Surveying bovine digital dermatitis and non-healing bovine foot lesions for the presence of Fusobacterium necrophorum, Porphyromonas endodontalis and Treponema pallidum.

BACKGROUND: Non-healing bovine foot lesions, including non-healing white line disease, non-healing sole ulcer and toe necrosis, are an increasingly important cause of chronic lameness that are poorly responsive to treatment. Recent studies have demonstrated a high-level association between these non-healing lesions and the Treponema phylogroups implicated in bovine digital dermatitis (BDD). However, a polymicrobial aetiology involving other gram-stain-negative anaerobes is suspected. METHODS: A PCR-based bacteriological survey of uncomplicated BDD lesions (n=10) and non-healing bovine foot lesions (n=10) targeting Fusobacterium necrophorum, Porphyromonas endodontalis, Dichelobacter nodosus and Treponema pallidum/T. paraluiscuniculi was performed. RESULTS: P. endodontalis DNA was detected in 80.0% of the non-healing lesion biopsies (p=<0.001) but was entirely absent from uncomplicated BDD lesion biopsies. When compared to the BDD lesions, F. necrophorum was detected at a higher frequency in the non-healing lesions (33.3% vs 70.0%, respectively), whereas D. nodosus was detected at a lower frequency (55.5% vs 20.0%, respectively). Conversely, T. pallidum/T. paraluiscuniculi DNA was not detected in either lesion type. CONCLUSION: The data from this pilot study suggest that P. endodontalis and F. necrophorum should be further investigated as potential aetiological agents of non-healing bovine foot lesions. A failure to detect syphilis treponemes in either lesion type is reassuring given the potential public health implications such an infection would present.

Evaluation of Bacteriological Profile in the Apical Root Segment of the Patients with Primary Apical Periodontitis.

INTRODUCTION: Apical periodontitis usually results from bacterial accumulation and contamination occurring in the root-canal system, and extending beyond the apical foramen to involve the periapical tissues. Literature has a paucity of the studies that stress on the division and analysis of the pulp canal segments. The reason for this disparity might be the technique used for collecting the samples from the pulp canals. Hence, we carried out the present study to evaluate the microbial flora in the apical part of the roots with necrotic pulp canals. MATERIALS AND METHODS: The present study included the assessment of 40 freshly extracted teeth that had necrotized pulpal tissue along with the presence of periapical periodontal lesions. Removal of the soft tissue lesions attached to the root portion of the teeth along with apical periodontal lesions was done with the help of scalpel blade, after rinsing them with a sterile solution of saline. Thorough cleaning of the root surfaces was done with hydrogen peroxide followed by rapid disinfection with the help of sodium hypochlorite at varying concentrations. Sectioning of the root portion of all the specimens with the help of a disk was done perpendicular to the long axis of the teeth at a distance of roughly 5 to 6 mm from the teeth's apicalmost point. Cryotubes were used for transferring the specimens of apical portions containing 1 mL of buffer and were subjected to immediate frozen processing at a temperature of -20°C. A 10 K-type file was used for the initial collection of the samples followed by subsequent incubation of the files and paper pints in the incubation cabinet. Subsequent deoxyribonucleic acid (DNA) extraction from the samples was done following the procedure described by Siqueira et al. Paster et al's modification of the reverse-capture checkerboard assay was used in the present study. Semiquantitative data were used for overcoming the difficulties arising due to obtaining the counts of the polymerase chain reaction (PCR)-based analysis of specimens. RESULTS: A positive result for the 16S ribosomal ribonucleic acid (rRNA) gene primer was observed only in two examined specimens of all the samples of the apical portion of the root canals in the present study. Negative result was shown by all the control group specimens, which were sterile samples. Presence of bacteria was confirmed by PCR in 38 out of 40 examined specimens. Amount of bacterial taxa, out of these 24 samples, ranged up to 6. Pseudoramibacter alactolyticus, Porphyromonas endodontalis, Dialister oral species, Bacteroidetes species, Streptococcus species, Olsenella uli, Synergistes species, Fusobacterium nucleatum, Parvimonas micra, Treponema denticola, and Filifactor alocis were the specific species detected. Bacteroidetes species was the only species that were detected at levels at or above 105. Heavy bacterial infections were noticed in more than 45% of the cases at the periradicular part of the root canals. CONCLUSION: Microbial flora of the apical segment of the root with necrotized pulp tissue comprises a vast variety of pathogenic bacteria. CLINICAL SIGNIFICANCE: For better prognosis of the treatment of such cases, adequate knowledge of the microbial flora of the root, especially the apical portion is necessary.

