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Aim: To analyse the effect of ultrasonic irrigant activation (UIA) and the GentleWave (GW) multisonic irrigation (GW) with minimal instrumentation on the root canal microbial diversity in an ex vivo model that used extracted molars with a history of pulp necrosis., Methodology: Twenty-three mandibular molars were prepared ex vivo for collection of superficial (surface control), pre-treatment and post-treatment samples 24 h after extraction. Samples were divided into two groups: UIA using 6% NaOCl (n = 11) and GW group (n = 12). All samples were processed using quantitative real-time polymerase chain reaction (qPCR) and 16S rRNA next-generation sequencing to measure microbial diversity before and after the antimicrobial treatment. For qPCR, a t-test (α = .05) was used to compare the log10 reduction. The Chao1 and Shannon indices evaluated alpha diversity. Differences in community composition (beta diversity) were evaluated by analysis of similarity (ANOSIM). Kruskal-Wallis test with Bonferroni corrections was performed to evaluate the differences in abundances genera in the samples., Results: Quantitative real-time polymerase chain reaction revealed an estimated 1.6 and 2.6 log10 reduction for UIA and GW groups respectively (p = .048). An average of 5 ± 4 and 3 ± 5 operational taxonomic units (OTUs) were found in surface's samples in the UIA and GW group respectively. These values were significantly lower (p < .001) compared to the number of preoperative OTUs in those groups (155 ± 79 and 187 ± 121). In assessing beta diversity, there were no significant differences found in pre-treatment samples (R = .090, p = .070 ANOSIM with Bonferroni corrections). Also, no significant differences in community composition were observed in post-treatment samples (R = -.05, p = .829). After treatment, there was a significant reduction of Eubacterium using conventional treatment with UIA and a significant reduction of Prevotella using minimal instrumentation with GW irrigation (p = .007 and p = .002 respectively)., Conclusion: Quantitative PCR analysis revealed a significant reduction in microbial load for GW group. Overall, diversity changes were similar between UIA and GW irrigation in this ex vivo model that used extracted teeth with a history of pulp necrosis. OTUs obtained from the surface sample were negligible and did not affect the statistical outcome of the study., (© 2023 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

Aim: This study was done to clinically investigate the efficacy of glycyrrhizin-based intracanal medication (ICM) compared to calcium hydroxide Ca(OH) 2 and Ledermix on the bacterial counts inside the root canals of necrotic teeth with chronic periapical periodontitis., Methodology: Thirty-six patients having single-rooted and single-canaled necrotic teeth with chronic periapical periodontitis were enrolled in this research. Aseptic control measures were taken before clinical steps. Access cavity preparation was done. First bacteriological samples (S1) were collected immediately after access cavity preparation and before cleaning and shaping inside the root canals. Cleaning and shaping were performed on the root canals. Patients were randomly allocated into 3 groups according to the type of intracanal medicament used [Ca(OH) 2 , Ledermix, Glycyrrhizin]. Second bacteriological samples (S2) were collected after 1 week from placing the ICMs. S1 and S2 were transferred to the lab of microbiology for culturing on blood agar dishes in anaerobic conditions, and the bacteria on the plates were enumerated as colony-forming units (CFUs) by the manual counting method. The anti-bacterial efficacy of the ICM was estimated by the percentage reduction in the bacterial colonies from S1 to S2., Results: All tested ICM documented a significant reduction in the CFUs from S1 to S2 ( p < 0.05). Regarding S1, there was no significant difference between the three tested materials ( p > 0.05). Regarding S2, Glycyrrhizin and Ledermix showed significantly lower CFUs than Ca(OH) 2 . There was no significant difference between Glycyrrhizin and Ledermix., Conclusion: Under the conditions of the present study, Glycyrrhizin and Ledermix showed comparable antimicrobial effects that were better than the commonly used Ca(OH) 2 ICM. Glycyrrhizin may be a promising ICM., Clinical Significance: Herbal medicine can be considered as an alternative antimicrobial material for root canal disinfection because it has many benefits, like low toxicity, absence of microbial resistance, and favorable antimicrobial efficacy. This study highlights the clinical efficacy of Glycyrrhizin as a promising ICM. How to cite this article: Eltantawi AR, Abdel-Razik GM, Elhawary YM, et al. Efficacy of Glycyrrhizin as an Intracanal Medicament on Bacterial Load Reduction in Primary Infected Root Canals: A Randomized Clinical Trial. J Contemp Dent Pract 2024;25(6):540-546.

After traumatic injuries to teeth, microorganisms may invade the compromised pulp tissue and initiate pulp infection and periapical inflammation. In addition to bone resorption that typically accompanies pulp necrosis, root resorption frequently occurs. Root resorption has several variants that may occur shortly after the trauma or at a later stage. The pathological changes seen after traumatic injuries to teeth are invariably linked to the presence of microbial irritants. The presence of bacterial biofilms in the dental pulp space can be treated with regenerative or therapeutic endodontic procedures. However, necrosis of periodontal ligament is usually terminal for the tooth involved. In this review, the sources of bacteria after traumatic injuries are discussed. The types and role of microorganisms involved in the pathogenesis of endodontic pathosis after traumatic injuries are presented, and contemporary approaches for the management of these conditions are reviewed. Contemporary antimicrobial strategies are discussed. The rationale for the use of systemic and topical antimicrobials is presented. Finally, novel approaches to the use of antimicrobial therapies, particularly in regenerative procedures, are reviewed., (© 2019 John Wiley & Sons A/S and American Association of Endodontists.)

