Quantitative analysis of association between herpesviruses and bacterial pathogens in periodontitis

Background and Objective: The development of human periodontitis may depend upon cooperative interactions among herpesviruses, specific pathogenic bacteria and tissue‐destructive inflammatory mediators. This study sought to identify associations among human cytomegalovirus, Epstein–Barr virus and six putative periodontopathic bacteria in periodontitis lesions. Material and Methods: Fifteen periodontitis patients (nine with aggressive periodontitis and six with chronic periodontitis) and 15 periodontally normal subjects were included in the study. In each study subject, a microbiological sample was collected, using a curette, from the deepest periodontal probing depth of the dentition. A real‐time TaqMan® polymerase chain reaction assay was employed to determine the subgingival counts of human cytomegalovirus, Epstein–Barr virus, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Campylobacter rectus. Statistical analysis was performed using the Student's t‐test, the Pearson correlation coefficient test and the single variable logistic regression test for odds ratio‐based risk calculation. Results: Human cytomegalovirus was detected in eight periodontitis lesions and in one normal periodontal site, Epstein–Barr virus was detected in nine periodontitis lesions and in two normal periodontal sites, and the study bacteria were detected in 6–15 periodontitis lesions and in 1–11 normal periodontal sites. Correlations were found between counts of human cytomegalovirus and Epstein–Barr virus, between counts of human cytomegalovirus and P. gingivalis, T. forsythia and C. rectus, and between counts of Epstein–Barr virus and P. gingivalis and T. forsythia. Human cytomegalovirus and Epstein–Barr virus counts were also positively associated with the level of periodontal attachment loss, probing pocket depth and gingival bleeding on probing. Conclusion: This study confirmed that periodontal human cytomegalovirus and Epstein–Barr virus are associated with major periodontopathic bacteria and with the severity of periodontal disease. The finding of abundant herpesviruses in periodontitis lesions redefines the pathogenic paradigm of the disease. Understanding the interplay between herpesviruses and specific bacterial species in the pathogenesis of periodontitis may form the basis for new approaches to preventing, reducing or delaying tissue breakdown from periodontal infections.     

Detection of Eight Periodontal Microorganisms and Distribution of Porphyromonas gingivalis fimA Genotypes in Chinese Patients With Aggressive Periodontitis

Background: The microbiologic feature of aggressive periodontitis (AgP) in Chinese patients has not yet been determined. This study aims to investigate the prevalence of eight periodontal microorganisms and the distribution of the Porphyromonas gingivalis fimA genotype in a cohort of Chinese patients with AgP. Methods: Saliva and pooled subgingival plaque samples were collected from 81 patients with AgP (25 with incisor–first molar type and 56 with generalized type [GAgP]) and 34 periodontally healthy controls. Eight periodontal microorganisms, including Aggregatibacter actinomycetemcomitans, P. gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, and Fusobacterium nucleatum were detected in these samples by the polymerase chain reaction (PCR). In addition, the distribution of fimA genotypes was assessed in P. gingivalis–positive individuals by PCR. Results: The prevalence of P. gingivalis, T. forsythia, T. denticola, C. rectus, P. intermedia, F. nucleatum, and A. actinomycetemcomitans in patients with AgP was significantly higher than that in healthy controls. The prevalence of A. actinomycetemcomitans in patients with GAgP was relatively low (30.4%) compared with other pathogens. Results of logistic regression analysis showed that younger patients were more likely to harbor A. actinomycetemcomitans (odds ratio = 2.85). Type II was the most prevalent fimA genotype of P. gingivalis in patients with AgP. Conclusions: P. gingivalis, T. forsythia, T. denticola, C. rectus, P. intermedia, and F. nucleatum were the predominant periodontal pathogens of patients with GAgP in China. Type II of fimA was the most prevalent genotype of P. gingivalis in patients with AgP. The prevalence of A. actinomycetemcomitans in patients with GAgP was relatively low.     

Periodontal Pathogens and Risk of Incident Cancer in Postmenopausal Females: The Buffalo OsteoPerio Study

