Periodontopathic Species Research Articles

Lipoprotein modifications by gingipains of Porphyromonas gingivalis.

Background and ObjectiveSeveral studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low‐density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro.Material and MethodsPlasma lipoproteins were isolated from whole blood treated with wild‐type and gingipain‐mutant (lacking either the Rgp‐ or Kgp gingipains) P. gingivalis by density/gradient‐ultracentrifugation and were studied using 2‐dimensional gel electrophoresis followed by matrix‐assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis‐induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid‐reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation.Results Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB‐100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high‐density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.Conclusion Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.

Transcriptome Analysis of B Cell Immune Functions in Periodontitis: Mucosal Tissue Responses to the Oral Microbiome in Aging.

Evidence has shown activation of T and B cells in gingival tissues in experimental models and in humans diagnosed with periodontitis. The results of this adaptive immune response are noted both locally and systemically with antigenic specificity for an array of oral bacteria, including periodontopathic species, e.g., Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. It has been recognized through epidemiological studies and clinical observations that the prevalence of periodontitis increases with age. This report describes our studies evaluating gingival tissue transcriptomes in humans and specifically exploiting the use of a non-human primate model of naturally occurring periodontitis to delineate gingival mucosal tissue gene expression profiles focusing on cells/genes critical for the development of humoral adaptive immune responses. Patterns of B cell and plasmacyte genes were altered in aging healthy gingival tissues. Substantial increases in a large number of genes reflecting antigen-dependent activation, B cell activation, B cell proliferation, and B cell differentiation/maturation were observed in periodontitis in adults and aged animals. Finally, evaluation of the relationship of these gene expression patterns with those of various tissue destructive molecules (MMP2, MMP9, CTSK, TNFα, and RANKL) showed a greater frequency of positive correlations in healthy tissues versus periodontitis tissues, with only MMP9 correlations similar between the two tissue types. These results are consistent with B cell response activities in healthy tissues potentially contributing to muting the effects of the tissue destructive biomolecules, whereas with periodontitis this relationship is adversely affected and enabling a progression of tissue destructive events.

S-nitrosoglutathione accelerates recovery from 5-fluorouracil-induced oral mucositis.

IntroductionMucositis induced by anti-neoplastic drugs is an important, dose-limiting and costly side-effect of cancer therapy.AimTo evaluate the effect of the topical application of S-nitrosoglutathione (GSNO), a nitric oxide donor, on 5-fluorouracil (5-FU)-induced oral mucositis in hamsters.Materials and MethodsOral mucositis was induced in male hamsters by two intraperitoneal administrations of 5-FU on the first and second days of the experiment (60 and 40 mg/kg, respectively) followed by mechanical trauma on the fourth day. Animals received saline, HPMC or HPMC/GSNO (0.1, 0.5 or 2.0 mM) 1 h prior to the 5-FU injection and twice a day for 10 or 14 days. Samples of cheek pouches were harvested for: histopathological analysis, TNF-α and IL-1β levels, immunohistochemical staining for iNOS, TNF-α, IL-1β, Ki67 and TGF-β RII and a TUNEL assay. The presence and levels of 39 bacterial taxa were analyzed using the Checkerboard DNA-DNA hybridization method. The profiles of NO released from the HPMC/GSNO formulations were characterized using chemiluminescence.ResultsThe HPMC/GSNO formulations were found to provide sustained release of NO for more than 4 h at concentration-dependent rates of 14 to 80 nmol/mL/h. Treatment with HPMC/GSNO (0.5 mM) significantly reduced mucosal damage, inflammatory alterations and cell death associated with 5-FU-induced oral mucositis on day 14 but not on day 10. HPMC/GSNO administration also reversed the inhibitory effect of 5-FU on cell proliferation on day 14. In addition, we observed that the chemotherapy significantly increased the levels and/or prevalence of several bacterial species.ConclusionTopical HPMC/GSNO accelerates mucosal recovery, reduces inflammatory parameters, speeds up re-epithelization and decreases levels of periodontopathic species in mucosal ulcers.