Association of Endodontic Lesions with Coronary Artery Disease.

An endodontic lesion (EL) is a common manifestation of endodontic infection where Porphyromonas endodontalis is frequently encountered. EL may associate with increased risk for coronary artery disease (CAD) via similar pathways as marginal periodontitis. The aim of this cross-sectional study was to delineate the associations between EL and CAD. Subgingival P. endodontalis, its immune response, and serum lipopolysaccharide were examined as potential mediators between these 2 diseases. The Finnish Parogene study consists of 508 patients (mean age, 62 y) who underwent coronary angiography and extensive clinical and radiographic oral examination. The cardiovascular outcomes included no significant CAD ( n = 123), stable CAD ( n = 184), and acute coronary syndrome (ACS; n = 169). EL was determined from a panoramic tomography. We combined data of widened periapical spaces (WPSs) and apical rarefactions to a score of EL: 1, no EL ( n = 210); 2, ≥1 WPS per 1 apical rarefaction ( n = 222); 3, ≥2 apical rarefactions ( n = 76). Subgingival P. endodontalis was defined by checkerboard DNA-DNA hybridization analysis, and corresponding serum antibodies were determined by ELISA. In our population, 50.4% had WPSs, and 22.8% apical rarefactions. A total of 51.2% of all teeth with apical rarefactions had received endodontic procedures. Subgingival P. endodontalis levels and serum immunoglobulin G were associated with a higher EL score. In the multiadjusted model (age, sex, smoking, diabetes, body mass index, alveolar bone loss, and number of teeth), having WPSs associated with stable CAD (odds ratio [OR] = 1.94, 95% confidence interval [95% CI] = 1.13 to 3.32, P = 0.016) and highest EL score were associated with ACS (OR = 2.46, 95% CI = 1.09 to 5.54, P = 0.030). This association was especially notable in subjects with untreated teeth with apical rarefactions ( n = 59, OR = 2.72, 95% CI = 1.16 to 6.40, P = 0.022). Our findings support the hypothesis that ELs are independently associated with CAD and in particular with ACS. This is of high interest from a public health perspective, considering the high prevalence of ELs and CAD.

Association of three putative periodontal pathogens with chronic periodontitis in Brazilian subjects.

Objective The aim of this study was to evaluate the association of Porphyromonas endodontalis, Filifactor alocis and Dialister pneumosintes with the occurrence of periodontitis. Material and Methods Thirty subjects with chronic periodontitis (ChP) and 10 with periodontal health (PH) were included in the study. Nine subgingival biofilm samples were collected as follows: i) PH group - from the mesial/buccal aspect of each tooth in two randomly chosen contralateral quadrants; ii) ChP group - from three sites in each of the following probing depth (PD) categories: shallow (≤3 mm), moderate (4-6 mm) and deep (≥7 mm). Checkerboard DNA-DNA hybridization was used to analyze the samples. Results We found the three species evaluated in a higher percentage of sites and at higher levels in the group with ChP than in the PH group (p<0.05, Mann-Whitney test). We also observed these differences when the samples from sites with PD≤4 mm or ≥5 mm of subjects with ChP were compared with those from subjects with PH (p<0.05, Mann-Whitney test). In addition, the prevalence and levels of D. pneumosintes, and especially of F. alocis were very low in healthy subjects (0.12x105 and 0.01x105, respectively). Conclusion F. alocis and D. pneumosintes might be associated with the etiology of ChP, and their role in the onset and progression of this infection should be further investigated. The role of P. endodontalis was less evident, since this species was found in relatively high levels and prevalence in the PH group.

