Interaction of oral bacteria with gingival epithelial cell multilayers

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air–liquid interface in low‐calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐6 and IL‐8 secretion; and F. nucleatum stimulated production of IL‐1β and TNF‐α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.     

Bactericidal effects of a high‐power,red light‐emitting diode on two periodontopathic bacteria in antimicrobial photodynamic therapy in vitro

Aim: Light‐emitting diodes have been investigated as new light activators for photodynamic therapy. We investigated the bactericidal effects of high‐power, red light‐emitting diodes on two periodontopathic bacteria in vitro. Methods: A light‐emitting diode (intensity: 1100 mW/cm², peak wavelength: 650 nm) was used to irradiate a bacterial solution for either 10 or 20 s. Bacterial solutions (Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans) at a concentration of 2.5 × 10⁶ c.f.u./mL were mixed with an equal volume of either methylene blue or toluidine blue O (0–20 μg/mL) and added to titer plate wells. The plate wells were irradiated with red light‐emitting diode light from a distance of 22 or 40 mm. The contents were diluted, and 50 μL was smeared onto blood agar plates. After 1 week of culturing, bacterial c.f.u. were counted. Results: The light‐emitting diode energy density was estimated to be approximately 4 and 8 J/cm² after 10 and 20 s of irradiation, respectively. Red light‐emitting diode irradiation for 10 s from a distance of 22 mm, combined with methylene blue at concentrations >10 μg/mL, completely killed Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Conclusion: High‐power, red light‐emitting diode irradiation with a low concentration of dye showed effective bactericidal effects against two periodontopathic bacteria.     

Detection of periodontal bacteria in atheromatous plaque by nested polymerase chain reaction

Background: In recent years, increasing evidence regarding the potential association between periodontal diseases and cardiovascular diseases has been identified. The available evidence underlines the importance of detecting periodontal pathogens on atheromatous plaque as the first step in demonstrating the causal relationship between the two conditions. The main aim of this investigation is to detect periodontitis‐associated bacteria from carotid artery atheromatous plaque from patients who received an endarterectomy using strict sample procurement and laboratory procedures. Methods: Atheromatous plaque from endarterectomies from carotid arteries were scraped and homogenized, and bacterial DNA was extracted. To obtain a representative concentration of amplicons, two amplifications of the bacterial 16S ribosomal‐RNA gene were carried out for each sample with universal eubacteria primers by a polymerase chain reaction (PCR). A nested PCR with specific primers for the target bacteria was performed next. Statistical tests included the χ² test. Results: Forty‐two atheromatous plaque were analyzed. All of them were positive for ≥1 target bacterial species. The bacterial species most commonly found was Porphyromonas gingivalis (78.57%; 33 of 42), followed by Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) (66.67%; 28 of 42), Tannerella forsythia (previously T. forsythensis) (61.90%; 26 of 42), Eikenella corrodens (54.76%; 23 of 42), Fusobacterium nucleatum (50.00%; 21 of 42), and Campylobacter rectus (9.52%; four of 42). The simultaneous presence of various bacterial species within the same specimen was a common observation. Conclusion: Within the limitations of this study, the presence of DNA from periodontitis‐associated bacteria in carotid artery atheromatous plaque retrieved by endarterectomy is confirmed.     

Combining Salivary Pathogen and Serum Antibody Levels Improves Their Diagnostic Ability in Detection of Periodontitis

Background: Initiation and progression of periodontitis correlates with increased quantities of periodontitis‐associated bacteria in periodontal biofilms. In the present study, the aim is to measure Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis amounts in saliva and their antibody (immunoglobulin [Ig]A and IgG) levels in serum and evaluate their diagnostic abilities, together or alone, in chronic periodontitis. Methods: The study population comprised 230 Finnish dentate adults: 84 with generalized chronic periodontitis (GCP), 65 with localized chronic periodontitis (LCP), and 81 controls without periodontitis. General and oral health information was obtained by questionnaires, interviews, and clinical and radiographic examinations. Salivary and serum samples were analyzed by quantitative single copy gene–based real‐time polymerase chain reaction and multiserotype enzyme‐linked immunosorbent assay, respectively. Results: Pathogen carriers suffered mostly from GCP and seldom from LCP. A. actinomycetemcomitans and P. gingivalis quantities in saliva were strongly associated with corresponding serum IgA and IgG values (P <0.001) and with severity of disease (P <0.001). P. gingivalis exhibited more straightforward associations among salivary bacterial burdens, corresponding antibody formation, and periodontitis severity than A. actinomycetemcomitans. The combination of information on age, sex, smoking, and P. gingivalis results provided an area under the curve of 0.817 (95% confidence interval 0.76 to 0.87, P <0.001) for GCP. Conclusion: The combination of saliva P. gingivalis quantity with pathogen‐specific host response may be used to diagnose periodontitis with high accuracy.     

Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans

Background and Objective: This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. Material and Methods: Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patient’s own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol‐dependent and isoluminol‐dependent chemiluminescence. Results: Polymorphonuclear neutrophils from patients with chronic (62.16 ± 19.39%) and aggressive (43.26 ± 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 ± 19.87%) at the 30‐min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. Conclusion: High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues.     

Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine‐specific cysteine proteinase (Rgp) and lysine‐specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT‐1 and KYT‐36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm‐destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co‐culture with P. gingivalis. However, this was not found after the addition of KYT‐36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.     

Subgingival Bacterial Burden in Relation to Clinical and Radiographic Periodontal Parameters

Background: This cross‐sectional study characterizes the association between subgingival bacterial profile and periodontal parameters in patients assigned to coronary angiography because of cardiologic problems, which may affect the oral microbiota. Methods: Pooled subgingival bacterial samples were collected from 477 dentate individuals during the oral examinations, along with periodontal probing depth (PD) and assessments of bleeding on probing (BOP) and radiographic alveolar bone loss (ABL). The checkerboard DNA–DNA hybridization assay was used to determine the levels of 29 oral bacteria, which were divided into three bacterial complexes. Results: All bacterial combinations from the etiologic bacterial group and each species from the red complex were significantly associated (P <0.001) with grade of ABL. The prevalence of the etiologic bacterial group and the level of each species were also associated strongly with the proportion of sites with PD 4 to 5 mm and ≥6 mm, BOP, and ABL, except Aggregatibacter actinomycetemcomitans. Levels of Gram‐negative oral bacteria correlated significantly with those of Gram‐positive species (r = 0.840, P <0.001). In multiple logistic regression analysis, the prevalence of the etiologic bacterial group, levels of Gram‐negative bacteria and Treponema denticola, and the prevalence of Porphyromonas gingivalis and T. denticola associated significantly with ABL, whereas other bacterial complexes and levels of Gram‐positive species did not. Conclusions: Although levels of Gram‐negative and ‐positive species paralleled periodontal parameters, only the species considered etiologic were associated with ABL.     

Antimicrobial Activity of Platelet‐Rich Plasma and Other Plasma Preparations Against Periodontal Pathogens

Background: In addition to releasing a pool of growth factors during activation, platelets have many features that indicate their role in the anti‐infective host defense. The antimicrobial activities of platelet‐rich plasma (PRP) and related plasma preparations against periodontal disease–associated bacteria were evaluated. Methods: Four distinct plasma fractions were extracted in the formulation used commonly in dentistry and were tested for their antibacterial properties against three periodontal bacteria: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. The minimum inhibitory concentration of each plasma preparation was determined, and in vitro time‐kill assays were used to detect their abilities to inhibit bacterial growth. Bacterial adhesion interference and the susceptibility of bacterial adherence by these plasma preparations were also conducted. Results: All plasma preparations can inhibit bacterial growth, with PRP showing the superior activity. Bacterial growth inhibition by PRP occurred in the first 24 hours after application in the time‐kill assay. PRP interfered with P. gingivalis and A. actinomycetemcomitans attachment and enhanced exfoliation of attached P. gingivalis but had no influences on F. nucleatum bacterial adherence. Conclusions: PRP expressed antibacterial properties, which may be attributed to platelets possessing additional antimicrobial molecules. The application of PRP on periodontal surgical sites is advisable because of its regenerative potential and its antibacterial effects.     

Carpegen® real‐time polymerase chain reaction vs. anaerobic culture for periodontal pathogen identification

Background/aims: The aim of this study was to compare two methods of microbiological diagnosis, anaerobic bacterial culture and real‐time polymerase chain reaction (PCR), for the detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, and Treponema denticola. Methods: Seventy‐two samples were collected from 18 patients who were suffering from aggressive periodontitis. The data obtained were compared for the two methods. Results: The results obtained with real‐time PCR were different from those obtained with bacterial culture. The detection differences were 3% for A. actinomycetemcomitans, 8.33% for P. intermedia, and 12.5% for F. nucleatum. However, the differences for P. gingivalis and T. forsythia were 51.39% and 36.11%, respectively. No comparison was possible for T. denticola because it cannot be identified in culture. The variations found were the result of the better detection level (10² pathogens) of the PCR probe. Unlike bacterial culture, PCR allows the detection of T. denticola, which does not forming colonies and is oxygen sensitive. For F. nucleatum, T. forsythia and P. gingivalis, the real‐time PCR technique was more sensitive than culture. Conclusion: Good results were obtained with the real‐time PCR technique for the six periopathogens targeted. This method seems to be indicated for its simplicity, rapidity and reproducibility but it cannot analyze data for an antibiotic susceptibility test. The periodontist must therefore choose one of these two methods according to his specific clinical objective: to obtain rapid, specific detection even with weak initial concentrations (but for targeted periopathogens only) or to be non‐specific and analyze the pathological activity with an antibiogram.     