After traumatic injuries to teeth, microorganisms may invade the compromised pulp tissue and initiate pulp infection and periapical inflammation. In addition to bone resorption that typically accompanies pulp necrosis, root resorption frequently occurs. Root resorption has several variants that may occur shortly after the trauma or at a later stage. The pathological changes seen after traumatic injuries to teeth are invariably linked to the presence of microbial irritants. The presence of bacterial biofilms in the dental pulp space can be treated with regenerative or therapeutic endodontic procedures. However, necrosis of periodontal ligament is usually terminal for the tooth involved. In this review, the sources of bacteria after traumatic injuries are discussed. The types and role of microorganisms involved in the pathogenesis of endodontic pathosis after traumatic injuries are presented, and contemporary approaches for the management of these conditions are reviewed. Contemporary antimicrobial strategies are discussed. The rationale for the use of systemic and topical antimicrobials is presented. Finally, novel approaches to the use of antimicrobial therapies, particularly in regenerative procedures, are reviewed., (Copyright © 2019 John Wiley & Sons A/S and American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To detect the unknown components of the oral microbiome and the effects of root canal treatment in a Turkish population and to evaluate the changes in microbial diversity in the root canals before and after treatment., Methodology: Single-rooted central and lateral maxillary incisors with one canal were chosen from 20 patients. Baseline samples of intact intracanal microbiota were collected from 20 root canals of permanent teeth with necrotic pulps using sterile paper points. After root canal preparation, the root canals were filled with a calcium hydroxide paste for 7 days. Calcium hydroxide was removed from root canal with 2.5% sodium hypochlorite and 17% EDTA using passive ultrasonic irrigation (PUI). A second bacteriologic samples were taken with sterile paper points prior to root filling. The samples were processes with DNase-I treatment using next-generation sequencing (NGS). Reduction in bacterial numbers during root canal treatment was evaluated using real-time quantitative PCR (qPCR). All statistical analyses were conducted using the MINITAB 17 software (Minitab Ltd. Co., Coventry, UK). A one-sample t-test was used to analyse the data. Statistical significance was accepted at P < 0.05., Results: Relative abundances of Mycoplasma sp., Paludibacter sp., Tannerella sp., Prevotella spp. and an uncultured species from the order Bacteroidales decreased with root canal preparation and medication (98.7%, 99.8%, 98.8%, 97.7% and 99.3%, respectively), whilst the relative abundances of Methylobacterium sp., Corynebacterium sp. and Streptococcus infantis increased (93.1%, 94.8% and 99.4%, respectively). Considerable numbers of Streptophyta species were detected before and after treatment. The ratio of Agrobacterium sp. in the treated teeth community and the ratio of order Streptophyta in the infected canals had negative correlations with the success of bacterial elimination., Conclusions: The combination of NGS and qPCR techniques resulted in detection of previously unknown components of the oral microbiome and the effects of root canal treatment on their relative abundance in a Turkish population., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Objective: This study aimed to analyze the correlation between crestal alveolar bone loss with the presence of some bacterial species in root canals and the apical lesion area of necrotic teeth., Design: Data from 20 patients with diagnosis of pulp necrosis and acute apical abscesses, without active periodontal diseases, were evaluated. Patients with history of antibiotic usage three months prior to the study, with exposed pulp cavity, and with probing depth >3 mm were not included. The root size, the distance between the bone crest to the tooth apex in the mesial and distal surfaces, and the apical lesion area were measured from standard periapical radiographies by a calibrated examiner. Root canal samples were collected using sterilized paper points. In multirooted teeth, the largest root canal was sampled. Culture, microbial isolation and identification by phenotypic methods were performed. Spearman correlation and exact Fischer test were calculated between higher/lower existing bone crests, according to the median and the presence of specific bacteria., Results: No statistically significant differences were found between occurrence of pathogenic bacteria, such as Porphyromonas gingivalis, Porphyromonas endodontalis, and Prevotella intermedia, and groups with higher/lower degree of bone loss (p > 0.05). A negative significant correlation was found between Parvimonas micra and periodontal bone loss (p = 0.02). Additionally, no statistically significant association was found between crestal bone loss and the apical lesion area., Conclusions: It was concluded that, in patients without active periodontitis, the presence of pathogenic bacteria in the root canal was not correlated with periodontal bone loss., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: To determine the presence of Prevotella species, the cfxA/cfxA2, blaZ and blaTEM genes associated with resistance to lactamic agents in different oral niches of children with pulp necrosis., Methodology: Children with pulp necrosis in primary teeth had samples of saliva, supragingival, pulp chamber and root canal biofilms collected and tested for Prevotella species (P. intermedia, P. nigrescens, P. tannerae) and for beta-lactam resistance genes (cfxA/cfxA2, blaZ and blaTEM). The presence of bacterial DNA was examined through PCR, with a specific primer directed to the 16S rRNA gene. Specific primers were used to detect the Prevotella species and beta-lactam resistance genes. The chi-square test was used to analyse associations between the presence of bacteria and clinical variables. The Cochran's Q test was used to assess whether the proportion of gene detection is the same between different sites., Results: Thirty-two teeth were sampled from 27 patients with a mean age of 5.5 years (±1.76). The total detection rate of Prevotella strains was 29.1%, 25%, 21.8% and 32.29% in saliva, supragingival, pulp chamber and root canal samples, respectively. P. nigrescens was the most commonly detected species in all oral niches. The previous use of antibiotics was associated with detection of P. nigrescens in saliva (P = 0.03). Pain was associated with the presence of P. nigrescens (P = 0.04) and P. tannerae (P = 0.01) in pulp chamber biofilm. blaTEM was detected in the four oral niches, being more frequent (23.8%) in supragingival biofilm (Cochran's Q test, P = 0.04). The presence of P. intermedia in SB and PC was associated with the detection of blaTEM in saliva (P = 0.04). The cfxA/cfxA2 and blaZ genes were not detected in any of the four oral niches., Conclusions: The oral cavity of children with pulp necrosis had a variable distribution of Prevotella strains in different niches. Saliva, supragingival biofilm, pulp chamber and root canals of primary teeth with necrotic pulps can harbour resistance genes to beta-lactams agents., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Introduction: We evaluated the radiopacity and antibacterial properties of various concentrations of double antibiotic paste (DAP) containing barium sulfate (BaSO 4 ) or zirconium oxide (ZrO 2 ) radiopaque agents., Methods: The radiopacity of 1, 10, and 25 mg/mL DAP containing 30% (w/v) BaSO 4 or ZrO 2 , DAP-free radiopaque pastes, and commercially available radiopaque calcium hydroxide (Ca[OH] 2 ) were evaluated according to ISO 6876/2001 with slight modifications (n = 6 per group). Dentin samples (n = 70) infected anaerobically for 3 weeks with bacterial biofilms obtained from a root canal of an immature tooth with pulpal necrosis were treated with similar experimental pastes or received no treatment (n = 7). After 1 week, the pastes were rinsed off, and biofilm disruption assays were conducted. To show the residual antibacterial effects, sterile dentin samples (n = 70) were pretreated for 1 week with the same pastes (n = 7). The pastes were rinsed off, and the samples were immersed in phosphate-buffered saline for 24 hours and infected anaerobically with the same bacterial biofilm mentioned earlier for 3 weeks before conducting biofilm disruption assays. Sterile dentin blocks were used in both antibacterial analyses as negative control groups (n = 7). Wilcoxon rank sum tests were used for statistical analyses., Results: No tested concentrations of BaSO 4 DAP or ZrO 2 DAP showed significant differences from Ca(OH) 2 in radiopacity. However, all tested concentrations of BaSO 4 DAP, ZrO 2 DAP, and Ca(OH) 2 exhibited significant direct antibacterial effects. ZrO 2 DAP at 1 mg/mL and Ca(OH) 2 did not show significant residual antibacterial effects., Conclusions: BaSO 4 DAP at 1 mg/mL provided significantly superior residual antibacterial effects and comparable radiopacity with the commercially available Ca(OH) 2 ., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to compare the Gram-negative pathogens identified in the root canals of primary teeth with irreversible inflammatory pulpitis and in teeth showing apical periodontitis. Methods: Samples were collected from 123 root canals of primary teeth from three- to seven-year-old patients. Root canals were assigned to either group one (irreversible inflammatory pulpitis; n equals 63) or group two (pulp necrosis and apical periodontitis; n equals 60). Total number of cells of selected Gram-negative microorganisms was determined by the checkerboard DNA-DNA hybridization technique. Demographic data were compared using either chi-squared or t tests. Total numbers of microorganisms were compared using the Mann-Whitney test ( α equals 0.05). Results: There were no significant intergroup differences in gender, age, and tooth group distribution (P>0.05). Among the 123 samples, 17 were discarded due to salivary contamination. The total numbers of Prevotella nigrescens, Treponema denticola, Fusobacterium nucleatum polymorphum, Fusobacterium nucleatum spp nucleatum, Aggregatibacter actinomycetemcomitans serotype a, Aggregatibacter actinomycetemcomitans serotype b, Porphyromonas gingivalis, Prevotella intermedia, and Prevotella melaninogenica were higher in teeth with apical periodontitis compared to those with irreversible inflammatory pulpitis (P<0.05). Conclusion: Higher numbers of Gram-negative bacteria were found in teeth with apical periodontitis compared to teeth with irreversible in- flammatory pulpitis.

Aim: The aim of this in vivo study is to evaluate the antimicrobial efficacy of calcium hydroxide (Ca(OH) 2 ), chlorhexidine (CHX), and metronidazole gel as intracanal medicaments against aerobic and facultative anaerobic microorganisms found in root canals of human primary teeth with necrotic pulp., Setting and Design: It is a double-blinded randomized clinical trial., Materials and Methods: Pulp canals of 45 single-rooted primary maxillary anterior teeth with pulp necrosis in 34 children were included in the study. They were divided into three groups of 15 samples each: Group I: Ca(OH) 2 ; Group II: 1% CHX gel; and Group III: 1% metronidazole gel. Microbial count was obtained from each tooth at two different stages - (1) after instrumentation and (2) after placement of the medication. Statistical analysis using the SPSS 10.0 software program (SPSS Inc., Chicago, IL, USA) with Wilcoxon signed-rank test after grouping the samples was performed., Results: Ca(OH) 2 , 1% CHX gel, and 1% metronidazole gel were ineffective in completely eliminating aerobic and facultative anaerobic microorganism from root canal of human primary teeth with necrotic pulp., Conclusion: None of the commonly used intracanal medicaments, that is Ca(OH) 2 , 1% CHX gel, and 1% metronidazole gel, was effective in completely eliminating aerobic and facultative anaerobic microorganism from root canal of human primary teeth with necrotic pulp. Ineffectiveness of these medicaments against aerobic and facultative anaerobic microorganism has opened new door of research regarding the manner of bacterial growth in unfavorable environmental and nutritional conditions, the way root canal environment favors biofilm formation and the use of suitable intracanal medicaments against single and multispecies biofilms., Competing Interests: There are no conflicts of interest

