Quantitative Assessment of the Efficacy of Two Different Single-file Systems in Reducing the Bacterial load in Oval-Shaped Canals: A Clinical Study.

INTRODUCTION: This randomized clinical study compared the in vivo antibacterial efficacy of Reciproc Blue (RB), XP-endo Shaper (XP-S), and XP-endo Shaper associated with XP-endo Finisher (XP-F) systems in infected oval-shaped root canals with primary apical periodontitis. METHODS: In this study, 28 human teeth with a single root and a single canal were randomly assigned to 2 groups according to the instrumentation technique: group 1, RB (n = 14) and group 2, XP-endo (XP-S and XP-F, n = 14). The single-rooted teeth were prepared by reciprocating and rotary nickel-titanium instruments with 5.25% sodium hypochlorite irrigation. Samples were collected from the canal at the baseline (S1), after chemomechanical preparation (S2), and after XP-F instrumentation (S3). The DNA extracts were subjected to quantitative analysis for total bacterial counts by quantitative real-time polymerase chain reaction. The data were analyzed using the analysis of variance test, and the level of significance was set at 5%. RESULTS: All samples tested positive for the presence of bacteria at baseline, and the bacterial counts substantially reduced after treatment procedures (P < .01). The results showed no statistical difference between RB and XP-S instrumentation with respect to the bacterial reduction (P > .05). A marked bacterial reduction was observed after the use of the XP-F instrument (P < .01). CONCLUSIONS: The XP-S and RB systems sharply reduced the bacterial load in oval-shaped root canals with primary apical periodontitis. XP-F used as a supplementary instrument to chemomechanical preparation promoted a significantly higher bacterial reduction.

Resinous adhesive systems differentially affect the expression of cytokines by human monocytes stimulated or not with Streptococcus mutans in vitro.

OBJECTIVES: The polymerization of adhesive systems is incomplete and the residual monomers that have been released have a cytotoxic capacity. Some teeth develop into pulp necrosis after composite resin restorations. Considering frequent pulpal inflammation in response to cariogenic bacteria, substances released from the patches could affect the cells of the inflammatory infiltrate and interfere with the mechanisms of defense against microorganisms and protection of pulpal tissue. The aim of this study was to evaluate the effect of substances released by different resinous adhesive systems on cell viability and cytokine expression by human monocytes stimulated in vitro with Streptococcus mutans. DESIGN: Peripheral blood mononuclear cells from 10 healthy subjects were stimulated with S. mutans and then incubated with supernatants obtained from the Single Bond Universal (SBU) or Clearfil SE Bond (CSEB) adhesive systems for eight hours. Staining with Annexin V and 7AAD for analysis of apoptosis were performed and detection of monocytes expressing cytokines IL-1α, IL-6, IL-8, IL-10, IL-12 and TNF-α were performed by flow cytometry. RESULTS: No treatment significantly affected apoptosis in monocytes. SBU supernatant increased the frequency of monocytes expressing IL-8 and decreased the monocytes expressing IL-10. Considering S. mutans-stimulated cells, while SBU increased the frequency of IL-8+ monocytes, CSEB reduced the frequency of IL-6 and TNF-α positive monocytes. CONCLUSIONS: Products released from SBU seem to induce proinflammatory effects on monocytes while those from CSEB show an anti-inflammatory outcome. These effects may interfere in the control of cytokine-mediated immunoinflammatory pulp reactions, both in the presence and absence of stimulation by cariogenic bacteria.

Exploring different photodynamic therapy parameters to optimize elimination of Enterococcus faecalis in planktonic form.

BACKGROUND: The failure of endodontic treatment is linked to the presence of Enterococcus faecalis in the root canals. The scope of this study was to evaluate the influence of the energy dose and frequency of photodynamic therapy (PDT cycles), as well as the volume of bacterial suspensions (BS) in the elimination of Enterococcus faecalis in planktonic form. METHODS: In four successive assays BS of Enterococcus faecalis ATCC 19433 were irradiated with a diode laser (40 mW) using the photosensitizer (PS) methylene blue (MB) (0.005 µg/mL). Group 1 - Effect of energy dose: 100 µâ€¯L of BS and 100 µâ€¯L of PS were irradiated by 1, 2.5, 5, 7.5 and 10 minute s. Group 2 - Effect of PDT cycles: The BS received 1, 2, 3 or 4 PDT cycles (in each cycle 100 m L of PS was added and irradiated by 2.5 minutes). Group 3 - Effect of energy dose and bacterial suspension volume: 10 µâ€¯L of BS and 10 µâ€¯L of PS were irradiated similar to group 1. Group 4 - Effect of energy dose, bacterial suspension volume and PDT cycles: 10 µâ€¯L of BS and 10 µL of PS were irradiated according to group 2. The laser source and MB isolated represented the controls. RESULTS: The mean log reduction after separate applying laser light and MB were 0.01 and 0.07, respectively. It was found that wells with 100 µâ€¯L of BS irradiated with 2.4 to 24 J of energy did not cause significant bacterial elimination (p > 0.05), on the other hand PDT cycles above 12 J increased significantly bacterial elimination (p < 0.05). In 10 µâ€¯L wells irradiation from 12 J of energy provided higher bacterial elimination (p < 0.05) which combined with PDT cycle resulted in the logarithmic elimination of E. faecalis (p < 0.05). CONCLUSIONS: The energy dose, the volume of the bacterial suspension and, especially, the PDT cycles optimized the bacterial elimination of Enterococcus faecalis in planktonic form.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Actinomyces israelii in radicular cysts: a molecular study.

