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Background: The association between the infection of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Treponema denticola in chronic periodontitis (CP) and the severity of periodontal disease remains to be elucidated. The aim of this study was to investigate the subgingival infection frequencies of three periodontopathic bacteria in Chinese CP patients and to evaluate the correlations between infection by these bacteria and periodontal destruction., Methods: A multiple PCR assay using primers derived from 16SrDNA genes of P. gingivalis, A. actinomycetemcomitans and T. denticola was established to measure simultaneously the presence of the three microbes in 162 subgingival samples from 81 Chinese CP patients., Results: The positive rates of P. gingivalis, A. actinomycetemcomitans and T. denticola in the subgingival samples were 84.6%, 83.3% and 88.3%, respectively. Of the subgingival samples, 68% revealed the coinfection of all the three microbes. The infection rates with P. gingivalis, A. actinomycetemcomitans or T. denticola alone was 5.9% (1/17), 17.6% (3/17) and 76.5% (13/17), respectively. A close association was present between the A. actinomycetemcomitans infection and gingival index (GI) (P < 0.01), but not between P. gingivalis or T. denticola infection and GI (P > 0.05). P. gingivalis and A. actinomycetemcomitans were more frequently detectable in middle and deep pockets than in shallow ones (P < 0.01), while T. denticola was found remarkably often in deep pockets (P < 0.05). The coinfection rate of the three microbes was significantly higher in sites with severe periodontitis than in those with mild periodontitis (P < 0.01)., Conclusions: The multiple PCR established in this study can be used as a sensitive and specific method to simultaneously detect all three microbes in subgingival samples. A. actinomycetemcomitans infection may be associated with CP and play an important role in the periodontal tissue destruction. The coinfection of P. gingivalis, A. actinomycetemcomitans and T. denticola can cause more serious periodontal destruction than infection of any one or two of the three microbes.

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.

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 microg/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.

A novel gene was identified in the Treponema denticola prcA-prtP protease operon. Strains with mutations in either the prcA-prtP or the msp region showed altered expression of a product(s) of the other locus. Together, these results provide information on the assembly of outer membrane complexes involved in T. denticola interaction with host cells and tissue.

Treponema pallidum and Treponema denticola encode within their genomes homologues of energy coupling and regulatory proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) but no recognizable homologues of PTS permeases. These homologues include (1) Enzyme I, (2) HPr, (3) two IIA(Ntr)-like proteins, and (4) HPr(Ser) kinase/phosphorylase (HprK). Because the Enzyme I-encoding gene in T. pallidum is an inactive pseudogene and because all other pts genes in both T. pallidum and T. denticola are actively expressed, the primary sensory transduction mechanism for signal detection and transmission appears to involve HprK rather than EI. We have overexpressed and purified to near homogeneity four of the five PTS proteins from T. denticola. Purified HprK phosphorylates HPr with ATP, probably on serine, while Enzyme I phosphorylates HPr with PEP, probably on histidine. Furthermore, HPr(His)-P can transfer its phosphoryl group to IIA(Ntr)-1. Factors and conditions regulating phosphoryl transfer prove to differ from those described previously for Bacillus subtilis, but cross-enzymatic activities between the Treponema, Salmonella, and Bacillus phosphoryl-transfer systems could be demonstrated. Kinetic analyses revealed that the allosterically regulated HPr kinase/phosphorylase differs from its homologues in Bacillus subtilis and other low G+C Gram-positive bacteria in being primed for kinase activity rather than phosphorylase activity in the absence of allosteric effectors. The characteristics of this enzyme and the Treponema phosphoryl-transfer chain imply unique modes of signal detection and sensory transmission. This paper provides the first biochemical description of PTS phosphoryl-transfer chains in an organism that lacks PTS permeases.

Treponema denticola and Porphyromonas gingivalis have been identified in atheromatous plaques of two patients suffering from atherosclerosis by PCR and fluorescence in situ hybridization (FISH). The use of the FISH technique suggested that these periodontopathic micro-organisms might be metabolically active within the wall of arteries, under the atherosclerotic lesion.

Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.