Association of three putative periodontal pathogens with chronic periodontitis in Brazilian subjects

ABSTRACT Objective The aim of this study was to evaluate the association of Porphyromonas endodontalis, Filifactor alocis and Dialister pneumosintes with the occurrence of periodontitis. Material and Methods Thirty subjects with chronic periodontitis (ChP) and 10 with periodontal health (PH) were included in the study. Nine subgingival biofilm samples were collected as follows: i) PH group - from the mesial/buccal aspect of each tooth in two randomly chosen contralateral quadrants; ii) ChP group - from three sites in each of the following probing depth (PD) categories: shallow (≤3 mm), moderate (4-6 mm) and deep (≥7 mm). Checkerboard DNA-DNA hybridization was used to analyze the samples. Results We found the three species evaluated in a higher percentage of sites and at higher levels in the group with ChP than in the PH group (p<0.05, Mann-Whitney test). We also observed these differences when the samples from sites with PD≤4 mm or ≥5 mm of subjects with ChP were compared with those from subjects with PH (p<0.05, Mann-Whitney test). In addition, the prevalence and levels of D. pneumosintes, and especially of F. alocis were very low in healthy subjects (0.12x105 and 0.01x105, respectively). Conclusion F. alocis and D. pneumosintes might be associated with the etiology of ChP, and their role in the onset and progression of this infection should be further investigated. The role of P. endodontalis was less evident, since this species was found in relatively high levels and prevalence in the PH group.

Combinations of bacterial species associated with symptomatic endodontic infections in a Chinese population.

AIM: To use microarrays to detect 11 selected bacteria in infected root canals, revealing bacterial combinations that are associated with clinical symptoms and signs of primary endodontic infections in a Chinese population. METHODOLOGY: DNA was extracted from 90 samples collected from the root canals of teeth with primary endodontic infections in a Chinese population, and the 16S rRNA gene was amplified by polymerase chain reaction (PCR). The PCR products were hybridized to microarrays containing specific oligonucleotide probes targeting 11 species, and the arrays were screened with a confocal laser scanner. Pearson's chi-squared test and cluster analysis were performed to investigate the associations between the bacterial combinations and clinical symptoms and signs using SAS 8.02. RESULTS: Seventy-seven samples (86%) yielded at least one of the 11 target species. Parvimonas micra (56%), Porphyromonas endodontalis (51%), Tannerella forsythia (48%), Prevotella intermedia (44%) and Porphyromonas gingivalis (37%) were the most prevalent taxa and were often concomitant. The following positive associations were found between the bacterial combinations and clinical features: P. endodontalis and T. forsythia with abscess; P. gingivalis and P. micra with sinus tract; P. gingivalis and P. endodontalis or P. micra and P. endodontalis with abscess and sinus tract; and the combination of P. endodontalis, P. micra, T. forsythia and P. gingivalis with sinus tract (P < 0.05). CONCLUSIONS: Various combinations of P. micra, P. endodontalis, T. forsythia and P. gingivalis may contribute to abscesses or sinus tracts of endodontic origin with bacterial synergism in a Chinese population.

TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens.

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855).

Proinflammatory Activity of Primarily Infected Endodontic Content against Macrophages after Different Phases of the Root Canal Therapy.

INTRODUCTION: This study investigated the presence of target bacterial species and the levels of endotoxins in teeth with apical periodontitis. Levels of inflammatory mediators (interleukin [IL]-1ß and tumor necrosis factor [TNF]-α) were determined after macrophage stimulation with endodontic content after different phases of endodontic therapy using different irrigants. METHODS: Thirty primarily infected root canals were randomly assigned into 3 groups according to the irrigant used for root canal preparation (n = 10 per group): GI: 2.5% sodium hypochlorite, GII: 2% chlorhexidine gel, and GIII (control group): saline solution. Root canal samples were taken by using paper points before (s1) and after root canal instrumentation (s2), subsequently to 17% EDTA (s3), after 30 days of intracanal medication (Ca[OH]2 + saline solution) (s4), and before root canal obturation (s5). Polymerase chain reaction (16S recombinant DNA) and limulus amebocyte lysate assay were used for bacterial and endotoxin detection, respectively. Macrophages were stimulated with the root canal contents for IL-1ß/TNF-α measurement using enzyme-linked immunosorbent assay. RESULTS: Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30), and Prevotella nigrescens (11/30) were the most prevalent bacterial species. At s1, endotoxins were detected in 100% of the root canals (median = 32.43 EU/mL). In parallel, substantial amounts of IL-1ß and TNF-α were produced by endodontic content-stimulated macrophages. At s2, a significant reduction in endotoxin levels was observed in all groups, with GI presenting the greatest reduction (P < .05). After a root canal rinse with EDTA (s3), intracanal medication (s4), and before root canal obturation (s5), endotoxin levels reduced without differences between groups (P < .05). IL-1ß and TNF-α release decreased proportionally to the levels of residual endotoxin (P < .05). CONCLUSIONS: Regardless of the use of sodium hypochlorite or CHX, the greatest endotoxin reduction occurs after chemomechanical preparation. Increasing steps of root canal therapy associated with intracanal medication enhances endotoxin reduction, leading to a progressively lower activation of proinflammatory cells such as macrophages.