Aim: To evaluate the association between the presence of selected bacterial species/groups in the apical root canal and expression of mediators of soft and bone tissue destruction in apical periodontitis lesions. Relationships between bacteria and some other features of apical periodontitis were also investigated., Methodology: Seventeen freshly extracted teeth with pulp necrosis and apical periodontitis were included. The apical root segment was sectioned and cryopulverized; DNA was extracted and evaluated for the presence of 9 bacterial species/groups using real-time polymerase chain reaction. Lesions were processed for histopathological and immunohistochemical analyses, which targeted matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), receptor activator of NFκB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). Associations of the target bacteria with expression of these mediators, presence of symptoms, lesion size and histopathological diagnosis were evaluated. Data were analysed using the chi-square, Fisher's exact, Mann-Whitney and Pearson tests. P values lower than 0.05 were considered significant., Results: All pulverized apical root samples were positive for bacteria. The most prevalent taxa were Actinobacteria (53%), Streptococcus species (35%), Fusobacterium species and Parvimonas micra (18%). The target mediators exhibited a high mean expression in the lesions (MMP-2: 82%; MMP-9: 73%; RANK: 78%; RANKL; 81%; OPG; 83%). Mean RANKL:OPG ratio was significantly higher in granulomas than cysts (P < 0.05, Mann-Whitney test). Actinobacteria were associated with granulomas, higher MMP-2 expression, lower OPG expression, and higher RANKL:OPG ratio (P < 0.05 for all, Fisher's exact test or Mann-Whitney test). No other significant associations were found., Conclusion: Actinobacteria may play an important role in the active phase of soft and bone tissue destruction in apical periodontitis., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Infections of the root canal have polymicrobial etiology. The main group of microflora in the infected pulp is bacteria. There is limited data that archaea may be present in infected pulp tissue. The aim of this study was to check the prevalence of archaea in necrotic root canal samples obtained from patients with primary or post-treatment infection. The prevalence of selected bacteria species (Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Synergistes sp.) in necrotic samples was evaluated as well. Sixty-four samples from root canal were collected for DNA and RNA extraction. A PCR assay based on the 16S rRNA gene was used to determine the presence of archaea and selected bacteria. Of the 64 samples, 6 were analyzed by semiquantitative reverse transcription PCR to estimate expression profiles of 16S rRNA, and another 9 were selected for direct sequencing. Archaea were detected in 48.4% samples. Statistical analysis indicated a negative association in coexistence between archaea and Treponema denticola (P < 0.05; Pearson's χ 2 test). The main representative of the Archaea domain found in infected pulp tissue was Methanobrevibacter oralis. Archaea 16S rRNA gene expression was significantly lower than Synergistes sp., Porphyromonas gingivalis, and Tannerella forsythia (P < 0.05; Student's t test). Thus, it can be hypothesized that archaea may participate in the endodontic microbial community.

Aims: To determine the efficacy of a single session protocol (SSP) in the reduction of septic content of primary teeth root canals and identify the persistence of bacterial species associated with unsuccessful treatment., Methods: Primary teeth root canals (16) with pulp necrosis and peri-radicular lesions were treated. Samples were collected at baseline (T1), and after chemo-mechanical preparation, before filling (T2). Identification of the microorganisms was determined using checkerboard DNA-DNA hybridisation., Statistical Analysis: Wilcoxon test was applied for comparison of mean number of species, proportion and mean count of each species between the evaluation times., Results: Significant reductions were found in the mean number of bacteria species between T1 and T2 (p < 0.05), but not for the reduction in proportion (p > 0.05). There was a reduction (6.0-4.6) of the mean number of species associated with failure, without statistical significance., Conclusion: The SSP was capable of significantly reducing the septic content, even though, many of the bacteria associated with failure persisted at the time of root canal filling.

Aim: This randomized clinical trial aimed to compare the effectiveness of ultrasonic activation with that of nonactivated irrigation on the removal of bacteria and endotoxin from root canals., Methodology: Fifty patients with necrotic pulps and asymptomatic apical periodontitis were randomly allocated into two groups according to the final irrigation protocol after root canal preparation: Group UI - ultrasonic irrigation (n = 25) and Group NI - needle irrigation (n = 25). The root canals were medicated with calcium hydroxide for 14 days. Microbiological sampling was performed before (S1) and after the root canal preparation (S2), after the irrigation protocols (S3) and after the removal of the intracanal medication (S4). Total bacteria counts were determined by qPCR and the endotoxin levels by the limulus amebocyte lysate assay. Intragroup analyses were performed using the Wilcoxon test for related samples, whereas intergroup analyses were performed using the Mann-Whitney U-test (P < 0.05)., Results: All S1 samples were positive for bacteria, with median numbers of 1.49 × 10 6 and 8.55 × 10 5 bacterial cells for the UI and NI groups, respectively. This number significantly decreased in S2 samples (UI: 1.41 × 10 4 ; NI: 3.53 × 10 4 ; both with P < 0.001). After final irrigation protocols, there was a significant decrease in bacterial load from S2 to S3 samples in both groups (UI: 4.29 × 10 3 ; NI: 1.08 × 10 4 ; P < 0.01). Intergroup analysis revealed a significant difference between irrigation methods regarding bacterial counts in S3 samples (P < 0.05). In contrast, no significant differences were observed between groups for endotoxin levels (P > 0.05)., Conclusions: Ultrasonic activation was more effective than nonactivated irrigation for reducing the number of bacteria but not the endotoxin levels in root canals of teeth with apical periodontitis., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Objective: This clinical study compared the effectiveness of 7- and 14-day intracanal medications in the reduction of bacteria/endotoxins from primarily infected root canals and determined their antigenicity against macrophages through the levels of cytokines., Methods: Seventy-two primarily infected teeth were randomly divided into six groups according to medication and time of application: 7-day groups = G1, Ca(OH) 2 + saline solution (SSL); G2, Ca(OH) 2 + 2% chlorhexidine (CHX) gel; and G3, 2% CHX gel and 14-day groups = G4, Ca(OH) 2 + SSL; G5, Ca(OH) 2 + 2% CHX gel; and G6, 2% CHX gel (all groups, n = 12). Bacterial and endotoxin samples were collected from root canals and inflammatory cytokines of macrophages supernatants. Culture techniques were used to determine bacterial counts and limulus amebocyte lysate (LAL) assay to quantify endotoxins. IL-1β, TNF-α, and PGE 2 were measured by ELISA-assay., Results: With regard to the bacterial reduction, no differences were found between all protocols tested (p > 0.05). The CHX protocols (G3 and G6) exhibited the lowest effectiveness against endotoxins (p < 0.05). All protocols were effective in lowering the levels of IL-1β, TNF-α, and PGE 2 (p < 0.05), with no difference between the medications tested on days 7 or 14 (p > 0.05). Particularly, the 7-day CHX-protocol (G3) exhibited the lowest effectiveness in lowering the levels of most cytokines compared to the 14-day protocols (G6) (p < 0.05)., Conclusions: All the 7- and 14-day intracanal medications were effective in reducing bacteria and endotoxins as well as in lowering the levels of inflammatory cytokines, with CHX showing limited effectiveness against endotoxins. Moreover, 7-day CHX-protocol exhibited the lowest effectiveness in lowering the levels of most cytokines compared to the 14-day protocols., Clinical Significance: Seven-day CHX protocol is the less effective protocol and should be carefully applied by the clinician.

Introduction: The rapid antibiotic sensitivity test (RAST) is a novel in-office culture and sensitivity system for endodontic infections. The purpose of this research was to validate the RAST system as a viable, in-office alternative to antibiotic sensitivity testing using turbidity to determine antibiotic sensitivities of endodontic infections., Methods: Aspirates were taken from the root canals of 9 necrotic human teeth at the initiation of root canal therapy. These samples were cultured in the RAST medium, and antibiotic sensitivity to 6 antibiotics was tested. Further analysis was performed using 16S ribosomal RNA (rRNA) gene sequencing., Results: Thirty-one bacterial phyla were identified as well as 2 phyla of the kingdom Archaea. Augmentin (Dr. Reddy's Laboratories Ltd, Hyderabad, India) and ampicillin performed identically at 24 hours, inhibiting turbidity in 100% of the samples. At 48 hours in anaerobic conditions, Augmentin outperformed ampicillin by 13%. Ciprofloxacin was the least efficacious antibiotic. At 48 hours, only 22% of anaerobic ciprofloxacin cultures affectively inhibited bacterial growth., Conclusions: The RAST medium is a viable in-office alternative to antibiotic susceptibility testing in an off-site laboratory. It is able to support the growth of a wide variety of microorganisms in both aerobic and anaerobic environments, and, in combination with 16S rRNA gene sequencing, it led to the identification of a new archaebacterial phylum, Crenarchaeota, as part of the endodontic infection microbiome., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: This clinical study was conducted to correlate the microbiological profile and levels of endotoxins found in primary endodontic infection with the presence of clinical features and to evaluate the removal of microorganisms and endotoxins using rotary, reciprocating, and hybrid systems for biomechanical preparation., Methods: Thirty single root canals with primary endodontic infection were evaluated with signs and symptoms and were randomly divided into 3 groups according to the instrumentation system used (n = 10) as follows: rotary Mtwo instruments (VDW, Munich, Germany) with 8 files, the reciprocating Reciproc system (VDW) with a single file, and Genius hybrid instruments with 3 files (1 rotary and 2 reciprocating files) with irrigation using 24 mL 2.5% sodium hypochlorite. Samples were collected before (S1) and after instrumentation (S2) before being submitted to microbiological culture (colony-forming units/mL) and the checkerboard DNA-DNA hybridization test. Endotoxins were quantified using the limulus amebocyte lysate assay., Results: Microbiological culture showed statistical differences in the reduction of colony-forming units/mL with all systems tested (P < .05), but no statistical difference was found among the groups. The most frequently detected species were Capnocytophaga ochracea (53%) and Fusobacterium nucleatum (53%) at S1 and F. nucleatum (50%) and Leptotrichia buccalis (50%) at S2. As for the reduction of endotoxins at S2, Mtwo presented the best results (95.05%) followed by the Genius (91.85%) and Reciproc (64.68%) groups, but no statistical difference was found among the groups. Previous pain, tenderness to percussion, and presence of a sinus tract were associated with specific microorganisms (P < .05)., Conclusions: Signs and symptoms were correlated with microorganisms. Endodontic treatment was effective in reducing bacteria and endotoxins but was not capable of completely removing them from the root canal., (Copyright © 2017. Published by Elsevier Inc.)