Biomarkers of periodontitis and inflammation in ischemic stroke: A case-control study

Periodontitis is a common infectious disease associated with increased risk for ischemic stroke though presently unclear mechanisms. In a case-control study, we investigated salivary levels of four periodontal pathogens, as well as systemic and local inflammatory markers. The population comprised 98 patients with acute ischemic stroke (mean ± SD, 68.2 ± 9.7 yrs; 45.9% women) and 100 healthy controls (69.1 ± 5.2 yrs; 47.0% women). Patients were more often edentulous and had fewer teeth than controls (13.8 ± 10.8 versus 16.6 ± 10.1). After adjusting for stroke risk factors and number of teeth, controls had higher saliva matrix metalloproteinase-8 (MMP-8), myeloperoxidase (MPO), IL-1β, Aggregatibacter actinomycetemcomitans, and serum LPS activity levels. Patients had higher serum MMP-8 and MPO, and they were more often qPCR-positive for A. actinomycetemcomitans (37.9% versus 19.0%) and for ≥3 periodontopathic species combined (50.0% versus 33.0%). We conclude that controls more often had evidence of current periodontal infection with higher periodontal pathogen amount, endotoxemia, local inflammation and tissue destruction. Stroke patients more often had evidence of end-stage periodontitis with edentulism and missing teeth. They were more often carriers of several periodontopathic pathogens in saliva, especially A. actinomycetemcomitans. Additionally, inflammatory burden may contribute to high systemic inflammation associated with elevated stroke susceptibility.

Distribution of periodontopathic bacterial species in dogs and their owners

ObjectivePresently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners. DesignDental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis, Porphyromonas gulae, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens were performed. ResultsP. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them. ConclusionsThese results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.

Periodontal vaccine: A short synopsis

Immunization using a vaccine against periodontitis decreases the number of periodontopathic bacteria in the subgingival flora and inhibits alveolar bone destruction in several animal models. The foremost step in vaccine development is identification of an antigenic component from various organisms that can provide immune protection. Identification of such an antigen is made difficult by the fact in that the periodontopathic species has a complex clonal structure with multiple serotypes, and no single type or groups of types have been documented to cause human periodontitis. This review highlights the importance of Porphyromonas gingivalis and its antigenic components as a potential candidate for periodontal vaccination.

Evaluation of the subgingival microbiota of alcoholic and non-alcoholic individuals

ObjectivesThe aim of this study was to evaluate the composition of the subgingival microbiota of alcoholic and non-alcoholic individuals. MethodsThe study was conducted with 49 alcoholic and 49 non-alcoholic males of the Philippe Pinel Institute, Rio de Janeiro, Brazil. The subjects were selected by convenience and two criteria were used to diagnose alcohol dependence: the CAGE (cut-down, annoyed, guilt, eyes-opener) questionnaire and the International Statistical Classification of Diseases, 10th edition (WHO). Subgingival biofilm samples were obtained from 4 sites, 2 with probing depth (PD)≥4mm and 2 sites with PD<4mm. The presence and levels of 45 bacterial taxa were analysed using the checkerboard technique. ResultsThe prevalence of bacterial species was not significantly different between groups. However, alcoholics showed significantly higher mean counts of Aggregactibacter actinomycetemcomitans, Fusobacterium nucleatum nucleatum, and Porphyromonas gingivalis (adjusted p<0.001). Moreover, alcoholics harboured significantly higher mean levels of Capnocytophaga sputigena, Fusobacterium nucleatum vincentii, F. nuc. nucleatum, Gemella morbillorum, Neisseria mucosa, P. gingivalis, Streptococcus gordonii, and Tannerella forsythia at sites with PD <4mm or ≥4mm compared to non-alcoholics (p≤0.001). Of interest, shallow sites of alcoholics presented significantly higher mean levels of F. nuc. vincentii, F. nuc nucleatum, P. gingivalis, and T. forsythia than sites with PD≥4mm of non-alcoholics (p≤0.001). ConclusionsAlcoholics and non-alcoholics present a diverse and complex microbiota; however, alcoholics harbour significantly higher levels of periodontopathic species in the subgingival microbiota than non-alcoholics.

Efficacy of taurolidine against periodontopathic species—an in vitro study

The antimicrobial effect of taurolidine was tested against periodontopathic species in comparison to chlorhexidine digluconate in the presence or absence of serum. Minimal inhibitory concentrations (MIC), microbiocidal concentrations (MBC), as well as killing were determined against 32 different microbial strains including 3 Porphyromonas gingivalis, 3 Aggregatibacter actinomycetemcomitans, and 15 potentially superinfecting species with and without 25% v/v human serum. The MIC(50) of taurolidine against the tested microbial strains was 0.025% and the MIC(90) 0.05%. The respective values for the MBCs were 0.05% and 0.1%. Addition of 25% serum (heat-inactivated) did not change the MIC and MBC values of taurolidine. In contrast, MICs and MBCs of chlorhexidine (CHX) increased by two steps after addition of serum. Taurolidine killed microorganisms in a concentration and time-dependent manner, the killing rate of 1.6% taurolidine was 99.08% ± 2.27% in mean after 2 h. Again, killing activity of taurolidine was not affected if serum was added, whereas addition of inactivated serum clearly reduced the killing rate of all selected bacterial strains by CHX. Therefore, taurolidine possesses antimicrobial properties which are not reduced in the presence of serum as a main component in gingival crevicular fluid and wound fluid. Taurolidine may have potential as an antimicrobial agent in non-surgical and surgical periodontal treatment.

