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.

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.

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.

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.

Extraradicular infection as the cause of persistent symptoms: a case series.

INTRODUCTION: This article describes 3 cases that presented persistent symptoms after appropriate endodontic treatment. Histopathologic and histobacteriologic investigation were conducted for determination of the cause. METHODS: Three cases are reported that presented with persistent symptoms after endodontic retreatment (cases 1 and 2) or treatment (case 3). Periapical surgery was indicated and performed in these cases. The biopsy specimens, consisting of root apices and the apical periodontitis lesions, were subjected to histopathologic and histobacteriologic analyses. RESULTS: Case 1 was an apical cyst with necrotic debris, heavily colonized by ramifying bacteria, in the lumen. No bacteria were found in the apical root canal system. Case 2 was a granuloma displaying numerous bacterial aggregations through the inflammatory tissue. Infection was also present in the dentinal tubules at the apical root canal. Case 3 was a cyst with bacterial colonies floating in its lumen; bacterial biofilms were also seen on the external apical root surface, filling a large lateral canal and other apical ramifications, and between layers of cementum detached from the root surface. No bacteria were detected in the main root canal. CONCLUSIONS: Different forms of extraradicular infection were associated with symptoms in these cases, leading to short-term endodontic failure only solved by periapical surgery.

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.

Microbial diversity in persistent root canal infections investigated by checkerboard DNA-DNA hybridization.

INTRODUCTION: The aim of the present study was to investigate the composition of the root canal microbiota in endodontic failures in order to identify and quantify these microorganisms. METHODS: Microbiological samples were taken from 36 root canals with persistent endodontic infection. The presence, levels, and proportions of 79 bacterial species were determined by checkerboard DNA-DNA hybridization. The Pearson correlation coefficient was used to investigate the relations between bacterial counts and clinical conditions (P ≤ .05). RESULTS: Enterococcus faecium (36%), Streptococcus epidermidis (36%), Eubacterium saburreum (28%), Parvimonas micra (28%), Streptococcus sanguis (28%), Capnocytophaga sputigena (28%), Leptotrichia buccalis (28%), Enterococcus faecalis (28%), and Staphylococcus warneri (28%) were the most prevalent species; and there was a low prevalence of Treponema socranskii (3%), Fusobacterium periodonticum (3%), Capnocytophaga gingivalis (3%), and Spiroplasma ixodetis (3%). The highest mean levels were found for the following species: E. faecium, Dialister pneumosintes, Staphylococcus epidermidis and Helicobacter pylori. There was a statistically significant difference between the levels of gram-negative species and gram-positive species (13.5 × 10(5) vs 6.5 × 10(5), respectively). A positive correlation was found between the area of the periapical lesion and the levels of gram-negative and rod species (P < .05). CONCLUSIONS: The microbiota from teeth with persistent apical periodontitis presents a mixed and complex profile, hosting E. faecium and S. epidermidis as the most highly prevalent species. No correlation was found between any of the species tested and clinical findings; however, periapical lesions with the largest areas presented higher counts of gram-negative and rod species.

Periapical inflammation subsequent to coronal inoculation of dog teeth root filled with resilon/epiphany in 1 or 2 treatment sessions with chlorhexidine medication.

INTRODUCTION: Therapeutic methods that inhibit microbial ingress into filled root canals are desirable. This in vivo study assessed the inhibition of periapical inflammation subsequent to coronal inoculation in canals medicated with 2% chlorhexidine gel and filled with Resilon/Epiphany (Pentron Clinical Technologies, Wallingford, CT). METHODS: Six Beagle dogs each had 10 two-rooted premolars treated. In group 1 (n = 36 roots), 1 root/tooth had the canal conditioned with Primer Epiphany, filled with Epiphany sealer and Resilon core in 1 session, and coronally sealed with PhotacFil. In group 2 (n = 36 roots), the second root/tooth had the canal medicated with 2% chlorhexidine gel for 1 week and then filled and coronally sealed as in group 1. After 3 weeks, canals were exposed to the oral environment for 7 days, inoculated with isologous plaque, and coronally sealed. Negative controls treated as groups 1 and 2 remained sealed. Positive controls had canals unfilled and exposed. Seven months after inoculation, dogs were euthanized; jaw blocks processed for histologic examination; and periapical inflammation (PI) recorded as none, mild, or severe. RESULTS: In groups 1 and 2, severe PI occurred in 5 of 65 roots (8%) and mild PI in 18 of 65 roots (28%) with a significantly higher (P = .031) PI incidence in group 2 than in group 1. Negative controls had only mild PI in 9 of 29 roots (31%). Roots medicated with 2% chlorhexidine gel had mild PI significantly more (P = .009) than roots filled in 1 session (more than 2-fold). CONCLUSIONS: Intracanal medication with 2% chlorhexidine gel and root filling with Resilon/Epiphany did not effectively inhibit apical periodontitis subsequent to coronal inoculation.