[Study of association between Parvimonas micra and pulp dominant pathogens in the infected root canals with chronic periradicular periodontitis].

OBJECTIVE: To study the prevalence of Parvimonas micra (Pm) and the associations between Pm and pulp dominant pathogens in order to reflect the colonization of Pm in the infected root canals with chronic periradicular periodontitis. METHODS: A total of 120 teeth diagnosed as chronic periradicular periodontitis from 104 patients were included into the study. The teeth were allocated into untreated (primary infectious) and root-canal- treated (secondary infectious) groups with 60 in either group. Samples were collected from the root canals using sterile files and paper points, and subsequent extraction of bacterial DNA was undertaken. The Pm 16S rDNA level was evaluated using 16S rDNA PCR. The prevalence of Pm in chronic periradicular periodontitis was determined accordingly. Then, the associations of Pm and Enterococcus faecalis (Ef), Porphyromonas endodontalis (Pe) as well as Porphyromonas gingivalis (Pg) were analysed. RESULTS: Pm was detected in 40% (24/60) of the samples from the primary infectious group, 5% (3/60) from the secondary infectious group. The prevalences of Pm from the two groups were different significantly (χ² = 21.06, P < 0.05). Significant correlations (untreated group OR = 5.98, root-canal-treated group OR = 33.50) between Pm and Pe were identified in both groups, while the correlations between Pm and Pg as well as Ef were not of significance, respectively. CONCLUSIONS: A significantly higher relevance ratio of Pm was estimated in the primary infectious group than the secondary infectious one. Pm and Pe were correlated significantly in the infected root canals, suggesting a symbiotic relation between these two bacteria.

Macrophage cell activation with acute apical abscess contents determined by interleukin-1 Beta and tumor necrosis factor alpha production.

INTRODUCTION: This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. METHODS: Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. RESULTS: Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). CONCLUSIONS: AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Microbial signature profiles of periodontally healthy and diseased patients.

AIM: To determine microbial profiles that discriminate periodontal health from different forms of periodontal diseases. METHODS: Subgingival biofilm was obtained from patients with periodontal health (27), gingivitis (11), chronic periodontitis (35) and aggressive periodontitis (24), and analysed for the presence of >250 species/phylotypes using HOMIM. Microbial differences among groups were examined by Mann-Whitney U-test. Regression analyses were performed to determine microbial risk indicators of disease. RESULTS: Putative and potential new periodontal pathogens were more prevalent in subjects with periodontal diseases than periodontal health. Detection of Porphyromonas endodontalis/Porphyromonas spp. (OR 9.5 [1.2-73.1]) and Tannerella forsythia (OR 38.2 [3.2-450.6]), and absence of Neisseria polysaccharea (OR 0.004 [0-0.15]) and Prevotella denticola (OR 0.014 [0-0.49], p < 0.05) were risk indicators of periodontal disease. Presence of Aggregatibacter actinomycetemcomitans (OR 29.4 [3.4-176.5]), Cardiobacterium hominis (OR 14.9 [2.3-98.7]), Peptostreptococcaceae sp. (OR 35.9 [2.7-483.9]), P. alactolyticus (OR 31.3 [2.1-477.2]), and absence of Fretibacterium spp. (OR 0.024 [0.002-0.357]), Fusobacterium naviforme/Fusobacterium nucleatum ss vincentii (OR 0.015 [0.001-0.223]), Granulicatella adiacens/Granulicatella elegans (OR 0.013 [0.001-0.233], p < 0.05) were associated with aggressive periodontitis. CONCLUSION: There were specific microbial signatures of the subgingival biofilm that were able to distinguish between microbiomes of periodontal health and diseases. Such profiles may be used to establish risk of disease.