OBJECTIVE: To investigate whether the microscopic filamentous aggregates observed in radicular cysts are associated with the molecular identification of Actinomyces israelii. Moreover, to verify whether this bacterium can be detected in radicular cyst specimens not presenting aggregates. STUDY DESIGN: Microscopic colonies suggestive of Actinomyces were found in 8 out of 279 radicular cyst samples (case group). The case and control groups (n = 12; samples without filamentous colonies) were submitted to the semi-nested polymerase chain reaction to test the presence of A israelii. DNA sequencing was performed to validate polymerase chain reaction results. RESULTS: Two and 3 samples in the case and control groups, respectively, did not present a functional genomic DNA template and were excluded from the study. A israelii was identified in all samples of the case group and in 3 out of 9 samples of the control group. CONCLUSIONS: Although A israelii is more commonly identified in radicular cysts presenting filamentous aggregates, it also appears to be detected in radicular cysts without this microscopic finding.

Monitoring the effectiveness of photodynamic therapy with periodic renewal of the photosensitizer on intracanal Enterococcus faecalis biofilms.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) can eliminate microorganisms in a root canal. However, the parameters for disinfection remain undefined. This study assessed the effectiveness of a PDT protocol against intracanal Enterococcus faecalis biofilms. MATERIALS AND METHODS: Root canals were contaminated with E. faecalis for 21 days. The instrumentation was associated to irrigation with 0.85% saline or an alternate irrigation (AI) with 5.25% NaOCl and 17% EDTA. Complementary treatments included saline/PDT and AI/PDT. Four PDT cycles were performed using a diode laser (660nm, 40mW) delivered through a tapered optical fiber. In each cycle, the root canal was filled with 1.56µM/mL methylene blue and irradiated for 150s. Microbiological samples were collected before (S1) and after (S2) instrumentation; after PDT (S3); and daily over the course of 14 days (S4-S17). Colony-forming units (CFUs) were counted, positive cultures verified, and data subjected to parametric and proportion's tests. RESULTS: The highest bacterial load reduction was observed in S2. In regard to S3, Saline/PDT reduced 1.3 log(10) CFU counts (p=0.000 for S2) and no CFUs were recovered after AI/PDT treatment. All canals were CFU-free on the 14th day for saline/PDT, AI and AI/PDT. Positive cultures were observed in 60% of saline-irrigated canals on the 14th day, whereas the saline/PDT, AI and AI/PDT treatments resulted in germ-free canals after 10, 5 and 2 days, respectively. CONCLUSION: Our findings suggest immediate and delayed antibacterial effects using the PDT protocol tested.

Sub-Inhibitory Concentration of Piperacillin-Tazobactam May be Related to Virulence Properties of Filamentous Escherichia coli.

Sub-inhibitory concentrations of antibiotics are always generated as a consequence of antimicrobial therapy and the effects of such residual products in bacterial morphology are well documented, especially the filamentation generated by beta-lactams. The aim of this study was to investigate some morphological and pathological aspects (virulence factors) of Escherichia coli cultivated under half-minimum inhibitory concentration (1.0 µg/mL) of piperacillin-tazobactam (PTZ sub-MIC). PTZ sub-MIC promoted noticeable changes in the bacterial cells which reach the peak of morphological alterations (filamentation) and complexity at 16 h of antimicrobial exposure. Thereafter the filamentous cells and a control one, not treated with PTZ, were comparatively tested for growth curve; biochemical profile; oxidative stress tolerance; biofilm production and cell hydrophobicity; motility and pathogenicity in vivo. PTZ sub-MIC attenuated the E. coli growth rate, but without changes in carbohydrate fermentation or in traditional biochemical tests. Overall, the treatment of E. coli with sub-MIC of PTZ generated filamentous forms which were accompanied by the inhibition of virulence factors such as the oxidative stress response, biofilm formation, cell surface hydrophobicity, and motility. These results are consistent with the reduced pathogenicity observed for the filamentous E. coli in the murine model of intra-abdominal infection. In other words, the treatment of E. coli with sub-MIC of PTZ suggests a decrease in their virulence.