Successful application of molecular biological technique for evaluation of changes in periodontopathic bacteria in Japanese children with developmental disabilities

Developments in molecular biological techniques enables rapid and easy identification of periodontopathic bacterial species in clinical specimens. However, there are few reports regarding their application for community dentistry. The aim of this study was to show successful application of a molecular biological technique for evaluation of changes in periodontal bacterial species in children at daycare centers. We studied 187 children who received oral examinations in 2009 and 186 who received examinations in 2010, among whom 102 were examined in both years. Clinical parameters regarding periodontal conditions were evaluated and the distribution of 10 periodontopathic species in dental plaque specimens were determined by polymerase chain reaction. Periodontal pocket depth values in the 2010 group were significantly smaller than those in 2009. When the subjects were divided into those with (positive group) and without (negative group) Porphyromonas gingivalis , Treponema denticola , Tannerella forsythia , or Campylobacter rectus in 2009, the positive group had significantly smaller periodontal pocket values than the negative group. In addition, the rate of subjects with P. gingivalis , T. denticola , T. forsythia , or C. rectus in the positive group in 2010 was significantly reduced. Our findings demonstrate that molecular biological methods provide more information as compared to a standard clinical examination when evaluating changes of periodontal conditions in the field of community dentistry.

Distribution of periodontopathic bacterial species in Japanese children with developmental disabilities

BackgroundRecent developments in molecular biological techniques have enabled rapid detection of periodontopathic bacterial species in clinical specimens. Accumulated evidence suggests that detection of specific bacterial species enables identification of subjects at high risk for the onset of periodontitis. We investigated the distribution of 10 selected periodontopathic bacterial species in dental plaque specimens obtained from children with disabilities who were attending daycare centers.MethodsA total of 187 children (136 boys, 51 girls) aged 1-6 years old and diagnosed with such disabilities as mental retardation, cerebral palsy, and autism, participated in the study. Subgingival dental plaque specimens were collected from the buccal side of the maxillary left second primary molar after a clinical examination. Bacterial DNA was extracted from the specimens and PCR analyses were carried out to detect 10 selected periodontopathic species using specific primers for each. In addition, statistical analyses were performed to analyze the correlations among clinical parameters and the detected species.ResultsThe most frequently detected species was Capnocytophaga sputigena (28.3%), followed by Aggregatibacter actinomycetemcomitans (20.9%) and Campylobacter rectus (18.2%). Eikenella corrodens, Capnocytophaga ochracea, and Prevotella nigrescence were detected in approximately 10% of the specimens, whereas Treponema denticola, Tannerella forsythia, and Prevotella intermedia were rarely found, and Porphyromonas gingivalis was not detected in any of the subjects. The total numbers of detected species were positively correlated with the age of the subjects. There were 10 subjects with positive reactions for T. denticola and/or T. forsythia, in whom the total number of bacterial species was significantly higher as compared to the other subjects. Furthermore, subjects possessing C. rectus showed significantly greater values for periodontal pocket depth, gingival index, and total number of species.ConclusionWe found that approximately one-fourth of the present subjects with disabilities who possessed at least one of T. denticola, T. forsythia, and C. rectus were at possible risk for periodontitis. Follow-up examinations as well as preventive approaches should be utilized for such individuals.

Perfiles Antimicrobianos de Bacterias Subgingivales en Pacientes con Periodontitis en Colombia