Detection rates of presumptive periodontal pathogens in subgingival plaque samples of untreated periodontitis using either four or six pooled samples

Aim: A comparison of the detection frequency and number of periodontal pathogens in patients with aggressive or generalized, severe chronic periodontitis using a gene‐probe analysis. Methods: In 16 aggressive and 34 generalized, severe chronic periodontitis patients, plaque was sampled from the deepest pockets per quadrant (MT4) and per sextant (MT6). After sampling two paper points simultaneously, one paper point from each pocket was pooled with three paper points of the other pockets (MT4). The remaining four paper points were pooled with two paper points from the deepest pockets from the two remaining sextants (MT6). Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected by 16S rRNA gene probes. Results: Log‐transformed counts for Aggregatibacter actinomycetemcomitans were statistically significantly higher with MT6 (aggressive: 3.21 ± 2.94; generalized, severe chronic: 2.22 ± 2.70) than MT4 (aggressive: 2.04 ± 2.74; generalized, severe chronic: 1.50 ± 2.37) (P < 0.05). The detection frequency and mean counts were high for Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (>95%/>6.0). Conclusion: Aggregatibacter actinomycetemcomitans was detected in higher numbers for MT6 than MT4. For both MT4 and MT6, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected in >95% of all patients and with mean log‐transformed numbers >6.0.     

Activation of toll‐like receptors 2 and 4 by gram‐negative periodontal bacteria

Background/aims: Periodontitis is a chronic infectious disease associated with a gram‐negative subgingival microflora. Bacterial components stimulate, among other receptors, Toll‐like receptor (TLR) 2 and/or TLR4. Accumulating evidence indicates that both qualitatively and quantitatively distinct immune responses result from the triggering of TLR2 as compared to TLR4 triggering. The aim was to study the interaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum and Veillonella parvula with TLR2 and TLR4. We investigated all known serotypes (K⁻, K1–K6) of P. gingivalis and A. actinomycetemcomitans serotype a–e strains for their potency to stimulate cytokine production. Methods: Human embryonic kidney (HEK) cells, stably transfected with CD14, CD14‐TLR2, or CD14‐TLR4 and whole blood were stimulated with bacterial sonicates. Cytokine production (interleukin‐6, ‐8, ‐10 and ‐12) was measured in the supernatant by enzyme‐linked immunosorbent assay. Results: All test bacteria stimulated HEK‐CD14‐TLR2, but only A. actinomycetemcomitans and V. parvula stimulated HEK‐CD14‐TLR4. No differences were found in the activation of HEK‐CD14‐TLR2/4, or cytokine production in whole blood between serotypes of P. gingivalis and A. actinomycetemcomitans. Conclusion: Gram‐negative periodontal bacteria predominantly stimulated TLR2, which may be of importance for the Th1/Th2 cell orientation of the immune response in periodontitis.     

Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro

Objectives: The aim of this study was to analyse the influence of the microtopography and hydrophilicity of titanium (Ti) substrates on initial oral biofilm formation. Materials and methods: Nine bacterial species belonging to the normal oral microbiota, including: Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus sanguinis were tested on Ti surfaces: pretreatment (PT [R_a<0.2 μm]), acid‐etched (A [R_a<0.8 μm]), A modified to be hydrophilic (modA), sand‐blasted/acid‐etched (SLA [R_a=4 μm]), and hydrophilic SLA (modSLA). Disks were incubated for 24 h in anaerobic conditions using a normal culture medium (CM) or human saliva (HS). The total counts of bacteria and the proportion of each bacterial species were analysed by checkerboard DNA–DNA hybridization. Results: Higher counts of bacteria were observed on all surfaces incubated with CM compared with the samples incubated with HS. PT, SLA, and modSLA exhibited higher numbers of attached bacteria in CM, whereas SLA and modSLA had a significant increase in bacterial adhesion in HS. The proportion of the species in the initial biofilms was also influenced by the surface properties and the media used: SLA and modSLA increased the proportion of species like A. actinomycetemcomitans and S. sanguinis in both media, while the adhesion of A. israelii and P. gingivalis on the same surfaces was affected in the presence of saliva. Conclusions: The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used. To cite this article:
Almaguer‐Flores A, Olivares Navarrete R, Wieland M, Ximénez‐Fyvie LA, Schwartz Z, Boyan BD. Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro.
Clin. Oral Impl. Res. 23 , 2012; 301–307.
doi: 10.1111/j.1600‐0501.2011.02184.x     

Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis

Background: The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine‐specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified. Methods: GCF was sampled from four sites per patient (one sample per quadrant using two samples per method) in 36 patients with chronic periodontitis. One week later, the procedure was repeated with alternative methods. Variables determined were loads of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and P. gingivalis, levels of interleukin‐6 and ‐8, activity of neutrophil elastase, and level of Rgps. Results: The detected cytokine levels were higher using paper strips compared to paper points. Bacteria were found in similar loads from paper strips and paper points. Rgps were only detectable in high quantities by washing the periodontal pocket. The level of Rgps correlated with the load of P. gingivalis. Conclusions: The use of paper strips was suitable for the simultaneous determination of microbial and immunologic parameters. Obtaining GCF by washing can be useful for special purposes. The gingipain concentration in periodontal pockets was directly determined to be ≤1.5 μM. This value indicated that most of the substrates of these proteases by in vitro assays identified until now can be easily degraded in P. gingivalis–infected sites.     

Interleukin‐1α stimulation in monocytes by periodontal bacteria: antagonistic effects of Porphyromonas gingivalis

Periodontal pathogenic bacteria are associated with elevated levels of interleukin‐1α (IL‐1α) but it is unclear if all species can induce cytokine production equally. Porphyromonas gingivalis may be able antagonize IL‐1α induced by other species through the activity of its proteases or lipopolysaccharide (LPS). Monomac‐6 cells and primary human monocytes were treated with culture supernatants from Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedius, Veillonella atypical and Prevotella nigrescens. IL‐1α protein levels were measured after 6 h of incubation. In addition, monocytes were co‐stimulated with supernatants from P. gingivalis and other bacteria. The role of P. gingivalis proteases was tested using Arg‐X and Lys‐X mutant strains. The role of LPS was investigated using purified P. gingivalis LPS and polymixin depletion. All species tested induced significant IL‐1α production, but P. gingivalis was the weakest. Co‐stimulation of monocytes with P. gingivalis antagonized the ability of other bacterial species to induce IL‐1α production. This effect was at its greatest with C. rectus (resulting in a 70% reduction). Gingipain mutant strains and chemical inhibition of protease activity did not reduce antagonistic activity. However, 100 ng/ml of P. gingivalis LPS can reproduce the antagonistic activity of P. gingivalis culture supernatants. Periodontitis‐associated bacterial species stimulate IL‐1α production by monocytes. P. gingivalis can antagonize this effect, and its LPS appears to be the crucial component. This study highlights the importance of mixed infections in the pathogenesis of periodontal disease because reduction of pro‐inflammatory cytokine levels may impair the ability of the host to tackle infection.     

Soluble Triggering Receptor Expressed on Myeloid Cells 1 (sTREM‐1) in Gingival Crevicular Fluid: Association With Clinical and Microbiologic Parameters

Background: Soluble triggering receptor expressed on myeloid cells 1 (sTREM‐1) belongs to the immunoglobulin superfamily and is involved in amplification of the inflammatory response to bacterial infection. This cross‐sectional study aims to investigate the levels of sTREM‐1 in gingival crevicular fluid (GCF) of individuals without periodontitis and with chronic periodontitis (CP) or generalized aggressive periodontitis (GAgP) and their association with the levels of key periodontal pathogens in subgingival plaque. Methods: GCF and subgingival plaque samples were obtained from healthy sites of participants without periodontitis (n = 20) and periodontitis sites of patients with CP (n = 22) and GAgP (n = 20). sTREM‐1 levels in GCF were measured by enzyme‐linked immunosorbent assay. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans levels in subgingival plaque were analyzed by quantitative real‐time polymerase chain reaction. Results: sTREM‐1 levels in GCF were higher in CP and GAgP than healthy sites by 3.6‐ and 4.4‐fold, respectively, with no significant differences between the two forms of periodontitis. Moreover, sTREM‐1 levels in GCF were positively correlated with site‐specific clinical periodontal parameters and levels of P. gingivalis, T. denticola, and T. forsythia, but not A. actinomycetemcomitans, in subgingival plaque. Conclusion: Increased GCF levels of sTREM‐1 at diseased sites and their positive correlation with clinical and microbiologic parameters strengthen the association of this inflammatory marker with periodontitis.     