Microbial evaluation of traumatized teeth treated with triple antibiotic paste or calcium hydroxide with 2% chlorhexidine gel in pulp revascularization.

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.

Signaling pathways activation by primary endodontic infectious contents and production of inflammatory mediators.

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.

Gingival fluid cytokine expression and subgingival bacterial counts during pregnancy and postpartum: a case series.

OBJECTIVES: The aim of this study was to assess gingival fluid (GCF) cytokine messenger RNA (mRNA) levels, subgingival bacteria, and clinical periodontal conditions during a normal pregnancy to postpartum. MATERIALS AND METHODS: Subgingival bacterial samples were analyzed with the checkerboard DNA-DNA hybridization method. GCF samples were assessed with real-time PCR including five proinflammatory cytokines and secretory leukocyte protease inhibitor. RESULTS: Nineteen pregnant women with a mean age of 32 years (S.D. ± 4 years, range 26-42) participated in the study. Full-mouth bleeding scores (BOP) decreased from an average of 41.2% (S.D. ± 18.6%) at the 12th week of pregnancy to 26.6% (S.D. ± 14.4%) at the 4-6 weeks postpartum (p < 0.001). Between week 12 and 4-6 weeks postpartum, the mean probing pocket depth changed from 2.4 mm (S.D. ± 0.4) to 2.3 mm (S.D. ± 0.3) (p = 0.34). Higher counts of Eubacterium saburreum, Parvimonas micra, Selenomonas noxia, and Staphylococcus aureus were found at week 12 of pregnancy than at the 4-6 weeks postpartum examinations (p < 0.001). During and after pregnancy, statistically significant correlations between BOP scores and bacterial counts were observed. BOP scores and GCF levels of selected cytokines were not related to each other and no differences in GCF levels of the cytokines were observed between samples from the 12th week of pregnancy to 4-6 weeks postpartum. Decreasing postpartum counts of Porphyromonas endodontalis and Pseudomonas aeruginosa were associated with decreasing levels of Il-8 and Il-1ß. CONCLUSIONS: BOP decreased after pregnancy without any active periodontal therapy. Associations between bacterial counts and cytokine levels varied greatly in pregnant women with gingivitis and a normal pregnancy outcome. Postpartum associations between GCF cytokines and bacterial counts were more consistent. CLINICAL RELEVANCE: Combined assessments of gingival fluid cytokines and subgingival bacteria may provide important information on host response.

Comparison of endotoxin levels found in primary and secondary endodontic infections.

INTRODUCTION: This clinical study was conducted to compare the levels of endotoxins (lipopolysaccharides [LPSs]) found in primary and secondary endodontic infections with apical periodontitis by correlating LPS contents with clinical/radiographic findings. In addition, the presence of target gram-negative anaerobic bacteria was also investigated. METHODS: Samples were taken from 15 root canals with primary infections and 15 with secondary infections by using paper points. The limulus amebocyte lysate assay was used to quantify endotoxins, and the polymerase chain reaction technique (16S rDNA) was used for bacterial investigation. RESULTS: Endotoxins were detected in 100% of the root canal samples collected from primary (15/15) and secondary (15/15) infections with median values of 7.49 EU/mL and 3.96 EU/mL, respectively (P < .05). The median value of endotoxins found in the presence of clinical symptoms was significantly higher than in asymptomatic teeth with primary infections (P < .05). A positive correlation was found between endotoxin contents and a larger size of the radiolucent area (>3 mm) (P < .05). Prevotella nigrescens (10/15, 4/15), Fusobacterium nucleatum (5/15, 1/15), Treponema denticola (3/15, 1/15), and Treponema socranskii (5/15, 1/15) were detected in teeth with primary and secondary infections, respectively. P. endodontalis was present only in teeth with primary infections (5/15). CONCLUSIONS: Teeth with primary endodontic infections had higher contents of endotoxins and a more complex gram-negative bacterial community than teeth with secondary infections. Moreover, the levels of endotoxins were related to the severity of bone destruction in periapical tissues as well as the development of clinical features in teeth with primary infections.