BackgroundIn vitro antimicrobial susceptibility of microorganisms associated to periodontitis has showed different results among world populations. In Latin America, few studies have described antimicrobial susceptibility of periodontopathogens. This study determined antimicrobial susceptibility of some bacteria isolated from healthy and periodontitis Colombian patients for six commonly used antimicrobials as adjunctive therapy for periodontal disease treatment. MethodsIsolates of Aggregatibacter actinomycetemcomitans (n=23), Porphyromonas gingivalis (n=74), Prevotella intermedia/nigrescens (n=38), Tannerella forsythia (n=8), Enterobacteriaceae (n=89) and non fermenters Gram negative rods (n=26) from subgingival samples of 127 patients with chronic periodontitis, 68 with aggressive periodontitis and 11 healthy /gingivitis patients were tested by means of the E-test®. The minimal inhibitory concentration (MIC) of tetracycline, ciprofloxacin, amoxicillin, metronidazole, azythromicin and clindamycin was tested for each isolate. ResultsHigh antimicrobial susceptibility of periodontopathogens was found to most of tested antimicrobials. However, A. actinomycetemcomitans showed resistance to metronidazole, amoxicillin and clindamycin and P. intermedia/nigrescens showed resistance to most of tested antimicrobials. No differences of susceptibilities to antimicrobials were observed between periodontal diagnoses. Gram negative rods were resistant to amoxicillin and susceptible to ciprofloxacin. ConclusionsAntimicrobial supportive therapy for the treatment of Colombian patients affected with periodontitis should be carefully administered considering that there is a great variability of susceptibility to antimicrobial agents among periodontopathic species.

Detection of Dialister pneumosintes in the subgingival biofilm of subjects with periodontal disease

Dialister pneumosintes has been indicated as a potentially new periodontopathic species. This study evaluated the prevalence of this microorganism in saliva and subgingival biofilm from subjects with different periodontal conditions. Subgingival biofilm and saliva samples from 48 subjects with periodontal health (PH) and 116 patients with chronic periodontitis (CP) were obtained. DNA was extracted from the samples and the presence of D. pneumosintes was determined by PCR. Differences in clinical parameters and frequency of D. pneumosintes between groups were sought by Mann–Whitney, Chi-square and Fisher's exact tests. Overall, D. pneumosintes was detected in 47.8% of the biofilm samples, but only in 3% of saliva samples. CP patients presented a significantly greater mean prevalence of this species in sites with periodontal health and periodontal infection (43.5±7.4% and 62.1±6.4%, respectively) than PH subjects (29.4±7.9%) (Mann–Whitney; p<0.01). Moreover, significant associations between the prevalence of D. pneumosintes and pocket depth ( p=0.001), attachment loss ( p=0.001) and bleeding on probing (GLM, p=0.014) were observed after adjusting for age and gender. These findings corroborate the association of D. pneumosintes with periodontitis.

Flow cytometry to monitor phagocytosis and oxidative burst of anaerobic periodontopathogenic bacteria by human polymorphonuclear leukocytes.

The reduced susceptibility to phagocytosis found among some periodontopathogenic anaerobes may account for the differences between invasive and non-invasive strains. We applied flow cytometry as a powerful tool to analyze and quantify phagocytosis using standardized cultures of oral anaerobes (Porphyromonas gingivalis, Prevotella intermedia, P. nigrescens, Capnocytophaga gingivalis, C. ochracea, C. sputigena, Fusobacterium nucleatum and Peptostreptococcus micros) and heparinized whole blood. Bacteria were labeled by a fluorescein-methylester and their esterase activity, resulting in green fluorescence. Ingested bacteria could be detected easily and quantified by a shift towards green fluorescence in the PMNL population involved and a concomitant decrease in the bacterial population. Furthermore, the oxidative burst of PMNLs was detected in parallel assays using the dye DHR123 which becomes fluorescent upon oxidation during the oxidative burst process. We found a great diversity in phagocytosis susceptibility determined by estimating the portion of phagocytosing PMNLs, ranging from 10.6% (strain W83) to > 99.4% (e.g. ATCC 33277T) in P. gingivalis and from 15.9% (strain MH5) to > 95% (ATCC 33563T) in P. nigrescens. In contrast, almost all P. intermedia strains as well as the representatives of the other anaerobic, putative periodontopathic species tested showed no or only moderate resistance in the phagocytosis assay. Comparison of clinical data of patients and the extent of phagocytosis resistance of the corresponding P. gingivalis strains suggests that this virulence factor may contribute to the clinical outcome.

The adherence of periodontopathogens to periodontal probes. A possible factor in intra-oral transmission?