Introduction: We investigated the direct and residual antibacterial effects of intracanal antimicrobials against bacterial biofilms obtained from infected mature and immature teeth with necrotic pulps., Methods: Sterile dentin slabs (n = 100) were inoculated with bacterial biofilms obtained from root canals of an immature or a mature tooth with pulpal necrosis and incubated anaerobically for 3 weeks (n = 50 per biofilm). Dentin infected with each type of biofilm received 1 week of treatment with 1 or 5 mg/mL double antibiotic paste (DAP) in methylcellulose hydrogels, calcium hydroxide, or placebo paste or received no treatment (n = 10). The pastes were removed, and biofilm disruption assays were performed. Additional dentin slabs (n = 100) were pretreated with the same treatments (n = 20). The pastes were rinsed off, and the samples were immersed in phosphate-buffered saline for 1 week. Thereafter, samples from the treatment groups were infected with bacterial biofilm from both clinical sources mentioned earlier (n = 10 per biofilm) and incubated anaerobically for 3 weeks before conducting biofilm disruption assays. Uninfected dentin slabs were used for both antibacterial experiments as negative control groups (n = 20)., Results: All antimicrobials showed significant direct antibacterial effects regardless of the biofilm source. Dentin pretreated with 5 mg/mL DAP provided significantly higher residual antibacterial effects in comparison with all other groups regardless of the source of biofilm. Dentin pretreated with calcium hydroxide did not show any residual antibacterial effects., Conclusions: Tested antimicrobials showed significant direct antibacterial effects. Only 5 mg/mL DAP exhibited significant residual antibacterial effects against bacterial biofilms from an infected root canal of an immature tooth., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The purpose of this study was to combine multiple displacement amplification and checkerboard DNA-DNA hybridization to qualitatively and quantitatively evaluate the microbiota present in infections refractory to endodontic treatment., Methods: The subjects of this study were 40 patients presenting with periapical lesions refractory to endodontic treatment. Samples were taken by scraping or filing root canal walls with a #10 K-type hand file. Sample DNA was amplified by multiple displacement amplification, and the levels of 107 bacterial taxa were analyzed by checkerboard DNA-DNA hybridization. The taxa were divided into 3 distinct microbial populations depending on their mean proportion in samples (% DNA probe counts ± standard error of the mean) as follows: dominant (≥3.0%), subdominant (>1.6%-3.0%), and residual (≤1.6%) populations. The significance of differences was determined using the Mann-Whitney test., Results: The taxa present with the highest mean proportions (constituting the dominant population) were Corynebacterium diphtheriae (8.03 ± 0.98), Porphyromonas gingivalis (5.42 ± 2.09), Streptococcus sobrinus (5.33 ± 0.69), and Stenotrophomonas maltophilia (4.72 ± 1.73). Among the subdominant population were Eubacterium saphenum (3.85 ± 1.06), Helicobacter pylori (3.16 ± 0.62), Dialister pneumosintes (3.12 ± 1.1), Clostridium difficile (2.74 ± 0.41), Enterobacter agglomerans (2.64 ± 0.54), Salmonella enterica (2.51 ± 0.52), Mobiluncus mulieris (2.44 ± 0.6), and Klebsiella oxytoca (2.32 ± 0.66). In the population of bacteria present at the lowest mean proportions (the residual population), Bacteroides ureolyticus (0.04 ± 0.01), Haemophilus influenzae (0.04 ± 0.02), and Prevotella oris (0.01 ± 0.01) were found at the lowest mean proportions. Enterococcus faecalis was detected in the residual population (0.52 ± 0.26)., Conclusions: The microbial climax community in teeth refractory to endodontic treatment not only harbors medically important species but also contains distinct microbial consortia present with different population levels., (Copyright © 2016. Published by Elsevier Inc.)

Introduction: The lack of mechanical debridement and reduced concentrations suggested for chemical debridement to maintain stem cell viability call into question the disinfection efficacy of current regenerative protocols. Current protocols vary in the concentration and type of antibiotic medicaments used. The aim of this study was to determine if simulated immature teeth infected with Enterococcus faecalis can be completely disinfected by following current standardized regenerative protocols and to evaluate the probable effects of residual bacteria on stem cell toxicity., Methods: Sixty-eight caries-free maxillary incisors were used. S1 sampling protocols were validated in both negative and positive control groups via culture, scanning electron microscopy, and confocal laser scanning microscopy. All teeth, except the negative controls, were inoculated with E. faecalis. The teeth were divided into the following groups: group 1, triple antibiotic paste (ciprofloxacin:metronidazole:minocycline) at concentrations of 10, 1, and 0.1 mg/mL; group 2, double antibiotic paste (ciprofloxacin:metronidazole) at concentrations of 10, 1, and 0.1 mg/mL; group 3: Ultracal XS calcium hydroxide (Ultradent, St Louis, MO); and controls, negative and positive controls. Current regenerative protocols recommended by the American Association of Endodontists were followed. S2 sampling was performed after 4 weeks and tested for bacterial presence via culturing, scanning electron microscopy, and confocal laser scanning microscopic analysis., Results: The data showed that calcium hydroxide and the current recommended antibiotic concentrations are not capable of completely eliminating bacteria from simulated necrotic immature permanent teeth., Conclusions: Overall, this study focuses on the need to re-evaluate the balance between stem cell toxicity and bacterial elimination in order to determine the appropriate concentrations and medicaments for successful regenerative endodontic procedures., (Published by Elsevier Inc.)

Introduction: Infective endocarditis (IE) is still associated with high mortality, and antibiotic prophylaxis strategies are under intense debate. We evaluated the incidence of bacteremia after root canal preparation in teeth with necrotic pulps and apical periodontitis., Methods: Blood samples were taken before and 5 and 30 minutes after endodontic treatment in teeth with apical periodontitis from individuals at high (n = 21) or no risk (n = 11) for IE. The former received prophylactic antibiotic therapy. Bacteriologic samples were taken from root canals before chemomechanical preparation to confirm pulp infection. Samples were subjected to aerobic and anaerobic culture and quantitative real-time polymerase chain reaction (qPCR), the latter to determine the total bacterial and streptococcal levels., Results: Culture revealed no bacteremia in all individuals. Analysis by qPCR showed that bacterial DNA occurred in all root canal samples. qPCR showed a similar incidence of bacteremia between patients who received or did not receive prophylactic antibiotic therapy (P > .05). In blood samples taken 5 minutes after endodontic procedures, bacteria were detected in 2 of 11 (18%) individuals not taking antibiotics and in 4 of 21 (19%) patients under prophylaxis. After 30 minutes, the incidence of bacteremia decreased to 2 of 21 (10%) in patients taking antibiotics and was undetectable in patients at no risk of IE. The incidence of bacteremia by streptococci was identical as that for total bacteria., Conclusions: No detectable bacteremia was evident by culture after treatment of infected root canals. Molecular analysis revealed bacterial DNA and streptococci in blood from some patients without a significant difference between individuals receiving or not receiving antibiotic prophylaxis., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Endodontic infections are characterized by their microbial polymorphism with the presence of pathogenic agents such as bacteria. The aim of this study was to investigate the presence of yeast in the root canals of teeth with pulp necrotic with or without apical periodontitis and to study the sensibility of these at disinfection at sodium hypochlorite to 2.5 %., Methods: Root canal samples, taken with paper points before and after disinfection with sodium hypochlorite dosed at 2.5 % on 50 single rooted teeth in 38 patients, were seeded in petri dishes containing Sabouraud Chloramphenicol (SC) (Conda Laboratories, Madrid). These stains were incubated in an incubator at 37°C for 48hours. The presence of yeast was confirmed by white colonies on the entire circumference of the tip paper. The identification of species was done by macroscopic and microscopic examinations associated with Blastese test., Results: The yeast colonies were isolated from 7 teeth (14 %) out of 50. All positive samples were from teeth with an open necrotic pulp (P<0.001). The only species found was Candida albicans. Immediate disinfection with sodium hypochlorite at 2.5 % did not show a complete elimination of yeasts., Conclusion: The results of this study show that the canal of necrotic teeth with or without apical periodontitis may shelter yeasts even after immediate disinfection with sodium hypochlorite 2.5 %., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The role of microbes and their antimicrobial susceptibilities in both acute and chronic infections of the dental pulp in humans has been well studied. Presently, no data are available on endodontic pathogens in cheetahs (Acinonyx jubatus). The aim of this study was to isolate and identify the bacteria found in the canine teeth of cheetahs, where the pulp was necrotic and exposed due to a complicated crown fracture. Thirty-six microbiologic samples were taken from root canals (RCs) of the canine teeth of 19 cheetahs: one pulp sample was taken from 10 cheetahs, four samples from 2 cheetahs, two samples from 3 cheetahs, and three samples from 4 cheetahs. Exposed pulps were cultured for aerobic and anaerobic bacteria; an additional screening with a 16S rRNA-specific polymerase chain reaction (PCR) was used for the last six samples. Antimicrobial susceptibility of isolates was determined by use of the Kirby-Bauer diffusion test. In total, 59 cultivable isolates belonging to 19 microbial species and 13 genera were recovered from the 36 RCs sampled. Only two samples yielded no cultivable bacteria. Thirty-two (54.49%) of the cultivable isolates were Gram positive and 27 (45.71%) were Gram negative. The maximum number of isolates cultivated from an individual RC was six. Facultative anaerobes (62.72%) were the most common bacteria of the RCs that yielded cultivable bacteria. Of the isolates, 28.81% were aerobic and 8.47% were strict anaerobes. The antimicrobials that showed the greatest efficacy in vitro against the different bacteria isolates were amikacin and gentamicin. The more common bacterial species isolated by PCR were anaerobes (60.8%), facultative anaerobes (30.2%), and aerobes (8.6%).