Background: Extraoral translocation of oral bacteria may contribute to associations between periodontal disease and cancer. The associations among the presence of three orange‐complex periodontal pathogens (Fusobacterium nucleatum, Prevotella intermedia, and Campylobacter rectus), two red‐complex periodontal pathogens (Porphyromonas gingivalis and Tannerella forsythia), and cancer risk were investigated. Methods: A total of 1,252 postmenopausal females enrolled in the Buffalo Osteoporosis and Periodontal Disease Study were followed prospectively. Baseline subgingival plaque samples were assessed for the presence of periodontal pathogens using indirect immunofluorescence. Incident cancer cases were adjudicated by staff physicians via review of medical records. Cox proportional hazards regression was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of periodontal pathogens with total cancer and site‐specific cancer risk in unadjusted and multivariable‐adjusted models. Results: Neither the presence of individual pathogens nor the presence of any red‐complex pathogens was associated with total cancer or site‐specific cancers. Borderline associations were seen among the presence of any orange‐complex pathogens (F. nucleatum, P. intermedia, and C. rectus), total cancer risk (HR = 1.35, 95% CI = 1.00 to 1.84), and lung cancer risk (HR = 3.02, 95% CI = 0.98 to 9.29). Conclusions: No associations were found between the presence of individual subgingival pathogens and cancer risk. However, there were suggestions of borderline positive associations of the presence of any orange‐complex pathogens with total cancer and lung cancer risk. The study is limited by the small number of cancer cases and the assessment of only five oral bacteria. Additional research is needed to understand the possible role of periodontal disease in carcinogenesis.     

Cross‐reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis

Background/aims: Chronic infections such as those caused by Chlamydia pneumoniae and periodontopathic bacteria such as Porphyromonas gingivalis have been associated with atherosclerosis, possibly due to cross‐reactivity of the immune response to bacterial GroEL with human heat shock protein (hHSP) 60. Methods: We examined the cross‐reactivity of anti‐GroEL and anti‐P. gingivalis antibodies with hHSP60 in atherosclerosis patients and quantified a panel of six pathogens in atheromas. Results: After absorption of plasma samples with hHSP60, there were variable reductions in the levels of anti‐GroEL and anti‐P. gingivalis antibodies, suggesting that these antibodies cross‐reacted with hHSP60. All of the artery specimens were positive for P. gingivalis. Fusobacterium nucleatum, Tannerella forsythia, C. pneumoniae, Helicobacter pylori, and Haemophilus influenzae were found in 84%, 48%, 28%, 4%, and 4% of arteries, respectively. The prevalence of the three periodontopathic microorganisms, P. gingivalis, F. nucleatum and T. forsythia, was significantly higher than that of the remaining three microorganisms. Conclusions: These results support the hypothesis that in some patients, cross‐reactivity of the immune response to bacterial HSPs including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may be a possible mechanism for the association between atherosclerosis and periodontal infection.     

Transmission of Periodontopathic Bacteria from Natural Teeth to Implants

Purpose: Prevention of peri‐implantitis is essential for the success of implant rehabilitation. Infection by periodontopathic bacteria is a major cause of peri‐implantitis. The aim of the present study was to identify the source of peri‐implant colonization by periodontopathic bacteria. Materials and Methods: Twenty‐one patients with implants were enrolled in the study. Subgingival plaque samples from the adjacent, occluding, and contralateral natural teeth were collected prior to second‐stage surgery. Samples from implant sulci were then obtained 2 weeks later. Detection of periodontopathic bacteria was performed by the polymerase chain reaction. Results: The detection rates for Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum in all subgingival samples from natural teeth were similar to that in the peri‐implant sulci. Multiple logistic regression analysis revealed an association between the detection of A. actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and F. nucleatum in the gingival crevices of adjacent teeth and that of the peri‐implant sulcus, but no association for Tannerella forsythia. Conclusions: The present findings suggest that colonization by A. actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and F. nucleatum at the implant sulcus was affected by these microorganisms in the gingival crevice of adjacent teeth rather than those on occluding and contralateral teeth.     

Influence of Periodontal Status and Periodontopathogens on Levels of Oral Human β‐Defensin‐2 in Saliva

Background: Expression patterns of human β‐defensin‐2 (HBD‐2) mRNA or HBD‐2 protein concentration and periodontal diseases have been a focus of scientific research. This study compares the salivary levels of HBD‐2 protein concentration of healthy patients and patients with gingivitis and chronic periodontitis (CP) and correlates these levels with the presence of periodontopathogens. Methods: A total of 89 patients were enrolled in this study: 31 periodontally healthy, 27 with gingivitis, and 31 with CP. Plaque and gingival indices, probing depth, and clinical attachment level were measured. The presence of Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Prevotella intermedia was evaluated qualitatively by conventional polymerase chain reaction. HBD‐2 quantification in saliva was performed using an immune enzymatic assay. Frequency of periodontopathogens and HBD‐2 protein concentration was assessed. Association between HBD‐2 protein concentration (≥100 pg/mL) and the simultaneous presence of one to two, three to four, or five to six periodontopathogens was tested. Results: Although periodontally healthy individuals and patients with gingivitis showed similar HBD‐2 levels, the CP group displayed an increased level of HBD‐2. P. gingivalis, P. intermedia, T. forsythia, and T. denticola were more prevalent in CP; however, their mere presence was not related to the increased levels of HBD‐2 (Pearson correlation and multinomial logistic regression model). Conclusions: Salivary HBD‐2 protein concentration was higher in patients with CP compared with healthy individuals or patients with gingivitis. These different protein concentrations were not related to the frequency of periodontopathogens. Clinical inflammatory profile had a higher impact on salivary HBD‐2 levels than bacteria.     

