Frequency of putative periodontal pathogens among type 1 diabetes mellitus: a case-control study.

OBJECTIVE: The aim of the present study is to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria viz., Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens. To fulfil the above objective, polymerase Chain reaction using the primers targeting 16S rRNA gene of the bacterial species was performed with the subgingival plaque collected from the permanent first molars of type 1 diabetic children and age matched healthy children. RESULTS: The prevalence of periodontal pathogens in diabetic and healthy children was 6% and 16% for E. corrodens, 18% and 36% for C. rectus, 2% and 2% for P. intermedia, 4% and 0%, for P. nigrescens respectively. Statistically, significant difference was not observed for the prevalence of all the four periodontal pathogens between type 1 diabetic and healthy children (P = 1.00). The results of the present study thus reveal a negative correlation of type I diabetes to periodontitis in association to Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens.

Microbial profile comparisons of saliva, pooled and site-specific subgingival samples in periodontitis patients.

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.

[Study of mutual dependence of periodontal and colonic microbiome in health and pathology using NSG-sequencing].

By using NGS-sequencing libraries of DNA from periodontal swabs with primers specific to V6 region of 16S rDNA prevalence of bacterial genera and species in periodontal and colonic microbiota of patients with periodontitis of different severity and healthy donors was analyzed. Hyper-colonization of the colon with Akkermansia muciniphila was found to be the most important maker of negative predisposition to periodontitis (t=133,7 at р=10(-6)). This result is in a good agreement with communications about positive impact of hyper-colonization of the colon with this species on type 2 diabetes, obesity, atopic dermatitis, and antibiotic-induced diarrhea associated with Clostridium dificile. Analysis of the periodontal protectors at the periodontium elucidated a number of close taxonomic relatives of the periodontal pathogens by Socransky, e.g. Aggregatibacter segnis and Aggregatibacter aphrophilus are closely related to Aggregatibacter actinomycetemcomitans; Treponema vencentii is a relative of Treponema denticola; Prevotella baroniae, Prevotella salivae and Prevotella spp. are relatives of Prevotella intermedia; Campylobacter concisus is a relative of Campylobacter jejuni, causative agent of enterocolitis.

Intraspecies Variability Affects Heterotypic Biofilms of Porphyromonas gingivalis and Prevotella intermedia: Evidences of Strain-Dependence Biofilm Modulation by Physical Contact and by Released Soluble Factors.

It is well known that strain and virulence diversity exist within the population structure of Porphyromonas gingivalis. In the present study we investigate intra- and inter-species variability in biofilm formation of Porphyromonas gingivalis and partners Prevotella intermedia and Prevotella nigrescens. All strains tested showed similar hydrophobicity, except for P. gingivalis W83 which has roughly half of the hydrophobicity of P. gingivalis ATCC33277. An intraspecies variability in coaggregation of P. gingivalis with P. intermedia was also found. The association P. gingivalis W83/P. intermedia 17 produced the thickest biofilm and strain 17 was prevalent. In a two-compartment system P. gingivalis W83 stimulates an increase in biomass of strain 17 and the latter did not stimulate the growth of P. gingivalis W83. In addition, P. gingivalis W83 also stimulates the growth of P. intermedia ATCC25611 although strain W83 was prevalent in the association with P. intermedia ATCC25611. P. gingivalis ATCC33277 was prevalent in both associations with P. intermedia and both strains of P. intermedia stimulate the growth of P. gingivalis ATCC33277. FISH images also showed variability in biofilm structure. Thus, the outcome of the association P. gingivalis/P. intermedia seems to be strain-dependent, and both soluble factors and physical contact are relevant. The association P. gingivalis-P. nigrescens ATCC33563 produced larger biomass than each monotypic biofilm, and P. gingivalis was favored in consortia, while no differences were found in the two-compartment system. Therefore, in consortia P. gingivalis-P. nigrescens physical contact seems to favor P. gingivalis growth. The intraspecies variability found in our study suggests strain-dependence in ability of microorganisms to recognize molecules in other bacteria which may further elucidate the dysbiosis event during periodontitis development giving additional explanation for periodontal bacteria, such as P. gingivalis and P. intermedia, among others, to persist and establish chronic infections in the host.