New bacterial composition in primary and persistent/secondary endodontic infections with respect to clinical and radiographic findings.

INTRODUCTION: The aim of the present study was to analyze the microbiota of primary and secondary/persistent endodontic infections of patients undergoing endodontic treatment with respect to clinical and radiographic findings. METHODS: Samples from the root canals of 21 German patients were taken using 3 sequential sterile paper points. In the case of a root canal filling, gutta-percha was removed with sterile files, and samples were taken using sterile paper points. The samples were plated, and microorganisms were then isolated and identified morphologically by biochemical analysis and sequencing the 16S rRNA genes of isolated microorganisms. RESULTS: In 12 of 21 root canals, 33 different species could be isolated. Six (50%) of the cases with isolated microorganisms were primary, and 6 (50%) cases were endodontic infections associated with root-filled teeth. Twelve of the isolated species were facultative anaerobic and 21 obligate anaerobic. Monomicrobial infections were found for Enterococcus faecalis and Actinomyces viscosus. E. faecalis was most frequently isolated in secondary endodontic infections (33%). Moraxella osloensis was isolated from a secondary endodontic infection that had an insufficient root canal filling accompanied by a mild sensation of pain. A new bacterial composition compromising Atopobium rimae, Anaerococcus prevotii, Pseudoramibacter alactolyticus, Dialister invisus, and Fusobacterium nucleatum was recovered from teeth with chronic apical abscesses. CONCLUSIONS: New bacterial combinations were found and correlated to clinical and radiographic findings, particularly to chronic apical abscesses. M. osloensis was detected in root canals for the second time and only in German patients.

Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth.

INTRODUCTION: To date, a variety of microbial species have been isolated from endodontic infections. However, endodontic clinical bacterial isolates have not been sufficiently characterized with regard to their capacity for antibiotic resistance and biofilm formation. In this study, antibiotic resistance and biofilm formation of 47 different aerobic and anaerobic bacterial isolates, belonging to 32 different species previously isolated from infected filled root canals, were studied. METHODS: Antibiotic sensitivity to 11 antibiotics including penicillin G, amoxicillin, clindamycin, gentamicin, vancomycin, tetracycline, doxycycline, fosfomycin, rifampicin, ciprofloxacin, and moxifloxacin was tested using the standardized Etest method (Bio Merieux, Marcy-1'Etoile, France). The antibiotic sensitivity of 4 control strains was also estimated in parallel. Additionally, the capacity to form biofilms was quantified using the microtiter plate test. RESULTS: Different aerobic and anaerobic bacterial species were either resistant against a number of antibiotics or showed high minimal inhibitory concentrations against clinically relevant antibiotics. Five aerobic and 2 anaerobic isolates, including Enterococcus faecalis, Streptococcus mutans, Lactobacillus fermentum, Actinomyces naeslundii, Actinomyces viscosus, Prevotella buccae, and Propionibacterium acidifaciens, were characterized as being high biofilm producers, whereas 8 aerobic and 3 anaerobic isolates were found to be moderate biofilm producers. Most isolates with resistance or markedly high minimal inhibitory concentration values were also either moderate biofilm producers or high biofilm producers. CONCLUSIONS: These results suggest that the clinical significance of endodontic infections could include that they serve as a reservoir for antibiotic resistance. Furthermore, endodontic treatment should consider the adhesion and biofilm formation by a variety of bacteria.

Analysis of the secondary endodontic lesions focusing on the extraradicular microorganisms: an overview.

The present study aimed at reviewing the literature on extraradicular infections of endodontically treated teeth, summarizing the main hypotheses on etiopathogenesis and describing the most suitable techniques to identify the composition of pathogenic extraradicular microorganisms. Medline database was searched using the keywords "Apical biofilm," "extraradicular infection," "secondary endodontic lesion," "endodontic retreatment," "biofilm" either alone or combined with AND. A further hand search was performed on the main endodontic journals. The most frequent bacterial species identified in different studies and with different techniques may vary considerably. Although the presence of some species of microorganisms seems to be determinant, the true origin of extraradicular infection is still undetermined. The literature analysis showed marked differences in methodology, materials, aims, and techniques adopted, which led to highly heterogeneous outcomes. The picture emerging from this review is that extraradicular infection is likely a multifactorial disease that requires further systematic investigation using standardized techniques.