Induction of calprotectin release by Porphyromonasgingivalis lipopolysaccharide in human neutrophils

Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils, monocytes/macrophages, and epithelial cells. This protein is known to be a marker for several inflammatory diseases and is detected in inflammatory gingival tissue with periodontal disease. Recently, we found that the calprotectin level in gingival crevicular fluid from periodontitis patients was significantly higher than that of healthy subjects. However, the regulation of calprotectin in periodontal disease is unclear. In the present study, we investigated the effect of lipopolysaccharides of periodontopathic bacteria on calprotectin release from human neutrophils. Neutrophils from healthy donors were treated with lipopolysaccharides from Porphyromonas gingivalis (P‐LPS), Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, and Escherichia coli. Calprotectin of neutrophil was identified by immunoblotting and calprotectin amount was determined by ELISA. Two subunits (10 and 14 kDa) of calprotectin were observed in the cell and medium fractions from neutrophils. P‐LPS increased calprotectin release from seven to 16 times the control level after 30 min and its effect appeared in a dose‐dependent manner (10–1000 ng/ml). Lipopolysaccharides from A. actinomycetemcomitans, P. intermedia, F. nucleatum, and E. coli also induced calprotectin release from neutrophils. These results suggest that lipopolysaccharides from periodontopathic bacteria induce calprotectin release from human neutrophils.     

Salivary biomarkers for predicting the progression of chronic periodontitis

ObjectivePredicting the progression of periodontitis would allow for targeted supportive periodontal therapy. The purpose of this study was to determine the usefulness of salivary biomarkers for predicting the progression of periodontitis.DesignEighty-five chronic periodontitis patients were enrolled in an 18-month longitudinal study. Amongst them, 57 experienced progression of periodontitis, indicated at the end of the 18 months by at least one site with >3 mm loss of attachment compared with baseline. We determined the levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free haemoglobin as biomarkers, as well as the counts of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, which represented the periodontal bacteria, in the stimulated saliva. The Mann–Whitney U test was used to compare patients with and without progression. After categorising the diagnostic values, the chi-square test was applied.ResultsCounts and ratios (ratio to total bacteria) of P. gingivalis and P. intermedia were found to be significant predictors of the progression of periodontitis. To increase prediction accuracy, combination analyses were performed. The combination of ALT level and the P. gingivalis ratio showed the highest likelihood (p < 0.001, sensitivity 0.40, specificity 0.96, likelihood 11.30).ConclusionOur findings suggest that salivary ALT level and the P. gingivalis ratio may be potential indicators for the progression of periodontitis. Such a salivary test could be a useful diagnostic tool for predicting periodontal disease progression.     

Quantitative analysis of microbiota in saliva, supragingival, and subgingival plaque of Chinese adults with chronic periodontitis

Objective

The aim of this study was to determine the profiles of periodontopathogenic bacteria in a Chinese population using quantitative real-time polymerase chain reaction (qRT-PCR).

Materials and methods

Twenty-four periodontally healthy Chinese subjects and 60 patients with chronic periodontitis (CP) were enrolled in this cross-sectional study. qRT-PCR was used to quantify Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia as well as total bacterial counts from 252 samples collected from the saliva, supragingival plaque, and subgingival plaque of all 84 subjects.

Results

The detection frequency of A. actinomycetemcomitans was less than 50%. F. nucleatum was detected in all subjects and CP patients had higher bacterial loads than healthy subjects. The median proportion of F. nucleatum was significantly higher in subgingival plaque than in supragingival plaque and saliva. P. gingivalis and P. intermedia had higher detection frequencies and bacterial loads in CP patients than in healthy subjects. The median proportion of P. gingivalis was significantly different among the three intraoral locations in the CP group and its proportion in subgingival plaque was 9.01%. Moreover, strong positive Spearman’s correlations were found in A. actinomycetemcomitans, P. gingivalis, and P. intermedia counts across the three intraoral locations.

Conclusion

The presence and bacteria loads of these four bacteria in this Chinese population are similar to those from other populations.

