Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Periodontal pathogens and associated factors in aggressive periodontitis: results 5-17 years after active periodontal therapy

OBJECTIVES To assess the association between presence of periodontal pathogens and recurrence of disease in patients with aggressive periodontitis (AgP) after active periodontal therapy (APT) and further influencing factors. MATERIAL & METHODS Microbiological samples were taken from 73 patients with AgP 5-17 years after APT at 292 sites (deepest site per quadrant). Real-time polymerase chain reactions were used to detect the periodontal pathogens Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. Uni- and multivariate analyses evaluated the associations between pathogens and recurrence of disease, smoking and adjunctive antibiotic therapy. RESULTS At re-examination A. actinomycetemcomitans could be detected in six patients (8.2%), P. gingivalis in 24 (32.9%), T. forsythia in 31 (42.5%) and T. denticola in 35 (48.0%). Increased levels of T. forsythia and T. denticola at re-examination were significantly associated with recurrence of disease in multivariate analyses (OR: 12.72, p < 0.001; OR 5.55, p = 0.002 respectively). Furthermore, high counts of T. denticola were found in patients with increased percentage of sites with clinical attachment levels (CAL) ≥ 6 mm compared to those with low counts (13.8% versus 3.2%, p = 0.005). CONCLUSION In patients with recurrence of disease T. forsythia and T. denticola were detected more frequently and in higher counts. Furthermore, T. denticola was found more frequently in patients with increased CAL.

A randomized, controlled clinical trial on the clinical, microbiological, and staining effects of a novel 0.05% chlorhexidine/herbal extract and a 0.1% chlorhexidine mouthrinse adjunct to periodontal surgery

BACKGROUND: Chlorhexidine (CHX) rinsing after periodontal surgery is common. We assessed the clinical and microbiological effects of two CHX concentrations following periodontal surgery. MATERIALS AND METHODS: In a randomized, controlled clinical trial, 45 subjects were assigned to 4 weeks rinsing with a 0.05 CHX/herbal extract combination (test) or a 0.1% CHX solution. Clinical and staining effects were studied. Subgingival bacteria were assessed using the DNA-DNA checkerboard. Statistics included parametric and non-parametric tests (p<0001 to declare significance at 80% power). RESULTS: At weeks 4 and 12, more staining was found in the control group (p<0.05 and p<0.001, respectively). A higher risk for staining was found in the control group (crude OR: 2.3:1, 95% CI: 1.3 to 4.4, p<0.01). The absolute staining reduction in the test group was 21.1% (9 5% CI: 9.4-32.8%). Probing pocket depth (PPD) decreases were significant (p<0.001) in both groups and similar (p=0.92). No rinse group differences in changes of bacterial counts for any species were found between baseline and week 12. CONCLUSIONS: The test CHX rinse resulted in less tooth staining. At the study endpoint, similar and high counts of periodontal pathogens were found.

Saliva/pathogen biomarker signatures and periodontal disease progression

The purpose of this study was to determine the role of saliva-derived biomarkers and periodontal pathogens during periodontal disease progression (PDP). One hundred human participants were recruited into a 12-month investigation. They were seen bi-monthly for saliva and clinical measures and bi-annually for subtraction radiography, serum and plaque biofilm assessments. Saliva and serum were analyzed with protein arrays for 14 pro-inflammatory and bone turnover markers, while qPCR was used for detection of biofilm. A hierarchical clustering algorithm was used to group study participants based on clinical, microbiological, salivary/serum biomarkers, and PDP. Eighty-three individuals completed the six-month monitoring phase, with 39 [corrected] exhibiting PDP, while 44 [corrected] demonstrated stability. Participants assembled into three clusters based on periodontal pathogens, serum and salivary biomarkers. Cluster 1 members displayed high salivary biomarkers and biofilm; 71% [corrected] of these individuals were undergoing PDP. Cluster 2 members displayed low biofilm and biomarker levels; 76% [corrected] of these individuals were stable. Cluster 3 members were not discriminated by PDP status; however, cluster stratification followed groups 1 and 2 based on thresholds of salivary biomarkers and biofilm pathogens. The association of cluster membership to PDP was highly significant (p < 0.0007). [corrected] The use of salivary and biofilm biomarkers offers potential for the identification of PDP or stability (ClinicalTrials.gov number, CT00277745).

