Aggregatibacter actinomycetemcomitans ; physiology ; Bacterial Adhesion ; Biofilms ; Periodontal Diseases ; microbiology ; Porphyromonas endodontalis ; physiology

OBJECTIVEThis study aims to assess and compare the prevalence of Porphyromonas endodontalis (P. endodontalis) in root canals associated with primary and secondary endodontic infections by using 16s rDNA PCR and real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR).

METHODSA total of 120 adult patients with one radiographically documented periapical lesion were included. Sixty teeth presented with primary endodontic infections and 60 with secondary endodontic infections requiring retreatment. P. endodontalis was identified by using 16s rDNA PCR techniques. The positive DNA expression of P. endodontalis in two types of infected root canals were quantitatively compared by using SYBR GREEN I RTFQ-PCR.

RESULTSThe prevalence of P. endodontalis in the root canals with primary endodontic infections was significantly higher than that in root canals with secondary endodontic infections (P = 0.001). However, RTFQ-PCR results showed no significant difference in DNA expression quantities between the primary and secondary endodontic infections root canals (P = 0.303).

CONCLUSIONP. endodontalis is more highly associated with root canals having primary endodontic infections, although P. endodontalis colonize in both root canals with primary and secondary chronic apical periodontitis.

Adult ; DNA, Bacterial ; Dental Pulp Cavity ; Gram-Negative Bacterial Infections ; Humans ; Polymerase Chain Reaction ; Porphyromonas endodontalis ; Retreatment

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-alpha from immune cells. Although monocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-alpha. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-alpha, and it was compared with Escherichia coli LPS. P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 microg/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)2 at 37degrees C for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4x106 cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10microg/ml) for 24 hours at 37degrees C in 5% CO2 incubator. The supernatants of cells were collected and the levels of IL-1alpha, IL-1beta and TNF-alpha were measured by enzyme-linked immunosorbent assay. The results were as follows; 1. The levels of IL-1alpha, IL-1beta, TNF-alpha from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p<0.05). 2. The levels of all three cytokines released from PMN stimulated with each calcium hydroxide treated LPS were significantly lower than those released from PMN stimulated with each untreated LPS (p<0.05), while they were not significantly different from those released from unstimulated PMN of the control group (p>0.05). 3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p<0.05), but not in PMN stimulated with each calcium hydroxide treated LPS (p>0.05). 4. The levels of all three cytokines released from PMN stimulated with P. endodontalis LPS were significantly lower than those released from PMN stimulated with E. coli LPS (p<0.05).
Calcium ; Calcium Hydroxide ; Centrifugation ; Cytokines ; Dental Pulp Cavity ; Escherichia coli ; Ficoll ; Humans ; Hydroxides ; Incubators ; Interleukin-1 ; Metrizoate ; Neutrophils ; Porphyromonas ; Porphyromonas endodontalis ; Tumor Necrosis Factor-alpha ; Ultrafiltration ; Water

The purpose of this study was to investigate the frequency of 7 putative pathogens in endodontic infections. The specimens were collected from infected pulpal tissue of patients who were referred for root canal treatment to the department of conservative dentistry, Chosun University. Samples were collected aseptically using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ml of 1 X PBS. DNAs were extracted from the samples by direct DNA extraction method using lysis buffer (0.5% EDTA, 1% Triton X-100). Identification of 7 putative pathogens was performed by PCR based on 16S rDNA. The target species were as follows: Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, and Treponema denticola. Our data revealed that the prevalence of P. endodontalis was found in 88.6% (39/54), P. gingivalis 52.3% (23/44), P. nigrescens 18.2% (8/44), P. intermedia 15.9% (7/44), B. forsythus 18.2% (8/44), A. actinomycetemcomitans 2.3% (1/44), T. denticola 25% (11/44) of the samples. The high prevalence of P. endodontalis and P. gingivalis suggests that they may play an important role in the etiology of endodontic infections.
Aggregatibacter actinomycetemcomitans ; Bacteroides ; Dental Pulp Cavity ; Dentistry ; DNA ; DNA, Ribosomal* ; Edetic Acid ; Humans ; Neptune ; Polymerase Chain Reaction* ; Porphyromonas endodontalis ; Porphyromonas gingivalis ; Prevalence ; Prevotella intermedia ; Prevotella nigrescens ; Treponema denticola