Coronal microleakage of endodontically treated teeth with intracanal post exposed to fresh human saliva.

OBJECTIVE: The aim of this study was to investigate the coronal microleakage of endodontically treated teeth prepared to receive an intracanal post and teeth with an intracanal post but without a prosthetic crown and exposed to contamination by fresh human saliva. MATERIAL AND METHODS: A mechanical-chemical preparation following the step-back technique was carried out in 35 extracted single-rooted human teeth. The teeth were randomly divided into five groups: G1=root canals instrumented, obturated, and prepared to receive an intracanal post (N=10); G2=root canals with cemented posts but without coronal sealing (N=10); PC1=positive control root canals instrumented and open (N=5); PC2=positive control 2 root canals without instrumentation and open (N=5); and NC=negative control healthy teeth (N=5). The crowns were removed except for the control group of intact teeth. The root canals were obturated and sterilized with cobalt 60 gamma irradiation and were then adapted in an apparatus using a Brain Heart Infusion (BHI) medium and fresh human saliva for contamination. Microbial growth was indicated by the presence of turbidity in the BHI liquid medium. RESULTS: Data were submitted to the Kaplan-Meier Survival Analysis and the Holm-Sidak statistic method, which observed an index of 90% of microleakage in root canals after 24 hours for G1 and 70% of microleakage in samples at the end of 40 days for G2. CONCLUSION: The results show that root canals with an intracanal post but without a prosthetic crown can be recontaminated when exposed to fresh human saliva in a short period.

Investigation of cultivable bacteria isolated from longstanding retreatment-resistant lesions of teeth with apical periodontitis.

INTRODUCTION: The objective of this research was to investigate the presence of viable bacteria in tissue samples from persistent apical lesions and to correlate the microbiological findings with the histopathological diagnosis of the lesion. METHODS: Twenty persistent apical lesions associated with well-performed endodontic retreatment were collected. Tissue samples were processed through culture techniques including serial dilution, plating, aerobic and anaerobic incubation, and biochemical tests for microbial identification followed by histopathological diagnosis. RESULTS: Cysts were more frequently diagnosed (13/20). Strict anaerobic species predominated in both cysts (80.4% of the species detected) and granulomas (65% of the species detected). Viable gram-positive bacteria were frequently recovered from apical lesions (cysts = 70.6%, granulomas = 84.4%). Gemella morbillorum and Propionibacterium acnes were the most frequently recovered species from cysts and granulomas, respectively. At least 1 gram-positive bacterial species was present in almost every sample (cysts = 12/13, granulomas = 7/7). No significant correlation was found between histologic findings and bacterial species. CONCLUSIONS: In conclusion, although cysts were more frequent than granulomas in cases of failure of endodontic retreatment, bacteria were isolated from both types of lesions, with a predominance of gram-positive species, suggesting that these species can survive outside the root canal and might be related to the persistence of the pathological process even after accurate endodontic retreatment.

Coronal microleakage of endodontically treated teeth with intracanal post exposed to fresh human saliva

OBJECTIVE: The aim of this study was to investigate the coronal microleakage of endodontically treated teeth prepared to receive an intracanal post and teeth with an intracanal post but without a prosthetic crown and exposed to contamination by fresh human saliva. MATERIAL AND METHODS: A mechanical-chemical preparation following the step-back technique was carried out in 35 extracted single-rooted human teeth. The teeth were randomly divided into five groups: G1=root canals instrumented, obturated, and prepared to receive an intracanal post (N=10); G2=root canals with cemented posts but without coronal sealing (N=10); PC1=positive control root canals instrumented and open (N=5); PC2=positive control 2 root canals without instrumentation and open (N=5); and NC=negative control healthy teeth (N=5). The crowns were removed except for the control group of intact teeth. The root canals were obturated and sterilized with cobalt 60 gamma irradiation and were then adapted in an apparatus using a Brain Heart Infusion (BHI) medium and fresh human saliva for contamination. Microbial growth was indicated by the presence of turbidity in the BHI liquid medium. RESULTS: Data were submitted to the Kaplan-Meier Survival Analysis and the Holm-Sidak statistic method, which observed an index of 90% of microleakage in root canals after 24 hours for G1 and 70% of microleakage in samples at the end of 40 days for G2. CONCLUSION: The results show that root canals with an intracanal post but without a prosthetic crown can be recontaminated when exposed to fresh human saliva in a short period. .

