Prevalence of Treponema denticola and Porphyromonas gingivalis in plaque from periodontally-healthy and periodontally-diseased sites

Intracrevicular plaque from periodontally-healthy individuals who had refrained from oral hygiene measures for 24 h prior to sampling, and subgingival plaque from diseased sites of patients with chronic periodontitis were screened by ELISA for the presence of Porphyromonas gingivalis and Treponema denticola. The samples were also subjected to the PerioScan test to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA). Of the 141 samples from periodontally-healthy sites, 73% contained T. denticola antigens and 78% P. gingivalis antigens, compared to 43% and 59%, respectively, in plaque samples from the 159 diseased sites. A positive reaction in the PerioScan test was obtained in 89% of plaque samples from diseased sites and in 60% of those from healthy sites. The correlation between the results of the two assays was poor in the case of intracrevicular plaque from healthy sites. However, with plaque samples from diseased sites, the results of the PerioScan test showed very strong correlation with those obtained with the ELISA, suggesting that the former may be a useful, rapid means of indicating the presence of T. denticola and P. gingivalis in such plaque samples.     

Declining prevalence of dental caries in school children in Singapore

Slime layers and capsules are common amongst medically relevant bacteria. We herein report that Treponema denticola, which has been associated with periodontitis, synthesizes or acquires an extracellular polysaccharide layer that we have observed through electron microscopy using the polysaccharide-specific dye Alcian blue and phosphotungstate. We have also visualized this extracellular layer by dark-field microscopy of Alcian blue-stained spirochete cells. A representative strain of each of the oral spirochete species T. denticola, Treponema vincentii and Treponema socranskii were differentiated by concanavalin A, phaseolus, lotus A and arachis lectins in a microtiter plate immunoassay for the detection of surface sugars.     

Low back pain in college athletes. A prospective study correlating lower extremity overuse or acquired ligamentous laxity with low back pain

This study compared the presence of 6 periodontopathic bacteria in whole saliva and subgingival plaque of 202 subjects. The test bacteria were identified using a 16S rRNA-based PCR detection method. Each study subject contributed a whole saliva sample and a paper point sample pooled from the deepest periodontal pocket in each quadrant of the dentition. The kappa test revealed a fair agreement between the presence of Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola in whole saliva and periodontal pocket samples (kappa > 0.4). The McNemar test showed that the differences between sample types were due to a more frequent detection of the 3 organisms in whole saliva than in periodontal pocket samples (P < 0.01). Prevotella nigrescens also was detected more frequently in whole saliva than in periodontal pocket samples (P < 0.01; McNemar test). Although little agreement between samples was found for Actinobacillus actinomycetemcomitans and Bacteroides forsythus (kappa < or = 0.4), neither whole saliva nor pocket samples showed better detection for these 2 species (P < 0.01, McNemar test). The results indicate that whole saliva is superior to pooled periodontal pocket samples to detect P. gingivalis, P. intermedia, P. nigrescens, and T. denticola in the oral cavity. The detection of oral A. actinomycetemcomitans and B. forsythus with reasonably good accuracy may require both whole saliva and periodontal pocket samples.     

Use of the bacille Calmette-Guérin vaccination for the prevention of tuberculosis: renewed interest in an old vaccine

A method for the enumeration of colony-forming units of oral anaerobic spirochetes in new oral spirochete agarose (NOS-A) medium was described recently. However, the high cost of agarose limits the extent to which large assays can be carried out. Accordingly, a search for an inexpensive gelling agent that remains molten at 37 degrees C and gels at 25 degrees C was undertaken. Varying amounts of Noble agar or Bacto agar (0.5 to 1.5%, w/v) were mixed with varying amounts of gelatin (0.5 to 1.0%, w/v) in NOS medium. NOS medium containing 0.5% gelatin-0.5% Noble agar (NOS-GN) or 0.5% gelatin-0.5% Bacto agar (NOS-GB) met the above criteria. NOS-GN and NOS-GB media yielded higher colony-forming units with Treponema denticola than NOS-A medium in that order. However, all three media, NOS-GN, NOS-GB and NOS-A, performed equally well in the recovery of viable counts of T. vincentii. The NOS-GN medium was not liquefied by subgingival bacteria or two gelatinase-producing species of bacteria, Bacillus subtilis and Staphylococcus aureus. Thus NOS-GN medium is the recommended medium both in cost and performance for obtaining colony counts of spirochetes.     

Fluoride and hard cheese exposure on etched enamel in neck-irradiated patients in situ

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.     