Microbiological composition associated with interleukin-1 gene polymorphism in subjects undergoing supportive periodontal therapy

BACKGROUND: Interleukin-1 gene polymorphism (IL-1 gene) has been associated with periodontitis. The present study examined the subgingival microbiota by IL-1 gene status in subjects undergoing supportive periodontal therapy (SPT). METHODS: A total of 151 subjects with known IL-1 gene status (IL-1A +4845/IL-1B -3954) (IL-1 gene) were included in this study. Clinical data and subgingival plaque samples (40 taxa) were collected. These taxa were determined by the checkerboard DNA-DNA hybridization method. RESULTS: Gender, smoking habits (n-par tests), age, and clinical periodontal conditions did not differ by IL-1 gene status. IL-1 gene-negative subjects had a higher total bacterial load (mean difference, 480.4 x 10(5); 95% confidence interval [CI], 77 to 884 x 10(5); P <0.02). The levels of Actinobacillus actinomycetemcomitans (mean difference, 30.7 x 10(5); 95% CI, 2.2 to 59.5 x 10(5); P <0.05), Eubacterium nodatum (mean difference, 4.2 x 10(5); 95% CI, 0.6 to 7.8 x 10(5); P <0.02), Porphyromonas gingivalis (mean difference, 17.9 x 10(5); 95% CI, 1.2 to 34.5 x 10(5); P <0.05), and Streptococcus anginosus (mean difference, 4.0 x 10(5); 95% CI, 0.2 to 7.2 x 10(5); P <0.05) were higher in IL-1 gene-negative subjects, an observation specifically found at sites with probing depths <5.0 mm. CONCLUSIONS: Bleeding on probing did not differ by IL gene status, reflecting clinical SPT efficacy. IL-1 gene-negative subjects had higher levels of periodontal pathogens. This may suggest that among subjects undergoing SPT, a lower bacterial load is required in IL-1 gene-positive subjects to develop the same level of periodontitis as in IL-1 gene-negative subjects.

Identification of pathogen and host-response markers correlated with periodontal disease

BACKGROUND: Periodontitis is the major cause of tooth loss in adults and is linked to systemic illnesses, such as cardiovascular disease and stroke. The development of rapid point-of-care (POC) chairside diagnostics has the potential for the early detection of periodontal infection and progression to identify incipient disease and reduce health care costs. However, validation of effective diagnostics requires the identification and verification of biomarkers correlated with disease progression. This clinical study sought to determine the ability of putative host- and microbially derived biomarkers to identify periodontal disease status from whole saliva and plaque biofilm. METHODS: One hundred human subjects were equally recruited into a healthy/gingivitis group or a periodontitis population. Whole saliva was collected from all subjects and analyzed using antibody arrays to measure the levels of multiple proinflammatory cytokines and bone resorptive/turnover markers. RESULTS: Salivary biomarker data were correlated to comprehensive clinical, radiographic, and microbial plaque biofilm levels measured by quantitative polymerase chain reaction (qPCR) for the generation of models for periodontal disease identification. Significantly elevated levels of matrix metalloproteinase (MMP)-8 and -9 were found in subjects with advanced periodontitis with Random Forest importance scores of 7.1 and 5.1, respectively. The generation of receiver operating characteristic curves demonstrated that permutations of salivary biomarkers and pathogen biofilm values augmented the prediction of disease category. Multiple combinations of salivary biomarkers (especially MMP-8 and -9 and osteoprotegerin) combined with red-complex anaerobic periodontal pathogens (such as Porphyromonas gingivalis or Treponema denticola) provided highly accurate predictions of periodontal disease category. Elevated salivary MMP-8 and T. denticola biofilm levels displayed robust combinatorial characteristics in predicting periodontal disease severity (area under the curve = 0.88; odds ratio = 24.6; 95% confidence interval: 5.2 to 116.5). CONCLUSIONS: Using qPCR and sensitive immunoassays, we identified host- and bacterially derived biomarkers correlated with periodontal disease. This approach offers significant potential for the discovery of biomarker signatures useful in the development of rapid POC chairside diagnostics for oral and systemic diseases. Studies are ongoing to apply this approach to the longitudinal predictions of disease activity.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Oral prophylaxis and its effects on halitosis-associated and inflammatory parameters in patients with chronic periodontitis