This article describes 2 cases that showed persistent intracanal exudation (wet canal) even after several visits of antimicrobial endodontic treatment. Histologic and histobacteriologic investigation was conducted for determination of the cause. The 2 cases involved teeth with apical periodontitis lesions, which presented persistent exudation refractory to treatment after several visits. In case 1, it was not possible to achieve a dry canal, and surgery had to be performed. In case 2, attempts to dry the canal succeeded and the canal was filled, but follow-up examination showed an enlarged apical periodontitis lesion and extraction was performed. Biopsy specimens consisting of the root apex and apical periodontitis lesion for case 1 and the whole root for case 2 were subjected to histologic and histobacteriologic analyses. Both cases showed complex bacterial infection in the apical root, affecting both the intraradicular space and the outer root surface. Case 1 showed bacterial biofilms in ramifications, on untouched walls, and extending to the external root surface to form a thick and partially mineralized structure with high bacterial density. Different bacterial morphotypes were evidenced. Case 2 had a ledge on the apical canal wall created during instrumentation, which was filled with necrotic debris, filling material, and bacteria. The walls of the apical portion of the canal were covered by a bacterial biofilm, which was continuous with a thick extraradicular biofilm covering the cementum and dentin in resorptive defects. The extraradicular biofilm showed areas of mineralization and was dominated by filamentous bacteria. The 2 cases with wet canals and treatment failure were associated with complex persistent infection in the apical part of the root canal system extending to form thick and partially mineralized biofilm structures (calculus) on the outer apical root surface., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: This in vivo study compared the antibacterial effectiveness of a reciprocating single-instrument system (Reciproc; VDW, Munich, Germany) and a rotary multi-instrument system (BioRaCe; FKG Dentaire, La Chaux-de-Fonds, Switzerland) during the preparation of infected root canals of teeth with primary apical periodontitis., Methods: Root canals from single-rooted teeth with necrotic pulps and apical periodontitis were instrumented using either Reciproc (n = 29) or BioRaCe (n = 30) instruments under irrigation with 2.5% sodium hypochlorite. DNA was extracted from samples taken before and after preparation and subjected to quantitative analysis of total bacteria and streptococci by using the quantitative real-time polymerase chain reaction., Results: All initial samples were positive for the presence of bacteria, with median numbers of 7.1 × 10(5) and 1.31 × 10(5) bacterial cells for the Reciproc and BioRaCe groups, respectively. After preparation with Reciproc and BioRaCe, 16 (55%) and 15 (50%) root canals still had detectable bacteria with median counts of 7.05 × 10(2) and 6.03 × 10(1), respectively. Both systems were highly effective in reducing the total bacterial counts (P < .001), and there were no significant differences between them (P > .05). Streptococci were highly frequent, and both systems succeeded in significantly reducing their levels (P < .001)., Conclusions: Both reciprocating single-instrument and rotary multi-instrument systems were highly effective in reducing the counts of total bacteria and streptococci in root canals of teeth with apical periodontitis. Regardless of the system used, approximately one half of the teeth still had detectable bacteria., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Purpose: The purposes of this study were (1) to assess the endotoxin levels in root canals of primary teeth with necrotic pulps and periapical lesions; and (2) to evaluate the correlation of endotoxin levels with the periapical lesion size., Methods: Twenty primary molars with necrotic pulps and periapical lesions were selected. Standardized radiographs were obtained and the lesions were measured. Material from each root canal was collected to quantify the bacterial endotoxin. The results were analyzed using Pearson's correlation, student's t test, and Fisher's exact test (α equals five percent)., Results: The initial amount of bacterial endotoxin in the root canals ranged from 0.089 to greater than 50.00 endotoxin units (EU)/mL and was detected in 100 percent of the samples. A positive but not significant correlation was observed between the amount of endotoxin and periapical lesion size in primary molars., Conclusion: Endotoxin was observed in 100 percent of root canals of primary teeth with a periapical lesion. However, there was no statistical significance between bacterial endotoxin levels and the periapical lesion area.

Introduction: This study was conducted to evaluate the microbiomes of endodontic-periodontal lesions before and after chemomechanical preparation (CMP)., Methods: Clinical samples were taken from 15 root canals (RCs) with necrotic pulp tissues and from their associated periodontal pockets (PPs) (n = 15) of teeth with endodontic-periodontal lesions before and after CMP. The Human Oral Microbe Identification using Next Generation Sequencing (NGS) protocol and viable culture were used to analyze samples from RCs and PPs. The Mann-Whitney U test and Benjamini-Hochberg corrections were performed to correlate the clinical and radiographic findings with microbial findings (P < .05)., Results: Bacteria were detected in 100% of the samples in both sites (15/15) using NGS. Firmicutes was the most predominant phylum in both sites using both methods. The most frequently detected species in the RCs before and after CMP using NGS were Enterococcus faecalis, Parvimonas micra, Mogibacterium timidum, Filifactor alocis, and Fretibacterium fastidiosum. The species most frequently detected in the PPs before and after CMP using NGS were P. micra, E. faecalis, Streptococcus constellatus, Eubacterium brachy, Tannerella forsythia, and F. alocis. Associations were found between periapical lesions ≤ 2 mm and Desulfobulbus sp oral taxon 041 and with periodontal pockets ≥ 6 mm and Dialister invisius and Peptostreptococcus stomatis (all P < .05, found in the RCs before CMP)., Conclusions: It is concluded that the microbial community present in combined endodontic-periodontal lesions is complex and more diverse than previously reported. It is important to note that bacteria do survive in some root canals after CMP. Finally, the similarity between the microbiota of both sites, before and after CMP, suggests there may be a pathway of infection between the pulp and periodontium., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of the present study was to evaluate the reduction in bacterial loading uing MTAD as an irrigating solution in pulpectomized primary teeth., Study Design: A randomized, controlled clinical trial was performed that included primary teeth with pulp necrosis. Sixty necrotic canals were included, 30 irrigated with MTAD (experimental group) and 30 with 1% Na0Cl solution (control group); in all cases, 2 microbiological samples from within the canals were taken with sterile paper points, the first after the canal opening and before the first irrigation, and the second after instrumentation and final irrigation, before obturation. All samples were evaluated by Agar plate method., Results: The results were statistically analyzed by student 't' test. After analyzing samples before and after irrigation in the control group (NaOCl), we found a significant decrease of bacterial load ( p = < 0.001). The same occurred in the MTAD group samples ( p = < 0.001). When both groups were compared post irrigation, a statistically significant difference was observed in favor of MTAD., Conclusion: MTAD can be suggested as an alternative irrigant for pulpectomy of necrotic teeth.

