Prevalence and virulence factors of haemolytic Enterococcus faecalis isolated from root filled teeth associated with periradicular lesions: A laboratory investigation in Thailand.

AIM: Previous endodontic research has provided limited understanding of the prevalence and roles of haemolytic and non-haemolytic Enterococcus faecalis strains in root filled teeth. This study aimed to determine the prevalence of these strains in root filled teeth with periradicular lesions and investigate their associated virulence factors. METHODOLOGY: A total of 36 root canal samples were collected from 36 subjects. The prevalence of E. faecalis was determined using culture and PCR methods. Antibiotic susceptibility of haemolytic and non-haemolytic E. faecalis strains was assessed using the broth dilution assay. The cytokine stimulation in periodontal ligament (PDL) cells and neutrophil migration were evaluated using real-time PCR and migration assay, respectively. Cell invasion ability of the strains was assessed using a cell culture model. Additionally, the virulence gene expression of the haemolytic and non-haemolytic strains was investigated using real-time PCR. The Mann-Whitney U and Spearman's ρ tests were used to examine the significant difference between the two strains and to analyse the correlation between phenotype and gene expression, respectively. RESULTS: Enterococcus faecalis was detected in 33.3% and 88.9% of samples by culture and real-time PCR, respectively. Haemolytic strains were found in 36.4% of subjects. Non-haemolytic strains exhibited susceptibility to erythromycin and varying susceptibility to tetracycline, while all haemolytic strains were resistant to both antibiotics. Haemolytic strains significantly upregulated the expression of IL-8, OPG and RANKL in PDL cells (p < .05). Notably, the fold increases in these genes were higher: IL-8 (556.1 ± 82.9 vs. 249.6 ± 81.8), OPG (2.2 ± 0.5 vs. 1.3 ± 0.2) and RANKL (1.8 ± 0.3 vs. 1.2 ± 0.1). Furthermore, haemolytic strains had a greater effect on neutrophil migration (68.7 ± 15.2% vs. 46.9 ± 11.4%) and demonstrated a higher level of internalization into oral keratinocyte cells (68.6 ± 0.4% vs. 33.8 ± 0.5%) (p < .05). They also showed enhanced expression of virulence genes associated with haemolysin, surface proteins, collagen-binding and aggregation substances. Gelatinase activity was only detectable in non-haemolytic strains. CONCLUSIONS: This study revealed that haemolytic strains E. faecalis possessed enhanced abilities in host invasion and a higher abundance of virulence factors, suggesting their potential contribution to more severe disease manifestations.

Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis.

AIM: To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach. METHODOLOGY: Apical lesion samples were collected from 12 patients (23-80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers. RESULTS: Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacterium acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples. CONCLUSION: Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species.

Antibacterial efficacy and drug-induced tooth discolouration of antibiotic combinations for endodontic regenerative procedures.

Elimination of microbial contamination from the root canal system is a precondition for successful root canal treatment. Teeth with immature root development, necrotic pulps and apical periodontitis present multiple challenges for successful treatment. Disinfection is achieved by irrigation followed by the placement of an intracanal medicament. A mixture of ciprofloxacin, metronidazole and minocycline (3-MIX S) has been shown to be very effective in eliminating endodontic pathogens in vitro and in vivo. Among the components of the mixture, minocycline can induce tooth discolouration after long-term oral use. Therefore, the elimination of minocycline from the above-mentioned combination has been suggested to prevent the occasion of this undesirable effect. The aim of this study was to investigate the potential antimicrobial efficacy of alternative antibiotic combinations [3-MIX C (clarithromycin); 3-MIX F (fosfomycin)] against bacteria from infected root canals. An additional objective was to evaluate their discolouration potential as possible alternatives to minocycline-based intracanal medicaments. Our in vitro results clearly demonstrated that 3-MIX C and 3-MIX F had a greater antimicrobial activity than 3-MIX S, underlying that clarithromycin still had a higher capacity to kill endodontic pathogens in vitro compared to fosfomycin. Both 3-MIX C and 3-MIX F were able to avoid the permanent staining effect of the crown.