OBJECTIVE A controlled clinical trial was conducted to evaluate the effects of oral prophylaxis on halitosis-associated, immunological and microbiological parameters. METHODS Thirty subjects were included in this controlled clinical trial (patients with generalized chronic periodontitis and controls without clinical attachment loss; each n = 15). Before oral prophylaxis and 14 days after (including tongue cleaning) volatile sulphur compounds (VSC), organoleptic scores and a tongue coating index were evaluated. The levels of IL-1β, IL-8, IL-10 and MMP-8 were measured in GCF, and also major periodontal pathogens were detected. Data were statistically analysed using anova and paired t-test. RESULTS Supragingival plaque and calculus removal with combined tongue cleaning was able to reduce significantly (P < 0.05) the VSC values in both groups (no significant differences between both groups). Two weeks after periodontal debridement, the VSC values were observed in the periodontitis group, but not in the control group, similar to the baseline values. The difference between the groups was statistically significant (P < 0.05). Only a repeated prophylaxis session in the periodontitis group was able to reduce VSC values significantly in comparison with baseline (P < 0.05). Organoleptic scores (10 and 30 cm) were significantly different (P < 0.05) between both groups before and after the treatment. Periodontal pathogens and host-derived markers were not significantly affected by a single prophylaxis session. CONCLUSIONS Oral prophylaxis may result in a significant decrease in VSC values. However, in periodontal diseases, a more complex treatment seems to be necessary.

A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System

Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium.

Short-term effects of systemic antibiotics during periodontal healing

OBJECTIVES: To investigate the short-term effects of nonsurgical therapy (scaling and root planing, SRP) on the subgingival microbiota in chronic (CP) and aggressive (AP) periodontal disease. METHOD AND MATERIALS: Ninety-seven CP and AP subjects underwent full-mouth SRP on 2 consecutive days. AP patients were randomly assigned to either receive systemic metronidazole plus amoxicillin (AP+AB) or were treated mechanically alone (AP). Pathogens were identified with 16S rRNA oligodeoxynucleotide probes and dot-blot hybridization before and at days 2, 3, 4, 7, 10, and 21 of healing. CP subjects were treated by scaling and root planing along with placebo tablets. RESULTS: Initially, AP cell counts were 69.9- (Porphyromonas gingivalis), 10.2- (Aggregatibacter actinomycetemcomitans), 5.7- (Tannerella forsythia), and 3.3-fold (Prevotella intermedia) enhanced compared to CP cell counts. Following SRP, immediate elimination occurred in single individuals of all three treatment groups at day 2. After SRP plus antibiotic therapy (AP+AB), the prevalence scores dropped beyond the levels of AP and CP, beginning at day 7, and remained low until day 21 (P =or< .05). Clinical healing statistically benefited from SRP with no differences among the three treatment groups. CONCLUSION: Nonsurgical therapy resulted in both a suppression and early elimination of single taxa immediately after completion of active treatment. Systemic antibiotics significantly accelerate the suppression of the periodontal microflora, but have limited effect on the elimination of target isolates during healing.

Bacterial profile and burden of periodontal infection in subjects with a diagnosis of acute coronary syndrome

BACKGROUND: Periodontitis has been identified as a potential risk factor in cardiovascular diseases. It is possible that the stimulation of host responses to oral infections may result in vascular damage and the inducement of blood clotting. The aim of this study was to assess the role of periodontal infection and bacterial burden as an explanatory variable to the activation of the inflammatory process leading to acute coronary syndrome (ACS). METHODS: A total of 161 consecutive surviving cases admitted with a diagnosis of ACS and 161 control subjects, matched with cases according to their gender, socioeconomic level, and smoking status, were studied. Serum white blood cell (WBC) counts, high- and low-density lipoprotein (HDL/LDL) levels, high-sensitivity C-reactive protein (hsC-rp) levels, and clinical periodontal routine parameters were studied. The subgingival pathogens were assayed by the checkerboard DNA-DNA hybridization method. RESULTS: Total oral bacterial load was higher in the subjects with ACS (mean difference: 17.4x10(5); SD: 10.8; 95% confidence interval [CI]: 4.2 to 17.4; P<0.001), and significant for 26 of 40 species including Porphyromonas gingivalis, Tannerella forsythensis, and Treponema denticola. Serum WBC counts, hsC-rp levels, Streptococcus intermedius, and Streptococcus sanguis, were explanatory factors to acute coronary syndrome status (Nagelkerke r2=0.49). CONCLUSION: The oral bacterial load of S. intermedius, S. sanguis, Streptococcus anginosus, T. forsythensis, T. denticola, and P. gingivalis may be concomitant risk factors in the development of ACS.