Clinical relevance

Examination of bacterial detection frequency and loads in Chinese adults may assist microbial studies of periodontal disease and will shed light on periodontal disease diagnosis and treatment using antibiotics in the Chinese population.     

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.     

Comparison of Two Different Sampling Methods for Subgingival Plaque: Subgingival Paper Points or Mouthrinse Sample?

Background: The collection of subgingival plaque samples with paper points is time‐consuming and accident‐sensitive. However, the collection of saliva is simple and contains pathogens of all intraoral surfaces. The aim of this study is to investigate whether a sampling strategy with mouthrinse (mouthrinse sample [MSP]; test) leads to results comparable with standard sampling method (multiple site test from the deepest pocket of each quadrant [MT4]; control). Methods: In 50 patients with periodontitis, subgingival plaque was sampled from the deepest pocket of each quadrant by using paper points and by gaining saliva with saline mouthrinse. Analysis was performed using a commercially available polymerase chain reaction test for 11 periodontal pathogens. Results: Detection frequency of Aggregatibacter actinomycetemcomitans (MT4/MSP: 42%/36%), Porphyromonas gingivalis (78%/66%), Tannerella forsythia (98%/84%), Treponema denticola (94%/74%), Parvimonas micra (86%/62%), Campylobacter rectus (90%/76%), Eubacterium nodatum (64%/30%), Prevotella intermedia (58%/54%), and Eikenella corrodens (90%/82%) was higher with MT4 than MSP. For Fusobacterium nucleatum (100%/100%), there was no difference between test and control. Only detection frequency of Capnocytophaga species (68%/74%) was higher with MSP than MT4. Differences were significant for P. gingivalis, T. forsythia, T. denticola, P. micra, C. rectus, and E. nodatum. Conclusions: There is no significant difference between MT4 and MSP for detection frequency of key pathogen A. actinomycetemcomitans. Key pathogens P. gingivalis, T. forsythia, T. denticola, P. micra, C. rectus, and E. nodatum show statistically higher detection frequencies with MT4.     

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.     

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.     

The periodontopathic bacteria in placenta,saliva and subgingival plaque of threatened preterm labor and preterm low birth weight cases: a longitudinal study in Japanese pregnant women

Objectives

This study determined the quantity of periodontopathic bacteria in saliva, subgingival plaque, and placenta on the threatened preterm labor (TPL) and preterm low birth weight (PLBW) subjects in order to identify specific periodontal pathogens with high association to adverse pregnancy outcomes.

Methods

We used real-time PCR with TaqMan probe and ELISA to detect the amount of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, and Prevotella intermedia in subgingival plaque, saliva, and placenta tissue, in addition to serum IgG titers against these bacteria in 28 patients with TPL and 36 healthy pregnant women.

Results

Thirteen of 64 births delivered PLBW infants. All 6 periodontopathic bacteria were detected in the placenta samples. The amount of F. nucleatum and detection frequency of T. denticola in placental samples was significantly higher in the TPL group than in the healthy group. Meanwhile, the age, anti-P. gingival IgG in serum, amount of P. gingivalis and T. forsythia in plaque samples, detection frequency of P. intermedia in saliva, and percentage of pocket probing depth ≥ 5 mm were higher in TPL-PLBW births than those in TPL-Healthy delivery (HD) group and/or in H-HD group. Ordinal logistic regression analysis revealed that the presence of F. nucleatum in placental tissues was significantly associated with TPL, while the maternal age was significantly associated with PLBW in TPL.

Conclusion

Our findings suggested all 6 bacteria may access the placenta. The increased presence of F. nucleatum in placenta might be related to TPL, while advanced maternal age might be associated with PLBW in TPL.

Clinical relevance

Periodontal therapy should be applied to reduce the deep periodontal pocket sites and the colonization of periodontal pathogens in high-risk population.

     

Azithromycin as an Adjunctive Treatment of Generalized Severe Chronic Periodontitis: Clinical,Microbiologic, and Biochemical Parameters