Aim: To investigate endotoxin levels from primary endodontic infections before and after chemomechanical preparation (CMP) and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of matrix metalloproteinases (MMPs)., Methodology: Twenty-four root canals with primary endodontic infection and apical periodontitis were selected. Samples were collected using paper points before (S1) and after chemomechanical preparation (CMP) (S2). The limulus amebocyte lysate assay was used for endotoxin measurement. Fibroblasts were stimulated with root canal contents for 24 h. Supernatants of cell cultures stimulated with root canal contents were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity using the zymography technique. Friedman and Wilcoxon tests were used to compare the amount of endotoxin before (S1) and after CMP (S2) (P < 0.05). Data obtained from gelatinolytic activity were analysed using anova and Tukey's tests (P < 0.05)., Results: Endotoxin was recovered in 100% of the samples. There was a significant reduction in endotoxin levels after CMP (P < 0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (P < 0.05). Root canal contents of initial samples (S1) induced significantly greater MMP-2 expression by fibroblasts when compared to S2 and the nonstimulated group (P < 0.05). No gelatinolytic activity of MMP-9 was observed in S1, S2 and control group., Conclusions: Root canal contents from primary endodontic infections had gelatinolytic activity for MMP-2. Moreover, CMP was effective in reducing endotoxin levels and their antigenicity against fibroblasts on gelatinolytic activity., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

The purpose of this clinical study was to determine the effect of 7 day intracanal dressing with calcium hydroxide on the amount of bacterial lipopolysaccharide (LPS; endotoxin) in human teeth with necrotic and infected pulp and apical periodontitis. Twenty-five single-rooted teeth with necrotic pulps and apical periodontitis were selected. Samples were collected before (S1), after root canal preparation (S2) and after 7 day intracanal dressing with calcium hydroxide (S3). The limulus amoebocyte lysate assay was used to quantify LPS. LPS was present in 100% of the root canals before (S1), after preparation (S2) and after 7 day intracanal dressing (S3). A significant reduction, equal to 29.54%, was found after root canal preparation (P < 0.05). A significant difference (equal to 25.26% reduction) was also detected between S2 and S3 (P < 0.05). Total endotoxin reduction (S3 compared with S1) was found to be 47.34%. Endotoxin concentration of the infected root canals was reduced after root canal preparation and also after 7 days of dressing of canals with calcium hydroxide; however, relatively high values of endotoxin remained in the root canals., (© 2013 Australian Society of Endodontology.)

Objectives: Biopure MTAD™, a new root canal irrigant has shown promising results against the most common resistant microorganism, E. faecalis, in permanent teeth. However, there is lack of studies comparing its antimicrobial effectiveness with NaOCl in primary teeth. The purpose of this study was to compare the in vivo antimicrobial efficacy of NaOCl 2.5% and Biopure MTAD™ against E. faecalis in primary teeth., Study Design: Forty non vital single rooted primary maxillary anterior teeth of children aged 4-8 years, were irrigated either with NaOCl 2.5% (n=15), Biopure MTAD™ (n=15) and 0.9% Saline (n=10, control group). Paper point samples were collected at baseline (S1) and after chemomechanical preparation (S2) during the pulpectomy procedure. The presence of E. faecalis in S1 & S2 was evaluated using Real time Polymerase Chain Reaction., Results: Statistical significant difference was found in the antimicrobial efficacy of NaOCl 2.5 % and BioPure MTAD™ when compared to saline (p>0.05). However, no statistical significant difference was found between the efficacies of both the irrigants., Conclusions: NaOCl 2.5% and BioPure MTAD™, both irrigants are equally efficient against E. faecalis in necrotic primary anterior teeth. MTAD is a promising irrigant, however clinical studies are required to establish it as ideal root canal irrigant in clinical practice.

Introduction: Revascularization outcome depends on microbial elimination because apical repair will not happen in the presence of infected tissues. This study evaluated the microbial composition of traumatized immature teeth and assessed their reduction during different stages of the revascularization procedures performed with 2 intracanal medicaments., Methods: Fifteen patients (7-17 years old) with immature teeth were submitted to the revascularization procedures; they were divided into 2 groups according to the intracanal medicament used: TAP group (n = 7), medicated with a triple antibiotic paste, and CHP group (n = 8), dressed with calcium hydroxide + 2% chlorhexidine gel. Samples were taken before any treatment (S1), after irrigation with 6% NaOCl (S2), after irrigation with 2% chlorhexidine (S3), after intracanal dressing (S4), and after 17% EDTA irrigation (S5). Cultivable bacteria recovered from the 5 stages were counted and identified by means of polymerase chain reaction assay (16S rRNA)., Results: Both groups had colony-forming unit counts significantly reduced after S2 (P < .05); however, no significant difference was found between the irrigants (S2 and S3, P = .99). No difference in bacteria counts was found between the intracanal medicaments used (P = .95). The most prevalent bacteria detected were Actinomyces naeslundii (66.67%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum, which were detected in 33.34% of the root canals. An average of 2.13 species per canal was found, and no statistical correlation was observed between bacterial species and clinical/radiographic features., Conclusions: The microbial profile of infected immature teeth is similar to that of primarily infected permanent teeth. The greatest bacterial reduction was promoted by the irrigation solutions. The revascularization protocols that used the tested intracanal medicaments were efficient in reducing viable bacteria in necrotic immature teeth., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Regenerative endodontic procedures for immature teeth with pulp necrosis have gained a lot of interest. Basic scientific research has documented the potential for dental pulp regeneration in preclinical studies. A number of case reports and case series have shown the control of clinical infection and increases in thickness and length of the roots. Preoperative infection is an important factor that predicts outcome in nonsurgical endodontic treatment in mature teeth and seems to also be an important determinant of outcome in treating immature teeth. However, antimicrobial strategies for the immature tooth in which pulp regeneration is contemplated are different from those used in mature cases. This is because of the interest in disinfecting the root canal to a higher level to promote tissue growth without disrupting the bioactive potential of root dentin and the viability of stem cells from which the regenerated tissue would develop. This review addresses the factors involved in making clinical decisions in this area in light of the information available on the microbiology of endodontic infections, the efficacy to antimicrobial strategies, and the outcomes of regenerative and alternative procedures., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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.

Introduction: This study investigated the bacterial community involved in primary endodontic diseases, evaluated its ability to activate the macrophage Toll-like receptor 4 receptor through p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways, and determined the levels of endotoxins and interleukins (interleukin [IL]-6 and -10) produced by endodontic content-stimulated macrophages., Methods: Samples were taken from 21 root canals by using sterile/apyrogenic paper points. Raw 264.7 macrophages were stimulated with root canal contents. Checkerboard DNA-DNA hybridization was used for bacterial analysis and the limulus amebocyte lysate assay for endotoxin measurement; p38 MAPK and NF-κB activation was determined by Western blot analysis. IL-6 and IL-10 were measured using the enzyme-linked immunosorbent assay., Results: Bacteria and endotoxins were detected in 100% of the samples (21/21). The most frequently observed species were Parvimonas micra (16/21, 76%), Fusobacterium nucleatum ssp. nucleatum (15/21, 71%), and Porphyromonas endodontalis (14/21, 66%). Correlations were found between endotoxins and IL-6 and IL-10 (P < .05); p38 phosphorylation had a peak at 60 minutes, and NF-κB was quickly activated after 10 minutes of stimulation., Conclusions: It was concluded that the complex bacterial community was shown to be a potent activator of TLR-4 determined by the p38 MAPK and NF-κB signaling pathways, culminating in a high antigenicity against macrophages through the levels of IL-6 and IL-10, all significantly affected by endotoxin levels., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To evaluate the efficacy of metronidazole gel versus metronidazole solution against Enterococcus faecalis in abscessed primary molars., Study Design: A clinical trial., Method: Twenty pulpally involved non-vital carious human primary mandibular second molars with furcal abscess were randomly allocated into two groups to evaluate the efficacy of metronidazole gel (3% w/v) and metronidazole solution (0.5% w/v) against E. faecalis. Subjects in the first experimental group were subjected to treatment with metronidazole gel (3% w/v) and subjects in the second experimental group were treated with metronidazole solution (0.5% w/v). Two microbial samples (pre-operative and post-operative samples) were obtained from the root canals of each subject from both the groups; sub cultured and efficacy of both the groups were evaluated., Results: Overall percentage reduction of the mean colony forming unit (CFU) count of metronidazole gel (3% w/v) group was 96.39% and metronidazole solution (0.5 % w/v) was 90.90%. Results of intergroup t test of the percentage difference of mean CFU counts between both the groups revealed a statistically highly significant difference, i.e. p value--0.008 (p < 0.01)., Conclusions: Metronidazole gel (3% w/v) was more effective than metronidazole solution (0.5% w/v) against E. faecalis.