Clinical and microbiological results following nonsurgical periodontal therapy with or without local administration of piperacillin/tazobactam

OBJECTIVES We assessed if adjunct administration of piperacillin/tazobactam added clinical and microbiological treatment benefits. MATERIALS AND METHODS Thirty-six subjects (mean age 52.1 years (SD ± 10.3)) (NS by group) with chronic periodontitis were randomly enrolled receiving subgingival debridement and the local administration of piperacillin/tazobactam (test group) or debridement alone (control group). Bleeding on probing (BOP), probing pocket depth (PPD), and microbiological counts of 74 species were studied by checkerboard DNA-DNA hybridization up to month 6 after treatment. RESULTS Mean PPD changes between baseline and month 6 in the test and control groups were 1.5 and 1.8 mm, respectively (NS between groups). BOP in both groups decreased from about 80 to 40 %. At 4 and 12 weeks, lower counts of the following bacteria were found in the test group (site level): Fusobacterium species, Parvimonas micra, Pseudomonas aeruginosa, Staphylococcus aureus, Tannerella forsythia, Treponema denticola, and a composite load of nine pathogens (p < 0.001). At week 26, subjects receiving local antibiotics had a lower prevalence at tested sites for Fusobacterium nucleatum sp. polymorphum, Fusobacterium periodonticum, P. micra, and T. denticola. CONCLUSIONS At 26 weeks, treatment with or without piperacillin/tazobactam resulted in similar BOP and PPD improvements. At week 26 and at the subject level, the prevalence of 4/74 pathogens was found at lower counts in the group receiving local antibiotics. CLINICAL RELEVANCE Administration of piperacillin/tazobactam reduces the prevalence of Fusobacterium, P. micra, and T. denticola to a greater extent than debridement alone but with no short-term differences in PPD or BOP.

Crevicular fluid biomarkers and periodontal disease progression.

AIM Assess the ability of a panel of gingival crevicular fluid (GCF) biomarkers as predictors of periodontal disease progression (PDP). MATERIALS AND METHODS In this study, 100 individuals participated in a 12-month longitudinal investigation and were categorized into four groups according to their periodontal status. GCF, clinical parameters and saliva were collected bi-monthly. Subgingival plaque and serum were collected bi-annually. For 6 months, no periodontal treatment was provided. At 6 months, patients received periodontal therapy and continued participation from 6 to 12 months. GCF samples were analysed by ELISA for MMP-8, MMP-9, Osteoprotegerin, C-reactive Protein and IL-1β. Differences in median levels of GCF biomarkers were compared between stable and progressing participants using Wilcoxon Rank Sum test (p = 0.05). Clustering algorithm was used to evaluate the ability of oral biomarkers to classify patients as either stable or progressing. RESULTS Eighty-three individuals completed the 6-month monitoring phase. With the exception of GCF C-reactive protein, all biomarkers were significantly higher in the PDP group compared to stable patients. Clustering analysis showed highest sensitivity levels when biofilm pathogens and GCF biomarkers were combined with clinical measures, 74% (95% CI = 61, 86). CONCLUSIONS Signature of GCF fluid-derived biomarkers combined with pathogens and clinical measures provides a sensitive measure for discrimination of PDP (ClinicalTrials.gov NCT00277745).

Improved detection of blood stream pathogens by real-time PCR in severe sepsis

Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures.

Significance of Periodontal Risk Assessment in the recurrence of periodontitis and tooth loss

Aim: To investigate the association of the Periodontal Risk Assessment (PRA) model categories with periodontitis recurrence and tooth loss during supportive periodontal therapy (SPT) and to explore the role of patient compliance. Material and Methods: In a retrospective cohort, PRA was performed for 160 patients after active periodontal therapy (APT) and after 9.5 ± 4.5 years of SPT. The recurrence of periodontitis and tooth loss were analysed according to the patient's risk profile (low, moderate or high) after APT and compliance with SPT. The association of risk factors with tooth loss and recurrence of periodontitis was investigated using logistic regression analysis. Results: In 18.2% of patients with a low-risk profile, in 42.2% of patients with a moderate-risk profile and in 49.2% of patients with a high-risk profile after APT, periodontitis recurred. During SPT, 1.61 ± 2.8 teeth/patient were lost. High-risk profile patients lost significantly more teeth (2.59 ± 3.9) than patients with moderate- (1.02 ± 1.8) or low-risk profiles (1.18 ± 1.9) (Kruskal–Wallis test, p=0.0229). Patients with erratic compliance lost significantly (Kruskal–Wallis test, p=0.0067) more teeth (3.11 ± 4.5) than patients compliant with SPT (1.07 ± 1.6). Conclusions: In multivariate logistic regression analysis, a high-risk patient profile according to the PRA model at the end of APT was associated with recurrence of periodontitis. Another significant factor for recurrence of periodontitis was an SPT duration of more than 10 years.