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Objectives: The purpose of this study was to compare microbial profiles of saliva, pooled and site-specific subgingival samples in patients with periodontitis. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens., Design: Site specific subgingival plaque samples (n = 54), pooled subgingival plaque samples (n = 18) and stimulated saliva samples (n = 18) were collected from 18 patients with generalized chronic periodontitis. Subgingival and salivary microbiotas were characterized by means of HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared using Spearman rank correlation coefficient., Results: Pronounced intraindividual differences were recorded in site-specific microbial profiles, and site-specific information was in general not reflected by pooled subgingival samples. Presence of Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Filifactor alocis, Tannerella forsythia and Parvimona micra in site-specific subgingival samples were detected in saliva with an AUC of 0.79 (sensitivity: 0.61, specificity: 0.94), compared to an AUC of 0.76 (sensitivity: 0.56, specificity: 0.94) in pooled subgingival samples., Conclusions: Site-specific presence of periodontal pathogens was detected with comparable accuracy in stimulated saliva samples and pooled subgingival plaque samples. Consequently, saliva may be a reasonable surrogate for pooled subgingival samples when screening for presence of periopathogens. Future large-scale studies are needed to confirm findings from this study.

High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R(2) and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/μl, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Chronic inflammation has earlier been detected in ruptured intracranial aneurysms. A previous study detected both dental bacterial DNA and bacterial-driven inflammation in ruptured intracranial aneurysm walls. The aim of this study was to compare the presence of oral and pharyngeal bacterial DNA in ruptured and unruptured intracranial aneurysms. The hypothesis was that oral bacterial DNA findings would be more common and the amount of bacterial DNA would be higher in ruptured aneurysm walls than in unruptured aneurysm walls., Materials and Methods: A total of 70 ruptured (n = 42) and unruptured (n = 28) intracranial aneurysm specimens were obtained perioperatively in aneurysm clipping operations. Aneurysmal sac tissue was analysed using a real-time quantitative polymerase chain reaction to detect bacterial DNA from several oral species. Both histologically non-atherosclerotic healthy vessel wall obtained from cardiac by-pass operations (LITA) and arterial blood samples obtained from each aneurysm patient were used as control samples., Results: Bacterial DNA was detected in 49/70 (70%) of the specimens. A total of 29/42 (69%) of the ruptured and 20/28 (71%) of the unruptured aneurysm samples contained bacterial DNA of oral origin. Both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively). There was no significant difference in the amount of bacterial DNA between the ruptured and unruptured samples., Conclusion: Dental bacterial DNA can be found using a quantitative polymerase chain reaction in both ruptured and unruptured aneurysm walls, suggesting that bacterial DNA plays a role in the pathogenesis of cerebral aneurysms in general, rather than only in ruptured aneurysms.

The examination was carried out of samplings of 110 patients with periodontitis (observation group) and 60 patients without pathology of periodont (comparison group). The polymerase chain reaction was used to analyze samples of saliva and contents of periodontal recesses for detecting species-specific DNA fragments of Porphymmonas gigngivalis, Streptococcus macacae, S. mutans, S. oralis, S. salivarius, S. sangis, S. sobrinus, Treponema denticola. In patients with periodontitis S. mutans, S. oralis S. sobrinus were reliably more often detected in the content of periodontal recesses and S. mutans, S. sobrinus i in saliva. In the observation group the rate of detection of association S. mutans--S. oralis--S. sangis--S. sobrinus was significantly exceeded (up to 15.6%, X2 = 9.1, p = 0.004). In ten days of effective treatment of periodontitis reliable decreastng of rate of detection of S. wasoralis, S. sobrinus was observed in contents of periodontal recesses but not in of saliva. The detection of S.sobrinus using technique of polymerase chain reaction in contents of periodontal recesses and/or saliva of patients with periodontitis has a diagnostic value. The detection of S.sobrinus in contents of periodontal recesses is significant both in monoculture and in association S. mutans--S. oralis--S. sangis--S.sobrinus. The absence of S. sobrinus in contents of periodontal recesses testifies effectiveness of treatment of main disease (periodontitis).