Aetiology, microbiology and therapy of periapical lesions around oral implants: a retrospective analysis.

OBJECTIVE: The aim of this study was: (i) to evaluate whether an endodontic pathology on the extracted tooth or adjacent teeth of an implant site has an influence on the emergence of a periapical lesion, (ii) to retrospectively analyse the outcome of different treatment strategies, (iii) to determine which bacteria were present in periapical lesions. METHODS: The endodontic status of the tooth at the implant site and the adjacent teeth was explored and linked to the periapical status of the implant. For all the lesions treated since 2000, their survival was assessed. Finally, microbial samples (culturing) from the periapical lesions, were analysed. RESULTS: If an endodontic treatment or a periapical lesion at the apex of a tooth is present, a periapical lesion around the implant can be detected in 8.2% up to 13.6% (OR 7.2). For periapical pathology at the adjacent teeth, the percentage rises to 25% (OR 8.0). The best treatment option could not be found. Bacteria were found in 9/21 lesions. The most prominent species was P. gingivalis. CONCLUSIONS: When an endodontic pathology is present on the extracted or neighbouring teeth, it is significantly more likely that a periapical lesion will develop around a future implant.

Investigation of six selected bacterial species in endo-periodontal lesions.

AIM: To investigate and determine possible associations of six tested bacteria belonging to 'orange' and 'green' complexes, in endo-periodontal lesions: Parvimonas micra, Fusobacterium nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens and Capnocytophaga sputigena. METHODOLOGY: Forty-six patients presenting with different types of endo-periodontal lesions were investigated. Clinical examinations, periapical radiographs and microbiological sampling from the canal system (endo) and periodontal pockets (perio) were performed. Qualitative and semiquantitative evaluation of bacteria was performed by polymerase chain reaction (PCR) and DNA-DNA hybridization (micro-IDent plus; Hain Lifescience, Germany). RESULTS: Extremely high bacterial loads in endodontic samples were recorded for P. micra, F. nucleatum and C. sputigena, while periodontal samples were often colonized by the same species, plus C. rectus. Significant association was recorded between F. nucleatum-endo and P. micra-endo (P = 0.03, Fisher's exact test). There was marginal evidence of associations between: (i) C. sputigena-endo and C. sputigena-perio (P = 0.06, Fisher's exact test); (ii) P. micra-endo and P. micra-perio (P = 0.05, Fisher's exact test). Sensitivity to percussion was associated with an increased chance of cases with P. micra-endo (P = 0.03, Pearson chi-square test). CONCLUSION: The findings suggest that F. nucleatum, P. micra and C. sputigena may play a role in the pathogenesis of endo-periodontal lesions.

[Microbial decontamination of the root canals of devitalized teeth]. / Mikrobiálna dekontaminácia korenových kanálikov devitálnych zubov.

The primary goal of endodontic therapy is the reduction or elimination of microorganisms and their by-products from the root canal system. Although a number of instrumentation and irrigation techniques exist, debris is often left behind in the root canal system and proper canal cleaning, shaping, and irrigation are needed to reduce significantly or sometimes even eliminate microorganisms from the canals. Residual microbes in the root canal system are the primary cause of post-treatment apical periodontitis that may persist in both poorly and properly treated cases. Apical periodontitis is a sequel to endodontic infection and manifests itself as the host defense response to microbial challenge emanating from the root canal system to the periapical tissue. It results in local inflammation, resorption of hard tissues, destruction of other periapical tissues, and eventual formation of various histopathological categories of apical periodontitis, commonly referred to as periapical lesions. When the root canal treatment is carried out properly, healing of the periapical lesion usually follows, with bone regeneration. In certain cases, post-treatment apical periodontitis still persists, the condition being commonly referred to as endodontic failure. It is widely acknowledged that such post-treatment apical periodontitis occurs when root canal treatment has not adequately controlled and eliminated the infection. However, complete elimination of microorganisms is not always achieved in clinical practice due to the anatomical complexities of root canals and consequent limitations in access by instruments and irrigants. The use of antimicrobial medication has been advocated to disinfect the root canal system. The recovery of Candida albicans and Enterococcus faecalis is common after failed root canal treatment. Therefore, when testing different antimicrobial agents for efficacy in endodontic treatment, 100% inhibition of the growth of the two microorganisms is required. The purpose of this article is to assess the antimicrobial action of intracanal medicaments and relevance of the root canal irrigation in endodontic therapy of devitalized teeth.