PURPOSE: Specific bacteria are believed to play an important role in chronic periodontitis. Although extensive microbial analyses have been performed from subgingival plaque samples of periodontitis patients, systemic analysis of subingival microbiota has not been carried out in a Korean population so far. The purpose of this study was to investigate the prevalence of 29 putative periodontal pathogens in Korean chronic periodontitis patients and evaluate which pathogens are more associated with Korean chronic periodontitis. MATERIAL AND METHODS: A total of 86 subgingival plaque samples were taken from 15 chronic periodontits(CP) patients and 13 periodontally healthy subjects in Korea. CP samples were obtained from the deepest periodontal pocket (>3 mm probing depth[PD]) and the most shallow periodontal probing site (< or =3 mm PD) in anterior tooth and posterior tooth, respectively, of each patient. Samples in healthy subjects were obtained from 1 anterior tooth and 1 posterior tooth. Polymerase chain reaction (PCR) of 16S ribosomal DNA (rDNA) of subgingival plaque bacteria was performed. Detection frequencies(% prevalence) of 29 putative periodontal pathogens were investigated as bacterium-positive sites/total sites RESULTS: With the exception of Olsenella profuse and Prevotella nigrescens, the sites of diseased patients generally showed higher prevalence than the healthy sites of healthy subjects for all bacteria analyzed. Tanerella forsythensis (B.forsythus), Campylobacter rectus, Filifactor alocis, Fusobacterium nucleatum, Porphyromonas endodontalis and Porphyromonas gingivalis were detected in more than 80% of sites with deep probing depths in CP patients. In comparison between the sites (deep or shallow PD) of CP patients and the healthy sites of healthy subjects, there was statistically significant difference(P <0.05) of prevalence in T.forsythensis (B.forsythus), C.rectus, Dialister invisus, F.alocis, P.gingivalis and Treponema denticola. CONCLUSION: Our results demonstrate that the four putative periodontal pathogens, T.forsythensis (B.forsythus), C.rectus, P.gingivalis and F.alocis are closely related with CP patients in the Korean population.
Bacteria ; Campylobacter rectus ; Chronic Periodontitis ; DNA, Ribosomal ; Fusobacterium nucleatum ; Humans ; Korea ; Metagenome ; Periodontal Pocket ; Periodontitis ; Polymerase Chain Reaction ; Porphyromonas endodontalis ; Porphyromonas gingivalis ; Prevalence ; Prevotella nigrescens ; Tooth ; Treponema

Black-pigmented bacteria have been implicated in the endodontic infections. This group of microorganisms includes Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens. The organisms display a wide variety of virulence factors that may be pertinent to acute endodontic infections. The aim of this study was to identify P. endodontalis, P. gingivalis, P. intermedia, and P. nigrescens by using special potency disk test, filter paper spot test, 16S rRNA gene-directed PCR, and API 32A. Microbial samples were collected from root canals of 33 intact teeth with necrotic pulp and/or apical periodontitis. Conventional laboratory methods were used for identification of the strains of black pigmented bacteria. Eighteen of 33 samples were positive for the growth of black-pigmented bacrteria. Five colonies were cultured from each pure cultured colonies from Brucella agar plate. Seventy seven colonies were positive for the growth of black-pigmented bacteria. Thirty three of 77(42.6%) were identifed as P. nigrescens, 10 of 77(12.9%) were P. gingivalis, 6 of 77(7.8%) were P. endodontalis, 10 of 77(12.9%) were P. intermedia. On the contrary the reference strains of P. nigrescens, experimental strains of P. nigrescens was sensitive to kanamycin in special potency disk test. 16S rRNA gene PCR and API test after rapid presumptative identification methods, such as special potency disk test and filter paper spot test, would be accurate detection methods for black-pigemented bacteria.
Agar ; Bacteria ; Brucella ; Dental Pulp Cavity ; Genes, rRNA ; Kanamycin ; Periapical Periodontitis ; Polymerase Chain Reaction ; Porphyromonas endodontalis ; Porphyromonas gingivalis ; Prevotella intermedia ; Prevotella nigrescens ; Tooth ; Virulence Factors

