation group (control) (43.67%) respectively. All agitation groups reduced the available chlorine and pH of NaOCl more than that in the passive irrigation group. SIGNIFICANCE: The 3D printing method provided a novel model to create a root canal simulation for studying and understanding a real-time biofilm removal under microscopy. Ultrasonic agitation of NaOCl left the least amount of residual biofilm in comparison to sonic and gutta-percha agitation methods.

Variation on a theme; an overview of the Tn916/Tn1545 family of mobile genetic elements in the oral and nasopharyngeal streptococci.

The oral and nasopharyngeal streptococci are a major part of the normal microbiota in humans. Most human associated streptococci are considered commensals, however, a small number of them are pathogenic, causing a wide range of diseases including oral infections such as dental caries and periodontitis and diseases at other body sites including sinusitis and endocarditis, and in the case of Streptococcus pneumoniae, meningitis. Both phenotypic and sequence based studies have shown that the human associated streptococci from the mouth and nasopharynx harbor a large number of antibiotic resistance genes and these are often located on mobile genetic elements (MGEs) known as conjugative transposons or integrative and conjugative elements of the Tn916/Tn1545 family. These MGEs are responsible for the spread of the resistance genes between streptococci and also between streptococci and other bacteria. In this review we describe the resistances conferred by, and the genetic variations between the many different Tn916-like elements found in recent studies of oral and nasopharyngeal streptococci and show that Tn916-like elements are important mediators of antibiotic resistance genes within this genus. We will also discuss the role of the oral environment and how this is conducive to the transfer of these elements and discuss the contribution of both transformation and conjugation on the transfer and evolution of these elements in different streptococci.

Chlorhexidine in endodontics.

Chemical auxiliary substances (CAS) are essential for a successful disinfection and cleanness of the root canals, being used during the instrumentation and if necessary, as antimicrobial intracanal medicaments. Different CAS have been proposed and used, among which sodium hypochlorite (NaOCl), chlorhexidine (CHX), 17% EDTA, citric acid, MTAD and 37% phosphoric acid solution. CHX has been used in Endodontics as an irrigating substance or intracanal medicament, as it possesses a wide range of antimicrobial activity, substantivity (residual antimicrobial activity), lower cytotoxicity than NaOCl whilst demonstrating efficient clinical performance, lubricating properties, rheological action (present in the gel presentation, keeping the debris in suspension); it inhibits metalloproteinase, is chemically stable, does not stain cloths, it is odorless, water soluble, among other properties. CHX has been recommended as an alternative to NaOCl, especially in cases of open apex, root resorption, foramen enlargement and root perforation, due to its biocompatibility, or in cases of allergy related to bleaching solutions. The aim of this paper is to review CHX's general use in the medical field and in dentistry; its chemical structure, presentation form and storage; mechanism of action; antimicrobial activity including substantivity, effects on biofilms and endotoxins, effects on coronal and apical microbial microleakage; tissue dissolution ability; interaction with endodontic irrigants; effects on dentin bonding, metalloproteinases and collagen fibrils; its use as intracanal medicament and diffusion into the dentinal tubules; its use as disinfectant agent of obturation cones; other uses in the endodontic therapy; and possible adverse effects, cytotoxicity and genotoxicity.

Chlorhexidine in Endodontics

Chemical auxiliary substances (CAS) are essential for a successful disinfection and cleanness of the root canals, being used during the instrumentation and if necessary, as antimicrobial intracanal medicaments. Different CAS have been proposed and used, among which sodium hypochlorite (NaOCl), chlorhexidine (CHX), 17% EDTA, citric acid, MTAD and 37% phosphoric acid solution. CHX has been used in Endodontics as an irrigating substance or intracanal medicament, as it possesses a wide range of antimicrobial activity, substantivity (residual antimicrobial activity), lower cytotoxicity than NaOCl whilst demonstrating efficient clinical performance, lubricating properties, rheological action (present in the gel presentation, keeping the debris in suspension); it inhibits metalloproteinase, is chemically stable, does not stain cloths, it is odorless, water soluble, among other properties. CHX has been recommended as an alternative to NaOCl, especially in cases of open apex, root resorption, foramen enlargement and root perforation, due to its biocompatibility, or in cases of allergy related to bleaching solutions. The aim of this paper is to review CHX's general use in the medical field and in dentistry; its chemical structure, presentation form and storage; mechanism of action; antimicrobial activity including substantivity, effects on biofilms and endotoxins, effects on coronal and apical microbial microleakage; tissue dissolution ability; interaction with endodontic irrigants; effects on dentin bonding, metalloproteinases and collagen fibrils; its use as intracanal medicament and diffusion into the dentinal tubules; its use as disinfectant agent of obturation cones; other uses in the endodontic therapy; and possible adverse effects, cytotoxicity and genotoxicity.