The efficacy of a cervical barrier in preventing microleakage of Enterococcus faecalis in endodontically treated teeth.

The clinical failure of coronal restorations can compromise the healthy periapical status of endodontically treated teeth. The purpose of the present ex vivo study was to assess the effectiveness of the cervical barrier in preventing bacterial microleakage in the eventual loss of the coronal restoration. Following removal of the smear layer and obturation to the cementoenamel junction using gutta-percha, 70 single-rooted mandibular premolars were randomly divided into five different groups: Group 1 received no additional treatment; Groups 2 and 3 had 2 mm and 3 mm of the obturation removed, respectively, followed by placement of a cervical barrier that was the same thickness as the obturation. In Group 4 (positive control), the root canals were filled only with gutta-percha, while in Group 5 (negative control), the root canals were completely impermeabilized following obturation. The filled root canals were incorporated into a split-chamber model system using Enterococcus faecalis as a microbial marker. The apices were suspended in the lower chambers. Leakage was determined daily for 60 days and evaluated for turbidity. According to Fisher's exact test, the cervical barrier in Groups 2 and 3 prevented the microleakage of E. faecalis into the root canals when compared with Groups 1 and 4. This result was similar to that for the completely sealed samples in Group 5 (p = 0.001).

Comparative evaluation of bactericidal potential of four root canal filling materials against microflora of infected non-vital primary teeth.

BACKGROUND AND OBJECTIVES: Since complete debridement of the root canals of the primary teeth is not practically possible due to the highly variable root canal anatomy, success of the endodontic therapy depends partly on the use ofantibacterial irrigating agents and root canal filling materials. Recent literature indicates that anaerobes comprise a majority of the bacteria in necrotic root canals ofprimary teeth. The study determined the antibacterial effectiveness of four root canal filling materials namely Calcium hydroxide, Zinc oxide eugenol, Vitapex and Metapex against microbial specimens obtained directly from necrotic root canals of primary teeth. METHOD: Microbial specimens were collected using sterile paper points, from 15 primary maxillary and mandibular posterior teeth of randomly selected children in the age group of 4-10 years with infected non vital primary teeth, requiring pulpectomy procedure. The microbial specimens collected were subjected to microbiological analysis and the antimicrobial potential of root canal filling materials were tested using Agar diffusion technique. RESULTS: were statistically analyzed using one-way ANOVA. Facultative/Aerobic organisms were isolated in all the cases, anaerobic organisms were isolated in 80% of the cases, and Candida albicans was isolated in 1 case. ZOE showed superior inhibitory activity against most of the organisms isolated followed by Vitapex, Calcium hydroxide and Metapex in descending order. CONCLUSION: Our data may be useful as a guide for relative antimicrobial effectiveness or non-effectiveness of the materials employed. In vivo studies are required to state the specific antimicrobial activity and merits and demerits of any of the test filling material.

Microflora of root filled teeth with apical periodontitis in Latvian patients.