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire.

UNLABELLED: Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼ 1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE: APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Effectiveness of chemomechanical preparation with alternating use of sodium hypochlorite and EDTA in eliminating intracanal Enterococcus faecalis biofilm.

INTRODUCTION: The elimination of microorganisms from root canals is a critical step in endodontic treatment. We aimed to evaluate the antimicrobial effectiveness of an alternating irrigation regimen during chemomechanical preparation (CMP). METHODS: During 21 days, root canals of extracted human teeth were infected with Enterococcus faecalis, and colonization was confirmed by scanning electron microscopy (SEM). Canals were irrigated with saline solution (control group), with 5.25% NaOCl followed by a final rinse with 17% EDTA (conventional irrigation group), or with the alternating use of NaOCl and EDTA (alternating irrigation [AI] group). Samples were taken before treatment (S1), after CMP (S2), and during the following 14 days. Two specimens/group were analyzed by SEM. RESULTS: The AI group yielded negative agar and liquid cultures from immediately after CMP and from the 5th day on, respectively. SEM confirmed several bacterium-free sites in the AI group. CONCLUSION: The irrigation regimen based on the alternating use of NaOCl and EDTA seems to be a promising endodontic tool because it promoted the elimination of root canal E. faecalis biofilms throughout the experimental period.

Composition analysis of dental solid waste in Brazil.

When developing proper waste management strategies, it is essential to characterize the volume and composition of solid waste. The aim of this work was to evaluate the composition of dental waste produced by three dental health services in Belo Horizonte, Minas Gerais State, Brazil. Two universities, one public and one private, and one public dental health service were selected. Waste collection took place from March to November 2007. During this period, three samples were collected from each dental health service. The total amount of dental waste produced in one day of dental work was manually separated into three categories: infectious and potentially infectious waste, accounting for 24.3% of the total waste; non-infectious waste, accounting for 48.1%; and domestic-type waste, accounting for 27.6% (percentages are for mean weights of solid waste). Our results showed that most of the waste considered as biomedical may be misclassified, consequently making the infectious waste amount appear much larger. In addition, our results suggest that the best waste minimization method is recycling, and they help to define an appropriate waste management system in all three of the dental health services involved in this study.

Antiproliferative activity of Actinobacillus (Haemophilus) actinomycetemcomitans and Fusobacterium nucleatum in peripheral blood mononuclear cells.

Several studies indicate Actinobacillus (Haemophilus) actinomycetemcomitans and Fusobacterium nucleatum as etiologic agents of periodontal disease. Immunosuppressive factors produced by microorganisms probably contribute to the initiation and evolution of this disease. This study evaluated the antiproliferative activity of ammonium precipitate fractions of A. (H.) actinomycetemcomitans and F. nucleatum isolates from humans and marmosets both with and without periodontal disease. All A. (H.) actinomycetemcomitans and most F. nucleatum strains inhibited PBMC proliferation in a dose-dependent manner. The degree of cell proliferative inhibition of each bacterial species differed among the strains and was independent of host clinical status. The in vitro inhibition of stimulated lymphocyte proliferation induced by different A. (H.) actinomycetemcomitans and F. nucleatum isolates demonstrated the importance of this phenomenon in bacterial virulence, playing a possible suppressor role in host defense mechanisms in vivo. Moreover, our findings pointed out a marked difference between A. (H.) actinomycetemcomitans and F. nucleatum cytoplasmic extracts in their antiproliferative activity, regarding the antigen concentration required for maximum inhibition and their vulnerability to heating and proteolytic treatment.

In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611.

Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.

Bacterial concentrations determine the ability to implant in the root canal system and translocate to lymph nodes in germ-free mice.

The purpose of this study was to evaluate the influence of the infecting dose on bacterial colonization in root canal systems (RCS) and translocation to the submandibular lymph node (SML) of gnotobiotic (germ-free) mice. RCS were inoculated with 10(2) and 10(1) colony-forming units (CFU) of Enterococcus faecalis (ATCC 4083) or 10(5), 10(4), 10(3), and 10(2) CFU of Prevotella nigrescens (ATCC 33563). E. faecalis implanted in 83.3% of the cases, for both inocula. Translocation was detected in 58.3% of lymph nodes for the 10(2) CFU inoculum and in 33.3% of lymph nodes for the 10(1) CFU inoculum. P. nigrescens implanted in 25%, 16.7%, 8.3%, and 0%, for the 10(5), 10(4), 10(3), and 10(2) CFU inocula, respectively, and did not translocate at any of the concentrations used. These results indicate that E. faecalis was able to implant in the RCS and translocate to the SML, whereas P. nigrescens implanted in lower percentages in the RCS and did not translocate to the SML.