Resumo Substâncias químicas auxiliares (SQA) são essenciais para o processo de limpeza e desinfecção dos canais radiculares, sendo utilizadas durante a instrumentação dos canais radiculares e, se necessário, como medicamentos intracanais. Diferentes SQA têm sido propostas e utilizadas, entre elas: hipoclorito de sódio (NaOCl), clorexidina (CHX), EDTA 17%, ácido cítrico, MTAD e solução de ácido fosfórico a 37%. CHX tem sido usada na endodontia como SQA ou medicação intracanal. CHX possui uma ampla gama de atividade antimicrobiana; substantividade (atividade antimicrobiana residual); menor citotoxicidade que NaOCl, demonstrando desempenho clínico eficiente; propriedades de lubrificação; ação reológica (presente na apresentação gel, mantendo os detritos em suspensão); inibe metaloproteinases; é quimicamente estável; não mancha tecidos; é inodora; solúvel em água; entre outras propriedades. CHX tem sido recomendada como uma alternativa ao NaOCl, especialmente em casos de ápice aberto, reabsorção radicular, perfuração radicular e durante a ampliação foraminal, devido à sua biocompatibilidade, ou em casos de alergia ao NaOCl. O objetivo deste trabalho é fazer uma revisão do uso da clorexidina na medicina e na odontologia; sua estrutura química; forma de apresentação e armazenamento; mecanismo de ação, atividade antimicrobiana, incluindo, substantividade, efeitos sobre biofilmes e endotoxinas; efeito sobre infiltração microbiana coronal e apical; capacidade de dissolução do tecido; interação com os irrigantes; efeitos sobre a união à dentina, metaloproteinases e fibrilas de colágeno; a sua utilização como medicamento intracanal e difusão ...

Complementary retrieval of 16S rRNA gene sequences using broad-range primers with inosine at the 3'-terminus: implications for the study of microbial diversity.

We evaluated the impact of the base analogue inosine substituted at the 3'-terminus of broad-range 16S rRNA gene primers on the recovery of microbial diversity using terminal restriction fragment length polymorphism and clonal analysis. Oral plaque biofilms from 10 individuals were tested with modified and unmodified primer pairs. Besides a core overlap of shared terminal restriction fragments (T-RFs), each primer system provided unique information on the occurrence of T-RFs, with a higher number generally displayed with inosine primers. All clones sequenced were at least 99% identical to publicly available full-length sequences. Analysis of the corresponding primer-binding sites showed that most sequence types were 100% complementary to the unmodified primers so that the characteristic of inosine to bind with all four nucleotides was not crucial for the observed increase in microbial richness. Instead, differences in community compositions were correlated with the identity of the nearest-neighbor 3' of the primer-targeting region. By influencing the thermal stability of primer hybridization, this position may play a previously unrecognized role in biased amplification of 16S rRNA gene sequences. In conclusion, the combined use of inosine and unmodified primers enables the complementary retrieval of 16S rRNA gene types, thereby expanding the observed diversity of complex microbial communities.

New methods for selective isolation of bacterial DNA from human clinical specimens.