OBJECTIVE: The aim of the present study was to investigate the microbial flora of root filled teeth with apical periodontitis and to determine the prevalence of ß-lactamase producing strains in isolated bacteria in Latvian patients. MATERIALS AND METHODS: 33 root filled teeth with asymptomatic persisting periapical lesions were selected for the present study. During nonsurgical endodontic retreatment, the root filling material was removed and canals were sampled. Determination of microbial species was based on series of biochemical tests using identification kits. All strains of bacteria were tested for ß-lactamase production by using chromogenic nitrocefin-impregnated slides. RESULTS: Bacteria were found in 32 (97%) of initial specimens from the teeth. The number of isolated microbial strains in the specimens ranged from one to six (mean 2.7). 79% of the isolated microbial species were Gram-positive bacteria. The most common isolates were Streptococcus (27%), Actinomyces (27%), Staphylococcus (18%), Enterococcus (18%) and Lactobacillus (18%) spp. Yeasts were found as four isolates in 3 cases (9%). ß-lactamase-producing bacterial strains were detected in 12 specimens, 36% of the patients. The most common enzyme-producing bacteria belonged to Actinomyces and Staphylococcus spp. CONCLUSIONS: The microbial flora in previously treated root canals with apical periodontitis is limited to a small number of predominantly Gram-positive microbial species. The most common isolates are Streptococcus, Actinomyces, Staphylococcus, Enterococcus and Lactobacillus spp. A moderately high prevalence of ß-lactamase producing bacterial strains was detected in patients with root filled teeth with apical periodontitis.

Proteomic analysis of endodontic infections by liquid chromatography-tandem mass spectrometry.

INTRODUCTION: Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the identification of proteins of bacterial origin present in endodontic infections. METHODS: Endodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected to tryptic in-solution digestion and analysed by reverse-phase nano-LC-MS/MS followed by a database search. RESULTS: Proteins, mainly of cell wall or membrane origin, from endodontic bacteria especially Enterococcus faecalis, Enterococcus faecium, Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola were identified from all the samples tested. Identified proteins included adhesins, autolysins, proteases, virulence factors, and antibiotic-resistance proteins. CONCLUSIONS: LC-MS/MS offers a sensitive analytical platform to study the disease processes in the root canal environment. The array of proteins expressed in endodontic infections reflects the complex microbial presence and highlights the bacterial species involved in the inflammatory process.

The mysterious appearance of enterococci in filled root canals.

In this narrative review, the potential reasons for the high occurrence of enterococci in filled root canals are explored. The pulpless root canal appears to be a habitat for these bacteria, particularly for Enterococcus faecalis. However, re-surveying the literature in caries research, it can be concluded that, contrary to earlier belief, enterococci are rare if ever found at the advancing front of dentinal lesions. The same is the case for true primary endodontic infections, but some uncertainty remains, because the coronal seal and the history of teeth harbouring enterococci have rarely been accurately investigated. Furthermore, from longitudinal studies with a known infection at the initiation of treatment, which was carried out under controlled asepsis, it is questionable whether enterococci are as difficult to eliminate from the canal system as is commonly held. A more likely explanation for the high occurrence of enterococci in filled root canals is that they enter after treatment, but from which source? The intriguing finding in this context is that enterococci do not appear to be colonizers of the oral cavity. They are merely transient oral bacteria, unless there is a predilection site such as the unsealed necrotic or filled root canal. The origin of this infection is most likely food. Using the example of enterococci in filled root canals, this paper highlights the possible importance of transient microorganisms in the oral cavity and changes in a microenvironment that can create favourable conditions for infection.

Photodynamic therapy in endodontic treatment of deciduous teeth.

The purpose of this study was to evaluate photodynamic therapy in deciduous teeth with necrotic pulp by means of fully quantifying viable bacteria, before and after instrumentation and after the use of photodynamic therapy. Radicular canal cultures were conducted (n = 10): the first one was performed right after access and location of the radicular canal; the second was performed after the conclusion of chemical-mechanical instrumentation, and the last one after photodynamic therapy. The photodynamic therapy was performed with 4 J/cm energy low-intensity diode together with toluidine blue. The results (log(10)) were submitted to a descriptive analysis and Wilcoxon test. The percentage of reduction was submitted to the Mann-Whitney test. The instrumentation resulted in a reduction of 82.59% of viable bacteria, and, after photodynamic therapy, the microbial reduction observed was 98.37% (P = 0.0126). Photodynamic therapy is recommended as adjunct therapy for microbial reduction in deciduous teeth with necrotic pulp.