Backgrounds: Numerous reports have demonstrated that periodontal bacteria are present in plaques from atherosclerotic arteries. Although periodontitis has recently been recognized as a risk factor for coronary artery disease, the direct relationship between periodontal bacteria and coronary artery disease has not yet been clarified. It has been suggested that these bacteria might contribute to inflammation and plaque instability. We assumed that if periodontal bacteria induce inflammation of plaque, the bacteria would be released into the bloodstream when vulnerable plaque ruptures. To determine whether periodontal bacteria are present in thrombi at the site of acute myocardial infarction, we tried to detect periodontal bacteria in thrombi of patients with acute myocardial infarction by polymerase chain reaction (PCR)., Methods: We studied 81 consecutive adults with ST-segment elevation acute myocardial infarction who underwent primary percutaneous coronary intervention (PCI). All patients underwent removal of thrombus with aspiration catheters at the beginning of percutaneous coronary intervention, and a small sample of thrombus was obtained for PCR., Results: The detection rates of periodontal bacteria by PCR were 19.7% for Aggregatibacter actinomycetemcomitans, 3.4% for Porphyromonas gingivalis, and 2.3% for Treponema denticola., Conclusions: Three species of periodontal bacteria were detected in the thrombi of patients with acute myocardial infarction. This raises the possibility that such bacteria are latently present in plaque and also suggests that these bacteria might have a role in plaque inflammation and instability., (Copyright © 2012 Mosby, Inc. All rights reserved.)

The oral spirochete Treponema denticola is associated with human periodontal disease. T. denticola ATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared to T. denticola ATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence of T. denticola ATCC 35405. In this report, we hypothesize that T. denticola ATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome of T. denticola ATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant of TDE0911 failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulate T. denticola, in particular ATCC 35405, and other strains that may carry similar endonucleases.

Periodontitis is a common chronic multibacterial infection in the tooth-supporting tissues. It has been shown that periodontitis patients carry higher number of disease-associated bacteria than healthy ones. The aim of this study was to generate a novel, single copy gene-based quantitative real-time PCR (qPCR) assay for five major periodontal pathogens - Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythia. The primer/probe sets were designed for conservative lipopolysaccharide-coding gene regions. They proved to be sensitive and able to detect strains representing different serotypes of the target bacteria. The specificity of designed primers was tested using 49 selected bacterial species and no false positive or negative results were observed. We validated the assay with a case-control population, including 165 saliva samples, and proved the diagnostic accuracy by Receiver Operating Characteristic (ROC) curves. All quantified pathogens alone were able to distinguish significantly between the subjects with and without periodontitis, and provided areas under the ROC curve larger than 0.5. The total pathogen burden comprising all five species associated with periodontitis with an area of 0.821 (95% CI, 0.758-0.885, P50.001). Our prominently sensitive and specific assay may have major importance in the diagnosis, prevention, and treatment of periodontitis.

Background: Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used to analyze microbial communities, including oral microflora. However, accurate identification of terminal restriction fragment (T-RF) origins is prevented by unpredictable errors in sizing, thus necessitating the clone library analysis. To minimize sizing errors, we proposed optimizing the size definition of internal standards., Methods: GeneScan-1000 ROX was regenerated as an internal standard by redefining the fragment sizes in terms of molecular weight (MW) based on their mobility relative to 6-carboxyfluorescein (FAM) -labeled restriction fragments derived from the 16S recombinant RNA gene of Porphyromonas gingivalis. Using the new size definition, the average sizing error among eight oral bacteria from six phyla was estimated and compared with that of the conventional method. Microbial communities isolated from saliva were analyzed using the new MW size definition. Bacterial species were assigned to peaks using TRFMA, a Web-based tool for T-RFLP analysis, and compared with those identified in a clone library analysis., Results: Using the new size definition, the average sizing error for 40 T-RFs was drastically reduced from 2.42 to 0.62 bases, and large sizing errors (more than two bases) were eliminated. More than 90% of the total bacterial clones detected by the clone library analysis were assigned by T-RFLP., Conclusion: The size definition of the newly constructed internal standards reduced fragment sizing errors and allowed for accurate assignment of bacteria to peaks by the T-RFLP analysis. This provided a more effective means for studying microbial communities, including the oral microflora.