Separation of bacterial DNA from human DNA in clinical samples may have an important impact on downstream applications, involving microbial diagnostic systems. We evaluated two commercially available reagents (MolYsis), Molzym GmbH & Co. KG, Bremen and Pureprove, SIRS-Lab GmbH, Jena, both Germany) for their potential to isolate and purify bacterial DNA from human DNA. We chose oral samples, which usually contain very high amounts of both human and bacterial cells. Three different DNA preparations each were made from eight caries and eight periodontal specimens using the two reagents above and a conventional DNA extraction strategy as reference. Based on target-specific real-time-quantitative PCR assays we compared the reduction of human DNA versus loss of bacterial DNA. Human DNA was monitored by targeting the beta-2-microglobulin gene, while bacteria were monitored by targeting 16S rDNA (total bacteria and Porphyromonas gingivalis) or the glycosyltransferase gene (Streptococcus mutans). We found that in most cases at least 90% of human DNA could successfully be removed, with complete removal in eight of 16 cases using MolYsis, and two (of 16) cases using Pureprove. Conversely, detection of bacterial DNA was possible in all cases with a recovery rate generally ranging from 35% to 50%. In conclusion, both strategies have the potential to reduce background interference from the host DNA which may be of remarkable value for nucleic-acid based microbial diagnostic systems.

Comparison of 2.5% sodium hypochlorite and 2% chlorhexidine gel on oral bacterial lipopolysaccharide reduction from primarily infected root canals.

INTRODUCTION: This clinical study was conducted to compare the efficacy of chemomechanical preparation with 2.5% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) gel on eliminating oral bacterial lipopolysaccharide (LPS) in teeth with pulp necrosis and apical periodontitis. METHODS: Fifty-four root canals were selected. Samples were collect before (s1) and after chemomechanical preparation (s2). Teeth were randomly divided into groups: GI, 2.5% NaOCl (n = 27) and GII, CHX gel (n = 27). Limulus amebocyte lysate assay was used to quantify endotoxins. RESULTS: Endotoxin was present in 100% of the samples investigated, with a median value of 272 endotoxin units (EU)/mL (GI) and of 152.46 EU/mL (GII). As a result of chemomechanical preparation, LPS content was reduced to a median value of 86 EU/mL (GI) and 85 EU/mL (GII). Higher percentage value of endotoxin reduction was found in GI (P < .05). CONCLUSIONS: The 2.5% NaOCl and 2% CHX gel were not effective in eliminating endotoxin from the primarily infected root canals.

Root canal filling with cements based on mineral aggregates: an in vitro analysis of bacterial microleakage.

The aim of this study was to assess bacterial microleakage through 2 different cements used as root canal filling materials: mineral-trioxide-aggregate (White MTA, Angelus, Londrina, PR, Brazil); and an experimental material containing calcium aluminate plus additives (EndoBinder, University of São Carlos [UFSCar-Brazil], patent number PI0704502-6). Forty incisors were divided into 4 groups: (1) white-MTA, (2) calcium aluminate cement, and (3) positive and (4) negative control group. Falcon tubes were prepared for this experiment, divided into 2 separated chambers; the lower part was filled with RTF-transport media so that only the root apex was in contact with the transport media, whereas the coronal part was immersed in BHI containing Enterococcus faecalis to investigate bacterial microleakage. The evaluated materials used as root canal filling materials showed an absence of microbial growth. Both materials when used as the root canal filling were efficient in sealing root canals and preventing E. faecalis microleakage for the 30-day evaluation period using an in vitro model.

Efficacy of sodium hypochlorite combined with chlorhexidine against Enterococcus faecalis in vitro.

The aim of this study was to investigate in vitro the efficacy of the combination of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) in different concentrations against Enterococcus faecalis compared with the antimicrobial activity of the same irrigating substances when applied alone. The substances tested were 2.0% CHX gel, 2.0% CHX liquid, 1% NaOCl, 2.5% NaOCl, 5.25% NaOCl, and combinations of them in the same proportions. Two methods were used: the agar diffusion test and the broth dilution test. The largest mean microbial growth zone was obtained by 2.0% CHX gel, and the smallest zones were obtained by 1% and 2.5% NaOCl (P < .05). 2.0% CHX (gel and liquid formulation), 5.25% NaOCl, and the combination of 2.0% CHX liquid and 5.25% NaOCl, all of which needed

Selective isolation of bacterial DNA from human clinical specimens.

We evaluated two DNA preparation strategies (MolYsis, Molzym GmbH & Co. KG, Bremen, Germany) and Pureprove, SIRS-Lab GmbH, Jena, Germany) to selectively extract bacterial DNA from human clinical samples. By testing 16 oral samples we found that human DNA could be largely eliminated while detectable levels of bacterial DNA were obtained with all samples. Both approaches hold great potential for microbial diagnostic systems.