Multiplex polymerase chain reaction detection of selected bacterial species from symptomatic and asymptomatic non-vital teeth with primary endodontic infections.

AIM: The aim of the present study was to investigate the presence of selective anaerobic microorganisms in primary root canal infections of symptomatic and asymptomatic non-vital teeth with periapical pathosis using multiplex polymerase chain reaction. METHODS: A total of 100 root canal samples (50 from symptomatic and 50 from asymptomatic teeth) were obtained from patients with primary endodontic infections. DNA extracted from the samples was amplified by using specific primers for the 16S rRNA gene of each bacterium, and semiquantification was done to analyze the prevalence of microorganisms and their correlation to clinical features. RESULTS: Treponema denticola (T. denticola) was present in 21 (42%) and 29 (58%) samples in the symptomatic and asymptomatic groups, respectively. Tannerella forsythia, Porphyromonas gingivalis (P. gingivalis), and Fusobacterium nucleatum (F. nucleatum) were significantly high (P < .05) in the symptomatic group, whereas Prevotella intermedia was significantly high (P < .05) in the asymptomatic group. The mean counts of T. denticola and F. nucleatum were significantly high (P < .05) in the symptomatic group. For symptoms, P. gingivalis, T. denticola, and F. nucleatum were significantly associated with clinical features. CONCLUSION: Significant differences exist in the bacterial composition between asymptomatic and symptomatic primary endodontic infections. As well as presence of pathogens, other factors, such as the phenotypic trait of bacteria and interactions among bacterial members, might play a determining role in the pathogenicity of primary endodontic infections.

Photodynamic Therapy with Pyoktanin Blue and Diode Laser for Elimination of Enterococcus faecalis.

BACKGROUND/AIM: Enterococcus faecalis is responsible for most cases of endodontic treatment failure. Despite various conventional disinfection methods, root canals are not completely free of microorganisms. Photodynamic therapy (PDT) is a new antimicrobial strategy that involves the use of a non-toxic photosensitizer (PS) and a light source. The aim of this study was to evaluate the antimicrobial effect of PDT using diode laser and pyoktanin blue (PB) and confirm the nontoxicity of PB as a PS. MATERIALS AND METHODS: Laser irradiation with an output power of 3 W was performed with PB as the PS to a bacterial solution containing E. faecalis. Then, the number of colony-forming units was counted. PB cytotoxicity was also assessed by the MTT assay. RESULTS: E. faecalis counts were reduced after laser irradiation, laser irradiation with PB, or the combination thereof compared to the control, non-irradiation or water. The 50% cytotoxic concentration value for adult human dermal fibroblasts incubated with PB for 1 min was 108 µg/ml. CONCLUSION: Diode laser irradiation in combination with PB as the PS is efficacious for the elimination of E. faecalis without toxic effects to human dermal fibroblasts. This strategy might be useful for root canal irrigants.

An in vitro comparison of bacterial leakage of three common restorative materials used as an intracoronal barrier.

The purpose of this in vitro study was to evaluate quantitatively the effectiveness of three different restorative materials used as an intracoronal barrier to prevent microleakage of endodontically treated teeth. Fifty-five extracted human single-canal teeth were used in this study. The teeth were endodontically prepared and obturated. Forty-five teeth were randomly assigned to three experimental groups: group 1: sealed with Ketac-Cem (3M ESPE, St Paul, MN) (n = 15), group 2: sealed with Clearfil Protect Bond/Clearfil AP-X (Kuraray, New York, NY) (n = 15), and group 3: sealed with Maxcem (Kerr, Orange, CA) (n = 15). Ten teeth were also randomly assigned to a negative control group (n = 5) and a positive control group (n = 5). Microleakage was tested by using a sterile two-chamber bacterial method and Enterococcus faecalis was used as a microbial marker. Samples were incubated aerobically at 37 degrees C for 120 days. Bacterial leakage was determined by change in turbidity in the medium. Statistical analysis was performed using a Wald chi-square test. No significant difference (p > 0.05) in bacterial leakage was found between the three experimental groups tested. All positive controls leaked within 60 days and broth of the negative control group remained clear throughout the entire experimental period.