Introduction: This clinical study was conducted to measure the endotoxin levels in infected root canals (RCs) and exudates related to acute apical abscesses (AAAs). In addition, the effectiveness of RC procedures in reducing the endotoxin levels in RCs was monitored., Methods: Paired samples of infected RCs and exudates from AAAs were collected from 10 subjects by using paper points. RCs samples were collected before (RCS1) and after chemomechanical preparation (CMP) (RCS2), after 17% EDTA (RCS3), and after 30 days of intracanal medication (Ca[OH]2 + chlorhexidine) (RCS4). A turbidimetric kinetic limulus amebocyte lysate assay was used for the measurement of endotoxins., Results: Endotoxins were detected in 100% of the baseline samples of AAAs and RCs (RCS1) with median values of 175 EU/mL and 41.5 EU/mL, respectively (P < .05). After CMP (RCS2), endotoxins were reduced to a median value of 0.54 EU/mL (P < .05). Subsequent irrigation with EDTA (RCS3) failed to present a significant effectiveness in reducing the endotoxin levels (median= 0.37 EU/mL) (P = .07). However, intracanal medication for 30 days (RCS4) reduced endotoxins to median values of 0.03 EU/mL (P < .01)., Conclusions: The present study revealed a strong association between the high levels of endotoxins found in AAAs and RCs collected from the same tooth. Moreover, the effectiveness of CMP in reducing the endotoxin levels from RCs in acute endodontic infection was improved by the use of RC medication., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: A significant portion of the bacteria taking part of the microbiome associated with apical periodontitis still remain to be cultivated and phenotypically characterized. This molecular study evaluated the prevalence of selected as-yet-uncultivated and difficult-to-culture bacterial taxa in infected root canals and their susceptibility to chemomechanical procedures., Methods: Root canals of single-rooted teeth with apical periodontitis were prepared using rotary nickel-titanium instruments and 2.5% sodium hypochlorite as the irrigant. DNA extracts from samples taken before (S1) and after (S2) chemomechanical preparation were surveyed for the presence of 7 as-yet-uncultivated phylotypes and 1 difficult-to-culture species using end-point polymerase chain reaction. Samples were also subjected to quantitative analysis of total bacteria and levels of the 2 most prevalent taxa., Results: Bacteroidaceae sp. HOT-272 (24%) and Fretibacterium fastidiosum (20%) were the most prevalent taxa in S1. Their mean counts in S1 were 8.25 × 10(3) and 2.13 × 10(3) rRNA gene copies, corresponding to 0.18% and 0.55% of the total bacteria. Chemomechanical debridement promoted a highly statistically significant reduction in total bacterial counts (P < .001), but 64% of the canals were still positive for bacterial presence. Of the target taxa, only Bacteroidaceae sp. HOT-272 and F. fastidiosum were detected in S2 (each one in 1 sample). The reduction in counts of both taxa was also highly significant (P < .001)., Conclusions: Findings confirmed that several as-yet-uncultivated and difficult-to-grow bacterial taxa can participate in the microbiome associated with apical periodontitis. Two of them were found in relatively high prevalence but rarely as a dominant species. Chemomechanical procedures were highly effective in completely eliminating these taxa or at least substantially reducing their numbers., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to assess the microbial content of root canals of human primary teeth with pulp necrosis and periradicular lesion., Methods: Microbial samples were collected from 24 canals of children treated at a pediatric dentistry clinic. Microbiological identification was performed using checker-board DNA-DNA hybridization for 40 different bacteria. Data were analyzed per canal based on the mean count and frequency of each bacterial species., Results: Detectable levels of bacterial species were observed for 35 probes (88%). The most frequent bacteria were Fusobacterium nucleatum sp. nucleatum, Fusobacterium periodonticum, Prevotella melaninogenica, Prevotella nigrescens, and Prevotella intermedia. Facultative species were identified in 20 root canals (83%), anaerobic species were identified in 24 root canals (100%), and aerobic species in 18 root canals (75%). Black-pigmented bacilli were found in 23 samples (96%). The number of different bacterial species detected per canal ranged from five to 33., Conclusion: Endodontic infection in primary teeth with pulp necrosis and periradicular lesion is multimicrobial, including aerobic, facultative, and anaerobic micro-organisms.

Introduction: For selective detection of viable bacteria with molecular methods, propidium monoazide (PMA) treatment has been successfully applied to a wide range of bacteria. The purpose of this study was to compare the quantity of live cells with the total amounts of both live and dead cells before and after chemomechanical preparation by using PMA in combination with real-time polymerase chain reaction (qPCR)., Methods: Twenty-one teeth with pulp necrosis and a periapical lesion were included. Bacterial sampling of the root canals was performed before (S1) and after (S2) chemomechanical root canal treatment. Each sample was separated into 2 different tubes. PMA was added to one of the tubes, and the other was left untreated. Then, DNA extraction and qPCR were performed. To evaluate the validity of the PMA treatment, the defined mixtures containing different ratios of live and dead cell suspensions of Enterococcus faecalis were either subjected to PMA treatment or not subjected to PMA treatment, followed by qPCR quantification., Results: A paired t test showed a highly significant difference in the mean threshold cycle values between S1 with and without PMA (P = .0002), and this difference (0.89) was similar to that (0.96) obtained from the samples consisting of 80% live cell suspension and 20% dead cell suspension of E. faecalis. The threshold cycle values between the S2 samples with and without PMA were also significantly different (P = .0134), and this difference (0.37) was similar to that obtained from the 100% live cell suspension of E. faecalis (0.42)., Conclusions: PMA in conjunction with qPCR appeared to be useful in analyzing the primary infections of root canals because there were significant amounts of dead bacteria in the root canals. Although the use of PMA treatment in post-preparation samples significantly reduced the detection of dead bacteria, this difference was still small, so further studies should be carried out to confirm the necessity of PMA treatment., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Clinical Significance: The apical extrusion of infected debris may have the potential to disrupt the balance between microbial aggression and host defense, resulting in incidents of acute inflammation. During preparation, irrigants and debris, such as bacteria, dentin filings and necrotic tissue may be extruded into the periradicular region leading to periapical inflammation and postoperative flare ups. Using an instrumentation technique that minimizes apical extrusion would be beneficial to both the practitioner and patient. The purpose of the study was to evaluate the weight of debris and volume of irrigant extruded apically from extracted teeth in vitro after endodontic instrumentation using four different rotary root canal instrumentation systems., Materials and Methods: Four groups of each 20 extracted mandibular premolars were instrumented using one of the four systems: ProTaper Universal (Dentsply Maillefer, Ballaigues, Switzerland)), Hero-shaper (MicroMega, Besancon, France), RaCe (FKG Dentaire, La-Chaux-de-Fonds, Switzerland) and K3 (SybronEndo, West Collins, CA). Debris and irrigant extruded from the apical foramen during instrumentation were collected in preweighed test tubes. Volume of irrigant extruded was noted. The containers were stored in incubator at 70° for two days to evaporate the moisture. Weight of dry debris was noted., Statistical Analysis: Data was analyzed using Kruskall-Wallis and Mann-Whitney U test at a significance of 0.001., Results: The results indicated that all of the instrumentation systems tested caused measurable apical extrusion of debris and irrigants. Higher extrusion was observed with Protaper system which was statistically significant with Hero-Shaper, RaCe and K3 systems. There were no statistical differences between Hero-shaper, K3 and RaCe systems (p < 0.05)., Conclusion: All instrumentation techniques apically extruded debris and irrigant. However, Hero-shaper, K3 and RaCe systems produced less extruded debris and irrigant than the Protaper system.