The aim of our research was to identify by bacterial genomic DNA analysis the prevalence of five different species of periodontopathogenic bacteria present in the subgingival biofilm, specifically: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Bacterioides forsytus (Bf), Treponema denticola (Td). For the analysis we used the systematic Multiplex-PCR-microdent kit with species-specific primers. We studied a group of 48 subjects, 18 males and 30 females, from 18 to 78 years of age. The initial clinical screening enabled us to select, among the group analysed, 24 subjects with signs of active periodontopathy (Group A) and 24 patients without identifiable clinical evidence of the disease used as the control group (Group B). Within the two experimental groups (A and B), the test was found to be positive in 75% of subjects from group A, whereas the test was found to be negative in all the subjects from group B. Our research shows that the Multiplex-PCR system is reliable. Furthermore, the sensitivity and simplicity of this technique, as well as the decrease in working times and the possibility of identifying non-culturable bacteria, since the presence of viable organisms is not essential, make this technique indicated for the simultaneous identification of periodontopathogenic bacteria and might, in perspective, provide a more effective clinical alternative to the techniques of bacterial typing of the subgingival plaque.

Objective: We analyzed the distribution of 10 periodontal bacteria species (Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea, Capnocytophaga sputigena, and Treponema denticola) in children, and then compared their distribution in those children and their mothers, with special attention given to three of the species known as the red complex (P. gingivalis, T. forsythensis, and T. denticola) whose presence has been shown to be associated with conditions related to periodontal diseases., Methods: One hundred thirteen pairs of children and their mothers were randomly selected from patients treated at the Pedodontic Clinic of Osaka University Dental Hospital. Saliva samples were taken at the second visit prior to receiving professional tooth brushing instruction. Genomic DNA was extracted from each saliva sample, followed by a polymerase chain reaction assay with species-specific sets of primers., Results: A. actinomycetemcomitans was the most frequently detected species in the mothers, followed by C. sputigena, P. gingivalis, and T. forsythensis, while C. sputigena had the highest detection rate, followed by A. actinomycetemcomitans and T. denticola in the children. The detection rate of the red complex species in children whose mothers possessed the same species was significantly higher than in those whose mothers did not possess them., Conclusions: Our results indicate a correlation between the presence of periodontal bacteria in children and their mothers, while the presence of red complex bacteria in children was highly associated with that in their mothers.

Background: Information on the impact of sample storage prior to analysis by DNA methods is limited., Aims: To investigate the effect of microbial sample storage on bacterial detection and proportional distribution of the red complex and its individual pathogens., Material and Methods: Subgingival plaque samples were analysed by (1) immediate processing, (2) after storage at +4 degrees C for 6 weeks, (3) after storage at -20 degrees C for 6 months or (4) after storage at -20 degrees C for 12 months using the checkerboard DNA-DNA hybridization., Results: Proportional distribution of the red complex did not differ between the first three protocols. However, the total bacterial DNA for pathogens studied decreased significantly in protocols 3 and 4. Relative amounts of Tannerella forsythensis, Porphyromonas gingivalis and Treponema denticola remained stable in the second protocols and changed in an unpredictable way if stored for 6 or 12 months., Conclusions: Results from samples stored for maximum 6 months at -20 degrees C with high proportional amounts of the red complex and T. denticola may be used as an indicator of persistence. All bacterial samples for DNA extraction should be processed following a standardized storage protocol (i.e. samples stored at +4 degrees C for maximum 6 weeks) in order to get comparable qualitative and quantitative results for total DNA, bacterial complexes and individual pathogens. Most representative results are yielded if processing and hybridization could be performed immediately after sampling.