Infection in a complex network of apical ramifications as the cause of persistent apical periodontitis: a case report.

INTRODUCTION: This article reports a case of persistent apical periodontitis lesion in a mesiobuccal root of a maxillary molar subjected to single-visit endodontic treatment. METHODS: The treatment protocol followed endodontic standards including using nickel-titanium instruments with working length ending 0.5-mm short of the apex, establishment and maintenance of apical foramen patency, irrigation with 5% NaOCl, smear layer removal, a final rinse with and ultrasonic agitation of chlorhexidine, and filling by the vertical compaction technique. Even so, the lesion in the mesiobuccal root became larger in size after follow-up examination at 1 year 6 months, and periradicular surgery was performed. Radiographic control after 11 months showed that periradicular healing was almost complete. The root apex and the lesion were analyzed histologically and histobacteriologically. RESULTS: The lesion was diagnosed as a "pocket cyst," and no bacteria were noted extraradicularly. The cause of continued disease was a heavy bacterial biofilm infection located in an intricate network of apical ramifications. Bacteria were also observed on the walls of one of the mesiobuccal canals packed between the obturation material and the root canal wall. CONCLUSIONS: This case report reinforces the need for treating the infected root canal as a complex system that possesses anatomic intricacies in which bacteria can spread and remain unaffected by treatment procedures.

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.

Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections.

INTRODUCTION: Enterococcus faecalis is a member of the mammalian gastrointestinal microbiota but has been considered a leading cause of hospital-acquired infections. In the oral cavity, it is commonly detected from root canals of teeth with failed endodontic treatment. However, little is known about the virulence and genetic relatedness among E. faecalis isolates from different clinical sources. This study compared the presence of enterococcal virulence factors among root canal strains and clinical isolates from hospitalized patients to identify virulent clusters of E. faecalis. METHODS: Multilocus sequence typing analysis was used to determine genetic lineages of 40 E. faecalis clinical isolates from different sources. Virulence clusters were determined by evaluating capsule (cps) locus polymorphisms, pathogenicity island gene content, and antibiotic resistance genes by polymerase chain reaction. RESULTS: The clinical isolates from hospitalized patients formed a phylogenetically separate group and were mostly grouped in the clonal complex 2, which is a known virulent cluster of E. faecalis that has caused infection outbreaks globally. The clonal complex 2 group comprised capsule-producing strains harboring multiple antibiotic resistance and pathogenicity island genes. On the other hand, the endodontic isolates were more diverse and harbored few virulence and antibiotic resistance genes. In particular, although more closely related to isolates from hospitalized patients, capsule-producing E. faecalis strains from root canals did not carry more virulence/antibiotic genes than other endodontic isolates. CONCLUSIONS: E. faecalis isolates from endodontic infections have a genetic and virulence profile different from pathogenic clusters of hospitalized patients' isolates, which is most likely due to niche specialization conferred mainly by variable regions in the genome.

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.

Microbial diversity in failed endodontic root-filled teeth.

BACKGROUND: Persistent/secondary infections of human root canals play an important role in the failure of endodontic treatment. This study used 16S rRNA sequencing to assess microbial diversity in root-filled teeth associated with failed endodontic treatment. METHODS: DNA was extracted from 15 teeth with persistent intraradicular infections, and the 16S rRNA of all present bacteria were amplified by PCR, followed by cloning and sequencing of the 16S rRNA amplicons. RESULTS: All sample extracts were positive for PCR amplification using the universal 16S rRNA gene primers. Negative control reactions yielded no amplicons. Sixty-five phylotypes belonging to seven phyla were identified from 760 clones; a mean of 9.4 phylotypes were detected in each sample (range 3 - 15). Twenty-eight phylotypes were detected in more than one sample, revealing a high inter-sample variability. Parvimonas micra (60%, 9/15), Solobacterium moore (47%, 7/15), Dialister invisus (33%, 5/15), Enterococcus faecalis (33%, 5/15), Filifactor alocis (27%, 4/15), and Fusobacterium nucleatum (27%, 4/15) were the prevalent species. Nineteen as-yet-uncultivated phylotypes were identified, comprising a substantial proportion of the bacteria in many cases. CONCLUSIONS: Persistent intraradicular infections were present in all root-filled teeth associated with failed endodontic treatment. The current observations reveal new candidate endodontic pathogens, including as-yet-uncultivated bacteria and phylotypes that may participate in the mixed infections associated with post-treatment apical periodontitis.