Molecular epidemiology of oral treponemes associated with periodontal disease

Periodontitis, a disease responsible for tooth loss worldwide, is characterized by chronic inflammation of the periodontium, eventually leading to destruction of periodontal ligaments and supporting alveolar bone. Spirochetes, identified by dark-field microscopy as being the most predominant bacteria in advanced lesions, are thought to play a causative role. Various spirochetal morphotypes were observed, but most of these morphotypes are as yet uncultivable. To assess the role of these organisms we designed oligonucleotide probes for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients. Subgingival plaque specimens taken from diseased sites (n = 200) and healthy control sites (n = 44) from 53 patients with rapidly progressive periodontitis (RPP) were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers. Spirochetes were found in all patients, but their distributions varied considerably. Parallel use of oligonucleotide probes specific for cultivable or so far uncultivable treponemes suggested the presence of novel yet unknown organisms at a high frequency. These uncultivable treponemes were visualized by fluorescence in situ hybridization, and their morphologies, sizes, and numbers could be estimated. All RPP patients included in this study harbored oral treponemes that represent either novel species, e.g., Treponema maltophilum, or uncultivable phylotypes. Therefore, it is necessary to include these organisms in etiologic considerations and to strengthen efforts to cultivate these as yet uncultivable treponemes.     

Identification of seven Treponema species in health- and disease-associated dental plaque by nested PCR

Species-specific nested PCR was used to detect Treponema amylovorum, Treponema denticola, Treponema maltophilum, Treponema medium, Treponema pectinovorum, Treponema socranskii, and Treponema vincentii in dental plaque. Subjects with periodontitis harbored all species, but T. pectinovorum and T. vincentii were not found in plaque from disease-free subjects.     

Tricuspid valve endocarditis in a non-drug addict associated with peliosis hepatis

The aims of the present study were to investigate whether the tachykinins substance P and neurokinin A were present in gingival crevicular fluid in both periodontal health and disease and to study the relationship with periodontal inflammation. Gingival crevicular fluid (GCF) was collected from a healthy, a gingivitis and a periodontitis site in 20 subjects with periodontitis and from a healthy site in 20 subjects without periodontitis. The volume of GCF was measured and each sample subsequently analysed for substance P and neurokinin A by radioimmunoassay. There were significantly increased levels of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) in gingivitis and periodontitis sites compared with healthy sites. Both tachykinins were significantly elevated in periodontitis affected subjects, with significantly more tachykinin-like immunoreactivity at healthy sites in periodontitis affected compared with periodontally-healthy subjects. Despite the considerable individual variation in the levels of SP-LI and NKA-LI, both tachykinins were present at levels at which they could have biological activity. It is concluded that substance P and neurokinin A may have a r?le in the pathogenesis of periodontal disease and that further investigations could prove useful in clarifying the mechanisms through which neuropeptides could modulate periodontal health and disease.     

Oral malodor in beagles: association with indicators of periodontal disease

The study of oral malodor continues to receive attention. Most bad breath is of oral origin and can be corrected with proper oral hygiene. Studies performed with saliva from people with periodontal disease and from healthy individuals showed that saliva from diseased patients produced a more objectionable odor faster than that of healthy people, and that the volatile sulfur components (VSC) produced may actually play a role in the etiology of periodontal disease. However, not all people or animals with bad breath have periodontal disease. The objectives of this study were to determine if a trained panel could discriminate between 10 dogs with clinically defined periodontal disease and those with relatively healthy periodontium. Second, this study attempted to establish a correlation between odor intensity and six clinical parameters of oral health. The judges were able to differentiate between the two groups of dogs based only on oral malodor (p < 0.02). There were strong associations of the intensity of oral malodor with oral health index scores. The correlations established between odor and gingivitis (r = 0.81) and between odor and furcation exposure (r = 0.88) were very high and statistically significant. Similarly, probing depth (r = 0.73), plaque (r = 0.07) and tooth mobility (r = 0.66) showed clear, positive relationships with oral malodor.     

Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects

This investigation compared the site prevalence of 40 subgingival species in 30 periodontally healthy (mean age 36+/-9 years), 35 elders with a well-maintained periodontium (mean age 77+/-5) and 138 adult periodontitis subjects (mean age 46+/-11). Subgingival plaque samples were taken from the mesial aspect of each tooth (up to 28 samples) in the 203 subjects at baseline. The presence and levels of 40 subgingival taxa were determined in 5003 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The % of sites colonized by each species (prevalence) was computed for each subject. Differences in prevalence and levels among groups were sought using the Kruskal-Wallis test. Commonly detected species, such as Actinomyces naeslundii genospecies 2, Streptococcus sanguis and Streptococcus oralis did not differ significantly among subject groups. After adjusting for multiple comparisons, 4 species were significantly elevated and at greater prevalence in the periodontitis group. Mean % of sites (+/-SEM) colonized by Bacteroides forsythus was 10+/-3, 12+/-2 and 40+/-2 (p<0.001) for healthy, elder and periodontitis groups respectively. The odds ratio was 14.4:1 that a subject had periodontitis when B. forsythus was detected at > or = 5% of sampled sites. Mean prevalence for Porphyromonas gingivalis in healthy, elder and periodontitis subjects was 4+/-2, 5+/-2 and 23+/-2 respectively (p<0.001); for Treponema denticola 12+/-4, 10+/-3 and 30+/-2 (p<0.001) and for Selenomonas noxia 6+/-2, 7+/-2 and 19+/-2 (p<0.01). Similar differences among subject groups were observed when only sites with PD 0-4 mm were analyzed. The data suggest an etiologic role for B. forsythus, P. gingivalis, T. denticola and S. noxia in adult periodontitis.     

Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.     

Prediction of the femoral neck-shaft angle from the length of the femoral neck

The purpose of this in vitro study was to compare the effects of the Sensonic. Oral-B Braun mechanical and Oral-B manual toothbrushes upon the morphology and cellular integrity of Treponema denticola. This spirochete was chosen because of its frequent isolation from active lesions of inflammatory periodontal disease and its pathogenic potential. T. denticola, strain ATCC 33421, was grown in an anaerobic nitrogen rich atmosphere in enhanced 1186 mycoplasma broth. 160, 5-ml aliquots of cultured microbes were assigned to 1 of 3 brushing treatment groups and a control group. Samples were further divided into 4 groups of 10 samples each and assigned to one of 4 brushing exposure times: 15, 30, 45, and 60 seconds. After treatment, 0.2 ml of each sample was applied to a millipore filter and examined by SEM. Intact microbes were counted from 10 non-overlapping fields at 4500x. Remaining treated samples were pelleted and examined by TEM. A statistically significant reduction of intact microbes for the Sensonic treatment group at each exposure time was found when compared to Oral-B Braun, Oral-B manual, and non-treated controls. TEM examination of Sensonic treated samples revealed separation of the outer membrane at lower exposure times and only cellular debris after exposures of 45 and 60 s. These results suggest that exposure to the sonic frequency generated by the Sensonic toothbrush is capable of severely disrupting the structural integrity of T. denticola.     

Subgingival microbiota in adult Chinese: prevalence and relation to periodontal disease progression

The "checkerboard" Dna-Dna hybridization technology was used to study the epidemiology of 18 microbial species associated with various states of periodontal health and disease, in a sample of 148 Chinese subjects never exposed to systematic dental therapeutic intervention, aged 30 to 39 and 50 to 59 years. Our aims were to: 1) describe the prevalence of these microorganisms; 2) correlate the microbiological and clinical profiles of the subjects; and 3) examine the association between the microbiological variables and the longitudinal changes of periodontal status that occurred over a preceding 10-year period. A maximum of 14 subgingival samples were obtained from each subject-1,864 in all. The frequency of occurrence of the 18 species examined was high in this Chinese population, on both the subject and the tooth site level. However, all species were not found equally capable of reaching high numbers in the subgingival samples and, as a rule, colonized heavily only limited proportions of tooth sites within each mouth. There was a profound increase of certain species such as Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus in deep pockets or progressing sites. Multivariate techniques using the subgingival profile could effectively discriminate between deep/shallow pockets and progressing/ stable tooth sites. The microbiological variables showed an enhanced discriminating potential when classifications were performed on the individual subject level. Colonization by P. gingivalis, B. forsythus, Campylobacter rectus, and T. denticola at levels exceeding certain thresholds entailed a significantly increased probability (odds ratios > 4) for an individual subject to harbor deep pockets or progressing tooth sites.     

Treponema denticola as a model for polar adhesion and cytopathogenicity of spirochetes

Recent studies suggest that the tip-oriented adhesion of Treponema denticola is a consequence of polar clustering of its adhesins on contact with the substratum and is compatible with viability. Such adhesion to cells stimulates a host of cytoskeletal and volume regulatory changes. These studies may give insight into mechanisms of colonization and cytopathogenicity of other pathogenic spirochetes.     