Detection of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia in primary endodontic infections in a Chinese population.

AIM: To assess the prevalence of three black-pigmented bacterial species (Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia) using microarray technology in root canals of teeth associated with primary endodontic infections in a Chinese population. METHODOLOGY: Microbial samples were taken from root canals of 80 teeth with pulp necrosis and primary endodontic infections in a Chinese population. DNA extracted from the samples was amplified by PCR with universal bacterial primers based on 16S rRNA gene sequences, and the products hybridized with the microarrays in which the specific oligonucleotide probes were added. The results of hybridization were screened by a confocal laser scanner. Pearson chi-square test and the two-sided Fisher exact test were used to analyse whether a significant association existed between the species and symptoms as well as in co-existence of two target organisms by a statistical software package (SAS 8.02). RESULTS: The 16S rRNA gene microarray detected at least one of the three test species in 76% of the study teeth. P. endodontalis, P. gingivalis and P. intermedia were found in 50%, 33% and 45%, respectively. A significant association was found in the presence of the pair P. endodontalis / P. gingivalis (P < 0.005). Both P. endodontalis (P <0.05) and P. gingivalis (P <0.005) had a statistically significant association with the presence of a sinus tract. The simultaneous presence of P. endodontalis and P. gingivalis was also associated with the presence of a sinus tract (P<0.005) and abscess formation (P<0.05). CONCLUSIONS: The three black-pigmented bacteria were prevalent in teeth with pulp necrosis and primary endodontic infections in a Chinese population. P. gingivalis and P. endodontalis were associated with the presence of sinus tract and abscess formation.

Molecular analysis of 16S rRNA genes identifies potentially periodontal pathogenic bacteria and archaea in the plaque of partially erupted third molars.

PURPOSE: Small subunit rRNA sequencing and phylogenetic analysis were used to identify cultivable and uncultivable microorganisms present in the dental plaque of symptomatic and asymptomatic partially erupted third molars to determine the prevalence of putative periodontal pathogens in pericoronal sites. MATERIALS AND METHODS: Template DNA prepared from subgingival plaque collected from partially erupted symptomatic and asymptomatic mandibular third molars and healthy incisors was used in polymerase chain reaction with broad-range oligonucleotide primers to amplify 16S rRNA bacterial and archaeal genes. Amplicons were cloned, sequenced, and compared with known nucleotide sequences in online databases to identify the microorganisms present. RESULTS: Two thousand three hundred two clones from the plaque of 12 patients carried bacterial sequences from 63 genera belonging to 11 phyla, including members of the uncultivable TM7, SR1, and Chloroflexi, and difficult-to-cultivate Synergistetes and Spirochaetes. Dialister invisus, Filifactor alocis, Fusobacterium nucleatum, Porphyromonas endodontalis, Prevotella denticola, Tannerella forsythia, and Treponema denticola, which have been associated with periodontal disease, were found in significantly greater abundance in pericoronal compared with incisor sites. Dialister invisus and F nucleatum were found in greater abundance in sites exhibiting clinical symptoms. The archaeal species, Methanobrevibacter oralis, which has been associated with severe periodontitis, was found in 3 symptomatic patients. CONCLUSIONS: These findings have provided new insights into the complex microbiota of pericoronitis. Several bacterial and archaeal species implicated in periodontal disease were recovered in greater incidence and abundance from the plaque of partially erupted third molars compared with incisors, supporting the hypothesis that the pericoronal region may provide a favored niche for periodontal pathogens in otherwise healthy mouths.

Clustering behavior in microbial communities from acute endodontic infections.