Detection and eradication of microorganisms in root-filled teeth associated with periradicular lesions: an in vivo study.

This study determined the presence of microorganisms by culture and polymerase chain reaction in asymptomatic root-filled teeth with periradicular lesions. Furthermore, a disinfecting regimen using sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), chlorhexidine digluconate (CHX) irrigation, and calcium hydroxide (Ca(OH)(2)) dressing was assessed. After removal of the root-filling material, specimens of 20 cases undergoing retreatment were sampled. Moreover, the canals were sampled after each step of the disinfecting regimen. Prevalence of microorganisms was 60% by culture and 65% by polymerase chain reaction. In four of those samples (31%), DNA of Enterococcus faecalis was found. After further root canal preparation and irrigation using NaOCl and EDTA, microorganisms could be detected in none of the teeth. Thus, CHX and Ca(OH)(2) could not show further disinfection. In contrast, microorganisms were found in two teeth after the interappointment dressing. It may be concluded that proper root canal preparation and irrigation using NaOCl and EDTA are sufficient for decontamination of the root canal system during endodontic retreatment.

Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol.

The present clinical study was conducted to assess the bacterial reduction after chemomechanical preparation using 0.12% chlorhexidine digluconate solution as an irrigant and the additive antibacterial effect of intracanal dressing with calcium hydroxide (Ca(OH)(2)) associated with 0.12% chlorhexidine digluconate gel. According to stringent inclusion/exclusion criteria, 13 teeth with primary intraradicular infections and chronic apical periodontitis were selected and followed in the study. Bacterial samples were taken at the baseline (before treatment) (S1), after chemomechanical preparation using chlorhexidine (CHX) as an irrigant (S2), and after a 7-day dressing with Ca(OH)(2)/CHX paste (S3). Cultivable bacteria recovered from infected root canals at the three stages were counted and identified by means of 16S ribosomal RNA gene sequencing analysis. At S1, all canals were positive for bacteria, with the mean number of 3.5 taxa per canal (range, 2-9). At S2, 7 cases (53.8%) still harbored cultivable bacteria, with a mean number of 1.7 taxon per canal (range, 1-4). At S3, only one case (7.7%) was positive for the presence of bacteria. The great majority of taxa found in posttreatment samples were gram-positive bacteria. A significantly high reduction in bacterial counts was observed between S1 and S2 and S1 and S3 (p<0.001). Also, significant differences were observed for comparisons involving S2 and S3 samples with regard to both quantitative bacterial reduction (p=0.014) and number of cases yielding negative cultures (p=0.01). It was concluded that chemomechanical preparation with 0.12% CHX solution as an irrigant significantly reduced the number of intracanal bacteria but failed to render the canal free of cultivable bacteria in about one half of the cases. Application of a 7-day intracanal dressing with Ca(OH)(2)/CHX paste further increased significantly the number of cases yielding negative cultures.

A big role for the very small--understanding the endodontic microbial flora.

Apical periodontitis, an inflammatory process around the apex of a tooth root, is primarily a sequel to microbial infection of the pulp space. The microbial flora is composed of a restricted group of the total oral flora, selected by environmental pressures of anaerobiosis, nutrition and competition with other species and inhabits the root canal as a biofilm of coaggregated communities in an extracellular matrix. The untreated infected canal is generally composed of a polymicrobial mix with approximately equal proportions of Gram-positive and Gram-negative species, dominated by obligate anaerobes. The type of microbial flora in the root-filled tooth with persistent apical periodontitis has very different characteristics. These infections are characterized by one or just a few species, predominantly Gram-positive micro-organisms with an equal distribution of facultative and obligate anaerobes. Enterococcus faecalis has been a conspicuous finding in most studies. Because the primary aetiological problem is infection, endodontic treatment is directed at control and elimination of the root canal flora by working in a sterile way. Based on current knowledge, the best available method for obtaining clean, microbe-free root canals is by instrumentation with antimicrobial irrigation reinforced by an intracanal dressing with calcium hydroxide.