Vascular endothelial growth factor in human periodontal disease

Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic cytokine of importance in inflammation and wound healing but its presence in chronic inflammatory periodontal disease has never been reported. The aims of this study were to investigate the presence of VEGF in human periodontal tissue and gingival crevicular fluid (GCF) in periodontal health and disease. VEGF in tissue was localized by immunohistochemistry. GCF and unstimulated saliva were collected from patients and clinically healthy subjects and VEGF was assessed by using an ELISA. VEGF was detected within vascular endothelial cells, neutrophils, plasma cells and junctional, pocket and gingival epithelium. In periodontitis patients, the volume of GCF and total amount of VEGF collected from diseased sites were both greater than from clinically healthy sites (Wilcoxon test p < 0.01). However, the concentration of VEGF per unit volume of GCF was higher at healthy sites compared with diseased sites (Wilcoxon test p < 0.05). Higher concentrations of VEGF were detected in healthy sites in patients compared with similar sites in clinically healthy subjects (Mann-Whitney U-test p < 0.05). A logistic regression approach indicated that there was variation in VEGF between subjects (p < 0.01), and that age (p < 0.05), plaque (p < 0.05) and pocket depth (p < 0.07) were explanatory variables. VEGF was also detected in all saliva samples and was significantly higher in patients than in healthy controls (p < 0.05). This study suggests that VEGF could be relevant to angiogenic processes in healthy as well as diseased periodontal tissue and that the periodontal status influences the salivary level of VEGF.     

Gingival crevicular pH in experimental gingivitis and occlusal trauma in man

The purpose of this study was to investigate the fluctuation of gingival crevicular pH during experimentally evoked gingivitis and occlusal trauma, and to examine the relationship between the pH and periodontal health status using a newly developed pH sensor. Maxillary first premolars with clinically healthy gingiva were selected as test teeth in 10 volunteers. In the first study phase, experimental gingivitis was evoked, and gingival index, plaque index, and crevicular pH were recorded during the study period. In the second study phase, experimental occlusal trauma was created with metal onlays having occlusal interferences in lateral movements, and tooth mobility and pH were recorded during the study period. In the gingivitis phase, GI, PlI, and pH values fluctuated significantly during the study period (P < 0.01) and positive correlations were observed between both GI and pH values (P < 0.05), and PlI and pH values (P < 0.01). In the occlusal trauma phase, significant fluctuation was found among tooth mobilities (P < 0.05) during the study phase, but not in pH values. Statistically significant correlations were not observed between tooth mobilities and pH values. These data suggest that the crevicular pH level may not be influenced by experimental occlusal trauma, but shifts toward alkaline with experimental gingivitis.     

Incidence of Prevotella intermedia and Prevotella nigrescens in periodontal health and disease

The incidence of black-pigmented rods (BPRs), especially Prevotella intermedia and Prevotella nigrescens, in periodontal health and disease were examined. Furthermore, the degradative enzyme activities of P. intermedia were compared among the strains from periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. The BPRs were found in 71.1% of periodontally healthy subjects (n=45), and in 47.1% of healthy sites (n=34) and 87.8% of active sites (n=41) among periodontally diseased patients. Porphyromonas gingivalis was detected only in active sites of periodontally diseased patients (17.8% of 180 strains). P. intermedia was the predominant BPR in both healthy and active sites (37.3 and 41.7%, respectively) of the patients. However, P. nigrescens was the predominant BPR (70.5% of 173 strains) in periodontally healthy subjects. The enzyme activities of esterase, esterase-lipase, acid-phosphatase and alpha-fucosidase of P. intermedia strains isolated from active sites in patients were significantly higher (P<0.05) than those of healthy subjects. The results suggest that P. intermedia might increase the activity of degradative enzymes under a certain condition and support the progression of periodontitis.     

Local metronidazole application in maintenance patients. Clinical and microbiological evaluation

The purpose of this investigation was to evaluate the clinical and microbiological effect of local antibiotic therapy in comparison with subgingival scaling and root planing in a randomized semi-masked study. Forty-six recall patients who completed systematic periodontal therapy 6 to 24 months prior to the study were enrolled. The inclusion requirements were at least one site with probing depth > or = 5 mm in each quadrant, no scaling, and no antibiotic therapy during the last 6 months. After randomization each patient received 2 different treatments: in 2 quadrants metronidazole 25% dental gel was applied subgingivally to the pockets at day 0 and day 7; scaling and root planing was carried out in the 2 other quadrants, one at day 0 and in the remaining quadrant at day 7. Subgingival microbiological samples were taken from each patient before treatment and on days 21, 91, and 175 after the treatment. The analyses were carried out by indirect immunofluorescence assay. At all treated sites probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were recorded on days 0, 21, 91, and 175. Both treatments resulted in PD reduction and CAL gain. PD reduction was statistically significant (P < 0.01) for both treatment modalities after 6 months. The CAL gain was not significant for either treatment. There was no statistical significance between scaling and antibiotic therapy. Treponema denticola, Porphyromonas gingivalis, and Prevotella intermedia were significantly reduced after therapy; however, there were no statistically significant differences between treatments. If Actinobacillus actinomycetemcomitans was present before therapy, it was also present after treatment in both groups. The conclusion is that, in recall patients, local application of metronidazole and scaling and root planing showed similar clinical and microbiological effects without statistically significant differences.