Investigation and quantification of key periodontal pathogens in patients with type 2 diabetes

Field CA, Gidley MD, Preshaw PM, Jakubovics N. Investigation and quantification of key periodontal pathogens in patients with type 2 diabetes. J Periodont Res 2012; 47: 470–478. © 2012 John Wiley & Sons A/S Background and Objective: Diabetes is a recognized risk factor for periodontitis. There are conflicting data regarding whether healthy diabetic patients or diabetic patients with chronic periodontitis have an altered subgingival microbiota compared with nondiabetic individuals. The aim of the present study was to detect quantitative differences in selected periodontopathogens in the subgingival plaque of diabetic patients using TaqMan quantitative PCR. Material and Methods:  Type 2 diabetes mellitus patients with (n = 9) or without chronic periodontal disease (n = 15) were recruited and matched to nondiabetic control subjects (n = 12 periodontally healthy, n = 12 chronic periodontitis). Subgingival plaque samples were collected from deep (> 4 mm probing depth) and shallow sites (≤ 3 mm probing depth) using paper points, and Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Porphyromonas gingivalis were quantified. Results: Forty‐eight subjects (69 samples) were recruited. Marked differences were seen in the levels of all three bacterial species, relative to the total bacterial population, according to periodontal health status. Using real‐time quantitative PCR, bacterial counts for P. gingivalis were significantly higher in deep pockets of diabetic and nondiabetic subjects compared with periodontally healthy subjects (p < 0.05) but did not differ significantly between diabetics and nondiabetics. A. actinomycetemcomitans was detected in all groups in low quantities, and counts did not differ significantly between groups (p > 0.05). F. nucleatum was abundant in all groups, with no clear significant differences between groups. P. gingivalis was found in higher quantities in periodontitis than in periodontally healthy subjects (p < 0.05). Statistically significant positive correlations were identified between pocket depth and counts for all three species tested (p < 0.05). Conclusion: A. actinomycetemcomitans, F. nucleatum and P. gingivalis were present in significantly different quantities and proportions in subgingival plaque, according to periodontal disease status. No significant differences were identified between the subgingival microbiota of type 2 diabetes mellitus patients compared with nondiabetic subjects.     

In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme‐labeled antigen method

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme‐labeled antigen method was applied to detecting plasma cells producing P. gingivalis‐specific antibodies in biopsied gingival tissue of periodontitis. N‐terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg‐pro, Arg‐hgp, Lys‐pro and Lys‐hgp) were prepared by the cell‐free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme‐labeled antigen method on paraformaldehyde‐fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme‐labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.     

Response of MG63 osteoblasts on bacterial challenge is dependent on the state of differentiation

The present in vitro study examines molecular processes that are relevant during bone homeostasis after Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infection with a focus on the differentiation level of osteoblasts. Regenerative processes are often hindered by the recurrence of bacterial infections, which can ultimately provoke a severe destruction of bone tissue. To obtain more detailed insights into such a complex scenario, we have used undifferentiated MG63 osteoblast‐like cells as an experimental paradigm to examine the impact of two oral pathogens, A. actinomycetemcomitans and P. gingivalis, on proliferation, cytotoxicity and osteogenic differentiation. Cell culture experiments were performed to analyze cellular behavior. The level of genes interfering with bone tissue integrity (matrix metalloproteinases and their tissue inhibitors) and osteogenic markers (alkaline phosphatase, Runx2, human β‐defensin‐2) was compared in undifferentiated versus differentiated MG63 cells using real‐time polymerase chain reaction. Functional activity of matrix metalloproteinases was quantified by zymography. Western blot analysis was used to verify the phosphorylation state of mitogen‐activated protein kinases p38 and extracellular‐signal‐regulated kinases 1/2. When co‐cultured with undifferentiated MG63 cells, oral pathogens provoked distinct cellular effects. Only A. actinomycetemcomitans reduced cell proliferation, increased cell death, and induced osteogenic differentiation. A comparison of matrix metalloproteinase network stability in the presence of oral pathogens revealed a partial sensitivity towards P. gingivalis but not A. actinomycetemcomitans. So, beside the proof of concept that MG63 cells co‐cultured with oral pathogens can serve as an in vitro model for mimicking destructive and regenerative events after bacterial infections, our data indicate that double infections might counterbalance otherwise positive effects.