Periodontal probes have previously been shown to harbor several bacterial types or species after probing periodontally diseased pockets. This study aims to identify and quantify periodontopathogens that may adhere to a periodontal probe by culturing techniques. It also examines the probe's roughness on its capability to collect bacteria, comparing Merrit-B probes (with deep indentations) with TPS probes (with smooth surfaces). From the differential phase contrast microscopy findings it was seen that, while paper-points harbored nearly 50% motile rods or spirochetes, the periodontal probes were just at, or below, the 20% threshold level for pathogenicity (23.6% for the Merrit-B probe and 11.3% for the TPS probe). The cultural data showed that paper-points had significantly higher (P < 0.05) numbers of anaerobic bacteria than the 2 probe types, which still harbored up to 10(7) CFU. No significant differences could be detected between the probes. When specific periodontopathic species were considered, it was seen that for all species, even for Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis, the detection frequency was comparable for the 3 sampling devices. However, the levels of Prevotella intermedia and Campylobacter rectus was significantly higher in samples from paper-points (P < 0.05), but still their numbers reached even 10(5) on the probes. Differences among the 2 probe types were again negligible. Periodontal probes harbor relatively high numbers of bacteria found in periodontal pockets and may be able to carry them over to other sites. Further studies are needed to determine if, and to what extent, transmission occurs during periodontal probing.

Detection of Two Anaerobic Periodontopathogens in Children by Means of the BANA and ELISA Assays

The mouths of young children become colonized by a variety of bacteria, but there have been only a few studies that have sought the presence of periodontopathic species in this population. Almost all of these studies used culturing techniques rather than the newer detection methodologies for various periodontopathogens. Studies in adults have shown that Treponema denticola and Porphyromonas (Bacteroides) gingivalis can be detected in dental plaque by use of the BANA and ELISA diagnostic tests. In the present study, plaque samples from four subgingival sites in each of 157 children (aged from two to 18 years) were tested for BANA hydrolysis with a BANA reagent card, and for T. denticola and P. gingivalis with an ELISA assay. Anaerobic periodontopathogens hydrolyzing the BANA substrate were found to be present in at least one of four plaque samples in 88 children (56%). T. denticola and/or P. gingivalis were detected by ELISA in at least one plaque sample in each of 135 children (86%). This study shows that children are widely colonized by these micro-organisms. A higher proportion of Black children than Caucasian children was colonized by these BANA-positive organisms. Also, children having a parent with a documented history of periodontal disease were more likely to be BANA-positive than were children of parents with unknown periodontal status.

Factors in Virulence Expression and Their Role in Periodontal Disease Pathogenesis

The classic progression of the development of periodontitis with its associated formation of an inflammatory lesion is characterized by a highly reproducible microbiological progression of a Gram-positive microbiota to a highly pathogenic Gram-negative one. While this Gram-negative microbiota is estimated to consist of at least 300 different microbial species, it appears to consist of a very limited number of microbial species that are involved in the destruction of periodontal diseases. Among these "putative periodontopathic species" are members of the genera Porphyromonas, Bacteroides, Fusobacterium, Wolinella, Actinobacillus, Capnocytophaga, and Eikenella. While members of the genera Actinomyces and Streptococcus may not be directly involved in the microbial progression, these species do appear to be essential to the construction of the network of microbial species that comprise both the subgingival plaque matrix. The temporal fluctuation (emergence/disappearance) of members of this microbiota from the developing lesion appears to depend upon the physical interaction of the periodontal pocket inhabitants, as well as the utilization of the metabolic end-products of the respective species intimately involved in the disease progression. A concerted action of the end-products of prokaryotic metabolism and the destruction of host tissues through the action of a large number of excreted proteolytic enzymes from several of these periodontopathogens contribute directly to the periodontal disease process.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in periodontal disease-associated microorganisms of subgingival plaque in prepubertal, pubertal and postpubertal children.

It is generally accepted that normal prepubertal children do not develop periodontitis, and that the severity of gingivitis in prepubertal children is usually less than that observed in children after puberty. One possible explanation is that the bacteria associated with periodontal diseases cannot become established in great numbers prior to puberty. Studies by Kornman and Loesche and others suggest that levels of black pigmented Bacteroides, especially B. intermedius, increase with increased levels of gonadotrophic hormones in pregnant women. Delaney and Kornman have found that there is a similar increase in levels of black pigmented Bacteroides with puberty. The present study involved cultural and microscopic characterization of the subgingival plaque flora of prepubertal, circumpubertal and postpubertal children with similar Silness and Löe plaque index scores. Puberty was confirmed through examination of wrist radiographs. Populations of black pigmented Bacteroides were very low in prepubescent children and were much higher in circumpubertal and postpubertal children. However, B. intermedius predominated only in circumpubertal plaques. Levels of total motile bacteria increased at each age level, but levels of spirochetes above 2% were observed only in the postpubertal group. These results support those of previous investigators who postulated a relationship between hormone levels and black pigmented Bacteroides levels in subgingival plaque and suggest that differences in the subgingival environment profoundly influence the proportions of suspected periodontopathic species in plaque.