Influence of the Apical Preparation Size and the Irrigant Type on Bacterial Reduction in Root Canal-treated Teeth with Apical Periodontitis.

INTRODUCTION: This clinical study evaluated the influence of the apical preparation size using nickel-titanium rotary instrumentation and the effect of a disinfectant on bacterial reduction in root canal-treated teeth with apical periodontitis. METHODS: Forty-three teeth with posttreatment apical periodontitis were selected for retreatment. Teeth were randomly divided into 2 groups according to the irrigant used (2.5% sodium hypochlorite [NaOCl], n = 22; saline, n = 21). Canals were prepared with the Twisted File Adaptive (TFA) system (SybronEndo, Orange, CA). Bacteriological samples were taken before preparation (S1), after using the first instrument (S2), and then after the third instrument of the TFA system (S3). In the saline group, an additional sample was taken after final irrigation with 1% NaOCl (S4). DNA was extracted from the clinical samples and subjected to quantitative real-time polymerase chain reaction to evaluate the levels of total bacteria and streptococci. RESULTS: S1 from all teeth were positive for bacteria. Preparation to the first and third instruments from the TFA system showed a highly significant intracanal bacterial reduction regardless of the irrigant (P < .01). Apical enlargement to the third instrument caused a significantly higher decrease in bacterial counts than the first instrument (P < .01). Intergroup comparison revealed no significant difference between NaOCl and saline after the first instrument (P > .05). NaOCl was significantly better than saline after using the largest instrument in the series (P < .01). CONCLUSIONS: Irrespective of the type of irrigant, an increase in the apical preparation size significantly enhanced root canal disinfection. The disinfecting benefit of NaOCl over saline was significant at large apical preparation sizes.

Diversity of Enterococcus faecalis Genotypes from Multiple Oral Sites Associated with Endodontic Failure Using Repetitive Sequence-based Polymerase Chain Reaction and Arbitrarily Primed Polymerase Chain Reaction.

INTRODUCTION: The aim of this study was to evaluate the diversity and similarity of Enterococcus faecalis genotype isolates from multiple oral sites using repetitive sequence-based polymerase chain reaction and arbitrarily primed polymerase chain reaction (AP-PCR). METHODS: Forty-two endodontically treated teeth with apical periodontitis were selected. A total of 126 microbial samples were collected from 3 different sites (saliva, pulp chamber, and root canals, all n = 42) during the nonsurgical retreatment procedures. After growth on m-Enterococcus agar, the colonies were isolated, characterized as gram-positive catalase negative cocci, and identified using an API 20 Strep kit (bioMérieux, Marcy-l'Etoile, France). Seventy-four colonies from 10 patients were confirmed as E. faecalis by polymerase chain reaction (16S ribosomal RNA). Repetitive sequence-based polymerase chain reactions using ERIC and AP-PCR using RW3A primers were performed in all 74 colonies. Fingerprints were analyzed and separated into genotypic groups based on the Dice coefficient percentage of similarity (82% or greater) as determined by ERIC reproducibility assays involving E. faecalis controls. RESULTS: Seven different E. faecalis genotypes (GTs) (GT1 = 27%, GT2 = 17.6%, GT3 = 1.3%, GT4 = 18.9%, GT5 = 9.5%, GT6 = 14.9%, and GT7 = 10.8%) were observed in different subjects and oral sites associated with endodontic failure. Remarkably, in 4 of 5 patients, the same GTs present in the infected root canals were also isolated from either the pulp chamber or the saliva samples. In particular, GT6 was detected in all 3 oral sites of patient 37. CONCLUSIONS: E. faecalis GTs isolated from saliva, the pulp chamber, and the root canal were similar using the Rep-PCR and AP-PCR methods. These findings suggest that coronal microleakage is a conceivable cause of endodontic failure.