It has been documented that periodontopathic bacteria are also implicated in endodontic infections. 16S rDNA gene-directed PCR was to examine the prevalence of periodontopathic bacteria including Actinobacillus actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Prevotella nigrescens (Pn), Porphyromonas gingivalis (Pg), Porphyromonas endodontalis (Pe), and Treponema denticola (Td) in the root canals of 36 endodontically infected teeth having apical lesions with or without clinical symptoms like pain, swelling, and fistula. 1. In 36 infected root canals, most frequently detected bacterial species was Pg (61.1%), followed by Td (52.8%) and Pe (38.9%). 2. Of 36 infected root canals, Aa was detected in 6 canals (16.7%) of the teeth, all of which showed clinical symptoms. 3. Of 36 infected root canals, Pi and Pn were found in 4 (13.9%) and 5 (33.3%), respectively. Notably, prevalence of Pn in the symptomatic teeth was 50.0%. 4. One of black-pigmented anaerobic bacteria (BPB) including Pi, Pn, Pe, and Pg was detected in all of the teeth that showed pain or especially swelling but not fistula. It was, however, found that prevalence of BPB in the asymptomatic teeth or the teeth with fistula was only 40%. 5. Pe and Pg were detected in the teeth regardless of the presence or absence of symptoms. 6. Td was detected in the teeth regardless of the presence or absence of symptoms. High prevalence of BPB in the symptomatic teeth but low in the asymptomatic teeth suggests that BPB may play an important role in the pathogenesis of periapical lesions.
Aggregatibacter actinomycetemcomitans ; Bacteria ; Bacteria, Anaerobic ; Dental Pulp Cavity ; DNA, Ribosomal ; Fistula ; Polymerase Chain Reaction ; Porphyromonas endodontalis ; Porphyromonas gingivalis ; Prevalence ; Prevotella intermedia ; Prevotella nigrescens ; Tooth ; Treponema denticola

During the last two decades, there has been an increasing interest in the impact of oral health on atherosclerosis and subsequent cardiovascular disease (CVD). To date, some periodontal pathogens including Porphyromonas gingivalis (P. gingivalis) have been reported to be relevant to CVD. Porphyromonas endodontalis (P. endodontalis), which shares approximately 87% sequence homology with P. gingivalis, is mostly found within infected root canals. However, recent studies reveal that this pathogen also resides in the dental plaque or periodontal pocket in patients with periodontitis. It has been shown that P. endodontalis invades human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC). To evaluate whether P. endodontalis can participate in the progression of atherosclerosis and CVD, we examined the changes in transcriptional gene expression profiles of HCAEC responding to invasion by P. endodontalis in this study. The following results were obtained. 1. Porphyromonas endodontalis was invasive of HCAEC. 2. According to the microarray analysis, there were 625 genes upregulated more than two-folds, while there were 154 genes downregulated by half. 3. Upregulated genes were relevant to inflammatory cytokines, apoptosis, coagulation and immune response. Enhanced expression of MMP-1 was also noticeable. 4. The transcription profiles of the 10 selected genes examined by real-time PCR agreed well with those observed in the microarray analysis. Thus, these results show that P. endodontalis presents the potential to trigger and augment atherosclerosis leading to CVD.
Apoptosis ; Atherosclerosis ; Cardiovascular Diseases ; Coronary Vessels ; Cytokines ; Dental Plaque ; Dental Pulp Cavity ; Endothelial Cells ; Gene Expression ; Humans ; Microarray Analysis ; Myocytes, Smooth Muscle ; Oral Health ; Periodontal Pocket ; Periodontitis ; Porphyromonas ; Porphyromonas endodontalis ; Porphyromonas gingivalis ; Real-Time Polymerase Chain Reaction ; Sequence Homology ; Transcriptome

OBJECTIVETo quantify the interleukin (IL)-1beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides ([PS) extracted from Porphyromonoas endodontalis (P. endodontalis) in osteoblasts, and to relate P. endodontalis LPS to the bone resorptive pathogenesis in the lesions of chronic apical periodontitis.

METHODSMG63 cells was pretreated with PD98059 or SB203580 for 1 h and then treated with P. endodontolis LPS for 6 h. The expression of IL-1beta mRNA and IL-6 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) technique.

RESULTSThe production of IL-1beta mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with PD98059. Both of the production of IL-1beta mRNA and JL-6 mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with SB203580.

CONCLUSIONThe synthesis of IL-1beta mRNA stimulated by Pendodontalis LPS in MG63 probably occur via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) signal transduction system. The synthesis of IL-6 mRNA stimulated by P.endodontalis LPS in MG63 probahly occur via p38MAPK signal transduction system.

Humans ; Imidazoles ; Interleukin-6 ; Lipopolysaccharides ; MAP Kinase Signaling System ; Osteoblasts ; Porphyromonas endodontalis ; Pyridines ; RNA, Messenger ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases

OBJECTIVETo investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process.

METHODSMC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h).

RESULTSThe level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control.

CONCLUSIONSPe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Animals ; Lipopolysaccharides ; pharmacology ; Macrophage Colony-Stimulating Factor ; drug effects ; physiology ; Mice ; NF-kappa B ; metabolism ; Nitriles ; Osteoblasts ; drug effects ; metabolism ; Porphyromonas endodontalis ; RNA, Messenger ; Signal Transduction ; Sulfones