INTRODUCTION: Acute endodontic infections harbor heterogeneous microbial communities in both the root canal (RC) system and apical tissues. Data comparing the microbial structure and diversity in endodontic infections in related ecosystems, such as RC with necrotic pulp and acute apical abscess (AAA), are scarce in the literature. The aim of this study was to examine the presence of selected endodontic pathogens in paired samples from necrotic RC and AAA using polymerase chain reaction (PCR) followed by the construction of cluster profiles. METHODS: Paired samples of RC and AAA exudates were collected from 20 subjects and analyzed by PCR for the presence of selected strict and facultative anaerobic strains. The frequency of species was compared between the RC and the AAA samples. A stringent neighboring clustering algorithm was applied to investigate the existence of similar high-order groups of samples. A dendrogram was constructed to show the arrangement of the sample groups produced by the hierarchical clustering. RESULTS: All samples harbored bacterial DNA. Porphyromonas endodontalis, Prevotella nigrescens, Filifactor alocis, and Tannerela forsythia were frequently detected in both RC and AAA samples. The selected anaerobic species were distributed in diverse small bacteria consortia. The samples of RC and AAA that presented at least one of the targeted microorganisms were grouped in small clusters. CONCLUSIONS: Anaerobic species were frequently detected in acute endodontic infections and heterogeneous microbial communities with low clustering behavior were observed in paired samples of RC and AAA.

Detection and clonal analysis of anaerobic bacteria associated to endodontic-periodontal lesions.

BACKGROUND: Microbial agents in root canal systems can induce periodontal inflammation. The aims of this study are to detect anaerobic microorganisms in endodontic-periodontal lesions, determine the genetic diversity among them, and assess the simultaneous colonization of the pulp and periodontal microenvironments by a single clone. METHODS: Twenty-seven teeth of patients with endodontic-periodontal lesions were selected. Samples were spread on an agar-blood medium, the detection of each species was performed using a polymerase chain reaction, and the determination of the simultaneous presence of the same species in the microenvironments by one or more clones was determined using arbitrarily primed PCR. RESULTS: Prevotella intermedia (Pi) was the most prevalent species of the colonies in periodontal pockets, whereas Porphyromonas gingivalis (Pg) and Pi were the more prevalent in root canals. Isolates of Pi and Pg were simultaneously identified in root canals and periodontal pockets. Eighteen percent of teeth exhibited the simultaneous colonization by Pg, Tannerella forsythia (previously T. forsythensis), and Porphyromonas endodontalis in the pulp and periodontal microenvironments. The presence of these species was noted even in niches from which no colonies were isolated. Seventeen different genotypes were found in periodontal and pulp sites, with the majority of sites colonized by one or two different genotypes. A high degree of genotype similarity was found for samples of Pg isolated from only one site as well as for those isolated from both microenvironments. CONCLUSION: Different clones of Pi and Pg with a high intraspecific genotype similarity were found to colonize the same anatomic sites in endodontic-periodontal infections.

Apical root canal microbiota as determined by reverse-capture checkerboard analysis of cryogenically ground root samples from teeth with apical periodontitis.

INTRODUCTION: Bacteria located in the apical root canal system potentially participate in the pathogenesis of apical periodontitis. Detection and identification of apical bacteria can be compromised because of limitations in conventional sampling and identification procedures. This study identified several bacterial taxa in the apical and middle/coronal segments of primarily infected root canal system by using pulverized root segments and a culture-independent molecular method. METHODS: Seventeen extracted teeth with attached apical periodontitis lesions were sectioned to obtain 2 root fragments (apical and middle/coronal segments). Root fragments were cryogenically ground, and DNA was extracted from samples. After multiple displacement amplification, DNA from samples was used as template in a reverse-capture checkerboard hybridization assay targeting 28 bacterial taxa. RESULTS: Bacterial DNA was detected in all samples. The most prevalent taxa in the apical root canal system were Olsenella uli (76.5%), Prevotella baroniae (71%), Porphyromonas endodontalis (65%), Fusobacterium nucleatum (53%), and Tannerella forsythia (47%). O. uli, P. endodontalis, and Propionibacterium acnes were as frequently detected in apical samples as they were in middle/coronal samples. P. baroniae, T. forsythia, and F. nucleatum were found more frequently in the apical part of the canal as compared with matched coronal segments. Streptococcus species were more prevalent in middle/coronal samples. The median and mean of shared bacterial taxa between matched apical and middle/coronal segments were 27% and 41%, respectively. CONCLUSIONS: Several candidate endodontic pathogens were very prevalent in the apical root canal system. The apical microbiota was usually complex and differed in species composition when compared with the microbiota of middle/coronal samples from the same tooth.