Background: This study examines the efficacy of azithromycin in combination with non‐surgical periodontal therapy on clinical and microbiologic parameters and gingival crevicular fluid (GCF) matrix metalloproteinases‐8 (MMP‐8) levels over 6 months in patients with severe generalized chronic periodontitis (CP). Methods: Twenty‐eight of 36 patients with severe generalized CP were included in this randomized, double‐masked, placebo‐controlled, parallel‐arm study. They were randomly assigned to azithromycin or placebo groups (500 mg, once daily for 3 days). Probing depth (PD), clinical attachment level, dichotomous presence or absence of supragingival plaque accumulation, and bleeding on probing were recorded. GCF samples were obtained from one single‐rooted tooth with PD ≥ 6 mm, whereas microbiologic samples were collected from two single‐rooted teeth with PD ≥ 6 mm. Microbiologic parameters were analyzed by quantitative real‐time polymerase chain reaction for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Prevotella intermedia, and total bacteria. GCF MMP‐8 levels were determined by immunofluorescence assay. Results: Azithromycin and placebo groups demonstrated similar but significant improvements in all clinical parameters (P <0.05). A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia, and total bacteria significantly decreased over the 6‐month period in both groups, whereas F. nucleatum was significantly reduced in all visits in the azithromycin group, with the levels also being lower compared with those of the placebo group (P <0.05). The azithromycin and placebo groups exhibited significant reduction in GCF MMP‐8 levels at the post‐treatment visit and at 2 weeks (P <0.05). Conclusion: On the basis of the present findings, it can be concluded that adjunctive azithromycin provides no additional benefit over non‐surgical periodontal treatment on parameters investigated in patients with severe generalized CP.     

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.     

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.     

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.     

Microbial Analysis of Subgingival Plaque Samples Compared to That of Whole Saliva in Patients With Periodontitis

Background: The detection of special bacterial species in patients with periodontitis is considered to be useful for clinical diagnosis and treatment. The collection of subgingival plaque samples is the common way for the determination of periodontopathic bacteria. However, recently, salivary analysis has been discussed as an advantageous future diagnostic method for periodontitis because it offers simple quantitative sampling and the possibility to assess various bacteria. The aim of this cross‐sectional study is to investigate whether there is a correlation between the results of different bacterial species in saliva and subgingival plaque samples from individuals with aggressive periodontitis (AgP) and chronic periodontitis (CP). Methods: Whole saliva and subgingival plaque samples from the deepest pocket of each quadrant were collected from 43 patients with CP and 33 patients with AgP. Twenty different bacterial species from both samplings were determined by the 16S ribosomal RNA‐based polymerase chain reaction with microarray technique. Results: All bacterial species were detected in salivary and subgingival plaque samples. For Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, as well as Actinomyces viscosus, Campylobacter rectus/showae, Prevotella intermedia, Parvimonas micra, Eubacterium nodatum, and Campylobacter gracilis, a significant positive correlation between salivary and subgingival plaque samples was detected in patients with both types of periodontitis. There were no significant differences in bacteria in salivary and subgingival plaque samples between AgP and CP. Conclusion: Salivary analysis might be discussed as a potential alternative to subgingival plaque sampling for microbiologic analysis in both AgP and CP.     

Prevotella nigrescens and Porphyromonas gingivalis are associated with signs of carotid atherosclerosis in subjects with and without periodontitis

Yakob M, Söder B, Meurman JH, Jogestrand T, Nowak J, Söder P.‐Ö. Prevotella nigrescens and Porphyromonas gingivalis are associated with signs of carotid atherosclerosis in subjects with and without periodontitis. J Periodont Res 2011; 46: 749–755. ©2011 John Wiley & Sons A/S Background and Objective: Oral microorganisms may be involved in the development of cardiovascular diseases, and Porphyromonas gingivalis is one of the periodontal microorganisms that has been found in carotid atheroma. The aim of this work was to study subgingival microorganisms and early carotid lesions in subjects with and without periodontitis. Material and Methods: Eighty‐eight subjects with periodontitis and 40 subjects without periodontitis underwent dental examinations in 2003. The presence of the periodontal microorganisms Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia was analyzed from subgingival plaque using PCR amplification. The common carotid artery was scanned using ultrasound and the calculated intima‐media area (cIMA) was measured. The association between periodontitis, the cIMA value and the presence of periodontal microorganisms, together with several confounders, was studied in a multiple logistic regression model. Results: Smoking [odds ratio (OR) = 5.64; p = 0.001), level of education (OR = 5.02; p < 0.05) and the presence of P. gingivalis (OR = 6.50; p < 0.05) were associated with periodontitis. Explanatory factors for the increased cIMA were periodontitis (OR = 4.22; p < 0.05), hypertension (OR = 4.81; p < 0.05), high body mass index (OR = 5.78; p < 0.01), male gender (OR = 3.30; p < 0.05) and poor socioeconomic status (OR = 4.34; p < 0.05). P. nigrescens (OR 4.08; p < 0.05) and P. gingivalis (OR 7.63; p < 0.01) also appeared as explanatory variables associated with increased cIMA values. Conclusion: This cross‐sectional study showed that P. nigrescens and P. gingivalis were significantly associated with increased cIMA values.