Aim: To compare the antibacterial efficacy of two instrumentation techniques, one using hand nickel-titanium (NiTi) instruments and the other using rotary NiTi instruments, in root canals of teeth with apical periodontitis., Methodology: Root canals from single-rooted teeth were instrumented using either hand NiTi instruments in the alternated rotation motion technique or rotary BioRaCe instruments. The irrigant used in both groups was 2.5% NaOCl. DNA extracts from samples taken before and after instrumentation were subjected to quantitative analysis by real-time polymerase chain reaction (qPCR). Qualitative analysis was also performed using presence/absence data from culture and qPCR assays., Results: Bacteria were detected in all S1 samples by both methods. In culture analysis, 45% and 35% of the canals were still positive for bacterial presence after hand and rotary NiTi instrumentation, respectively (P > 0.05). Rotary NiTi instrumentation resulted in significantly fewer qPCR-positive cases (60%) than hand NiTi instrumentation (95%) (P = 0.01). Intergroup comparison of quantitative data showed no significant difference between the two techniques., Conclusion: There was no significant difference in bacterial reduction in infected canals after instrumentation using hand or rotary NiTi instruments. In terms of incidence of positive results for bacteria, culture also showed no significant differences between the groups, but the rotary NiTi instrumentation resulted in more negative results in the more sensitive qPCR analysis., (© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Introduction: A 3-antibiotic combination (3Mix) is widely used in endodontics for root canal disinfection, particularly in pulp revascularization procedures. However, the cytotoxicity of 3Mix has not been evaluated. The purpose of this study was to determine the cytotoxicity and antibacterial efficacy of 3Mix and each single antibiotic component of 3Mix., Methods: For the cytotoxicity test, human dental pulp cells and apical papilla cells were exposed to either 3Mix or to each single antibiotic component of 3Mix using concentrations of 0.024, 0.097, 0.39, 1.56, 6.25, and 25.00 μg/mL for 1, 3, 5, and 7 days. Cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. For the antibacterial test, 25.00 μg/mL and 0.39 μg/mL 3Mix or single antibiotic were tested on bacteria isolated from necrotic teeth by measuring bacterial recovery on blood agar., Results: The 0.024-μg/mL concentration of all experimental groups generated the highest dental pulp cell or apical pulp cell viability at all time periods. On day 7, 0.39 μg/mL 3Mix produced more than 90% cell viability; 25.00 μg/mL 3Mix completely eliminated isolated bacteria, whereas 0.39 μg/mL was unable to eradicate all bacteria. However, the overall bacterial reduction was significantly different compared with the control group (P < .01)., Conclusions: All drugs except metronidazole induced cytotoxicity on cultured cells. 3Mix generated higher cytotoxicity compared with a single drug. The cytotoxicity increased in a concentration- and time-dependent manner; 0.39 μg/mL 3Mix had less cytotoxicity and was able to significantly reduce bacteria isolated from necrotic teeth., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To evaluate clinically the antibacterial effects of root canal treatment procedures using molecular microbiology analyses., Methodology: Samples were taken from 14 necrotic root canals of teeth with apical periodontitis before (S1) and after instrumentation with NaOCl irrigation (S2), a final rinse with chlorhexidine (CHX) (S3) and then one-week interappointment medication with calcium hydroxide/CHX paste (S4). The parameters examined included the following: incidence of positive broad-range PCR results for bacterial presence; impact on bacterial community structures evaluated by PCR-Denaturing Gradient Gel Electrophoresis (DGGE); quantitative bacterial reduction determined by real-time PCR; and identification of bacterial persisters by cloning and sequencing. Data from the different tests were subjected to statistical analyses and diversity indicator calculations., Results: All S1 samples were positive for bacteria in all tests. Treatment procedures promoted a decrease in microbial diversity and significantly reduced the incidence of positive results and the bacterial counts (P < 0.05). In general, each subsequent treatment step improved disinfection. No specific taxon or community pattern was associated with post-treatment samples., Conclusion: Supplementary steps consisting of a final rinse with CHX followed by calcium hydroxide interappointment medication promoted further decrease in the bacterial bioburden to levels significantly below those achieved by the chemomechanical procedures alone. Because the long-term outcome of root canal treatment is dependent upon maximal bacterial reduction, the present results are of clinical relevance., (© 2012 International Endodontic Journal.)

Aim: this study was conducted to evaluate the antimicrobial effectiveness of 6 root canal filling materials and a negative control agent against 18 strains of bacteria isolated from infected root canals of primary molar teeth using agar diffusion assay., Materials: Aloevera with sterile water Zinc oxide and Eugenol, Zinc oxide-Eugenol with aloevera, Calcium hydroxide and sterile water, Calcium hydroxide with sterile water and aloevera, Calcium hydroxide and Iodoform (Metapex) and Vaseline (Control). MIC and MBC of aloevera was calculated., Results: All materials except Vaseline showed varied antimicrobial activity against the test bacterias. The zones of inhibition were ranked into 4 inhibition categories based on the proportional distribution of the data. All the 18 bacterial isolates were classified under 2 groups based on Gram positive and Gram negative aerobes. Statistical analysis was carried out to compare the antimicrobial effectiveness between materials tested with each of the bacterial groupings., Conclusion: Aloevera + Sterile Water was found to have superior antimicrobial activity against most of the microorganisms followed by ZOE + Aloevera, calcium hydroxide + Aloevera, ZOE, calcium hydroxide, Metapex in the descending order and Vaseline showed no inhibition.

One of the major objectives in endodontic therapy is to disinfect the entire root canal system. This goal may be achieved using mechanical instrumenation and chemical irrigation in conjunrction with medication of the root canal between treatment sessions. Microorganisms and their by-products are considered to be the major cause of pulpal and periradicular patholic. In order to reduce or eliminate bacteria and popular tissue remnants, the use of various irrigation solution during treatment have been suggested. Sodium hypochlorite (NaOCI), the most common irrigant, is an excellent nonspecific proteolytic and antimicrobial agent. The purpose of this paper is to review the antimicrobial activity of sodium hypochlorite.

Different microbial identification methods have shown that the microbial community profiles in endodontic infections are diverse and assorted. The aim of this study was to evaluate the frequency of selected endodontic pathogens in the pulp chambers (PCs) and root canals (RCs) of infected primary teeth using PCR methods. Paired PC and RC samples were collected from 15 subjects and analyzed by PCR for the presence of Filifactor alocis, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, Tanerella forsythia, Treponema denticola, and Treponema socranskii. The frequency of each species was determined in the PC and RC of each case. The species most frequently detected in PCs were P. nigrescens (86.7%), P. gingivalis (73.3%), and F. alocis (73.3%). Of the PC samples, 13.3% contained P. micra and T. denticola, and 6.7% contained T. forsythia. The species most frequently detected in RCs were P. gingivalis (100%) and P. nigrescens (93.3%). P. tannerae, P. micra, and T. denticola were found in 40% of the RC samples; T. forsythia was found in 26.7% of the RC samples. The "red complex", which comprises P. gingivalis, T. denticola, and T. forsythia, was not found in the PC of any tooth but was found in 30% of the RC samples. The detection of P. nigrescens in the PC was statistically associated with the presence of P. nigrescens in the RC (p = 0.04). The results suggest high heterogeneity among the samples, even among those from the same subject.

Objective: To compare via a split-mouth randomized clinical trial the efficacy of 1% chlorhexidine gel, calcium hydroxide/camphorated paramonochlorophenol (Callen PMCC), and a one-visit endodontic treatment against mutans streptococci and anaerobic bacteria found in primary molars with necrotic pulps and to assess clinical success rates after 12 months., Method and Materials: Pre- and posttreatment intracanal samples were collected from 37 teeth (from 21 children) for analysis of the number of mutans streptococci and anaerobic bacteria. Clinical and radiographic criteria were analyzed to determine treatment outcome., Results: Chlorhexidine gel significantly reduced mutans streptococci levels (P = .010), whereas Callen PMCC significantly reduced the levels of anaerobic bacteria (P = .002). No differences in the reduction of mutans streptococci (P = .187) and anaerobes (P = .564) were observed between groups. The clinical success rates were 85.71% (Callen PMCC), 78.57% (chlorexidine gel), and 77.77% (one-visit treatment)., Conclusion: Teeth treated with Callen PMCC presented the highest clinical success rate. The 1% chlorhexidine gel, as well as calcium hydroxide/camphorated paramonochlorophenol, presented limited efficacy in reducing bacteria from necrotic primary root canals.

Introduction: This in vivo study used molecular microbiology methods to evaluate the effects of passive ultrasonic irrigation (PUI) as a supplementary disinfecting step after root canal preparation., Methods: Samples were taken from 10 necrotic root canals of teeth with apical periodontitis before (S1) and after rotary nickel-titanium instrumentation using 2.5% NaOCl as the irrigant (S2) and then after PUI for NaOCl activation (S3). The parameters examined included the incidence of positive broad-range polymerase chain reaction (PCR) results for bacterial presence, the impact on bacterial diversity evaluated by PCR-denaturing gradient gel electrophoresis (DGGE), the quantitative bacterial reduction determined by real-time PCR, and the identification of persistent species by clone library analysis., Results: All S1 samples were positive for bacteria in all tests. Treatment procedures were significantly effective in reducing the incidence of positive results for bacteria, the number of bacterial cells (infectious bioburden), and the bacterial diversity (number of species and abundance). However, the supplementary PUI approach did not succeed in significantly enhancing disinfection beyond that achieved by chemomechanical preparation. Several bacterial species/phylotypes were identified in post-treatment samples that were positive for bacteria., Conclusions: Findings from this clinical study including a small sample size suggest that PUI can be ineffective in significantly improving disinfection of the main root canal after chemomechanical procedures., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects., Materials and Methods: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively., Results: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples., Conclusions: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals., (© 2012 John Wiley & Sons A/S.)