Subgingival microbiome in patients with healthy and ailing dental implants.

Dental implants are commonly used to replace missing teeth. However, the dysbiotic polymicrobial communities of peri-implant sites are responsible for peri-implant diseases, such as peri-implant mucositis and peri-implantitis. In this study, we analyzed the microbial characteristics of oral plaque from peri-implant pockets or sulci of healthy implants (n = 10), peri-implant mucositis (n = 8) and peri-implantitis (n = 6) sites using pyrosequencing of the 16S rRNA gene. An increase in microbial diversity was observed in subgingival sites of ailing implants, compared with healthy implants. Microbial co-occurrence analysis revealed that periodontal pathogens, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, were clustered into modules in the peri-implant mucositis network. Putative pathogens associated with peri-implantitis were present at a moderate relative abundance in peri-implant mucositis, suggesting that peri-implant mucositis an important early transitional phase during the development of peri-implantitis. Furthermore, the relative abundance of Eubacterium was increased at peri-implantitis locations, and co-occurrence analysis revealed that Eubacterium minutum was correlated with Prevotella intermedia in peri-implantitis sites, which suggests the association of Eubacterium with peri-implantitis. This study indicates that periodontal pathogens may play important roles in the shifting of healthy implant status to peri-implant disease.

Comparative genome analysis of Prevotella intermedia strain isolated from infected root canal reveals features related to pathogenicity and adaptation.

BACKGROUND: Many species of the genus Prevotella are pathogens that cause oral diseases. Prevotella intermedia is known to cause various oral disorders e.g. periodontal disease, periapical periodontitis and noma as well as colonize in the respiratory tract and be associated with cystic fibrosis and chronic bronchitis. It is of clinical significance to identify the main drive of its various adaptation and pathogenicity. In order to explore the intra-species genetic differences among strains of Prevotella intermedia of different niches, we isolated a strain Prevotella intermedia ZT from the infected root canal of a Chinese patient with periapical periodontitis and gained a draft genome sequence. We annotated the genome and compared it with the genomes of other taxa in the genus Prevotella. RESULTS: The raw data set, consisting of approximately 65X-coverage reads, was trimmed and assembled into contigs from which 2165 ORFs were predicted. The comparison of the Prevotella intermedia ZT genome sequence with the published genome sequence of Prevotella intermedia 17 and Prevotella intermedia ATCC25611 revealed that ~14% of the genes were strain-specific. The Preveotella intermedia strains share a set of conserved genes contributing to its adaptation and pathogenic and possess strain-specific genes especially those involved in adhesion and secreting bacteriocin. The Prevotella intermedia ZT shares similar gene content with other taxa of genus Prevotella. The genomes of the genus Prevotella is highly dynamic with relative conserved parts: on average, about half of the genes in one Prevotella genome were not included in another genome of the different Prevotella species. The degree of conservation varied with different pathways: the ability of amino acid biosynthesis varied greatly with species but the pathway of cell wall components biosynthesis were nearly constant. Phylogenetic tree shows that the taxa from different niches are scarcely distributed among clades. CONCLUSIONS: Prevotella intermedia ZT belongs to a genus marked with highly dynamic genomes. The specific genes of Prevotella intermedia indicate that adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of Prevotella intermedia.

Colony polymerase chain reaction of heme-accumulating bacteria.

Colony PCR of anaerobic black-pigmenting Bacteroidetes species Porphyromonas gingivalis and Prevotella intermedia was modified by addition of bovine serum albumin to reverse the inhibitory action of accumulated heme.

Genome-wide association study of periodontal pathogen colonization.

Pathological shifts of the human microbiome are characteristic of many diseases, including chronic periodontitis. To date, there is limited evidence on host genetic risk loci associated with periodontal pathogen colonization. We conducted a genome-wide association (GWA) study among 1,020 white participants of the Atherosclerosis Risk in Communities Study, whose periodontal diagnosis ranged from healthy to severe chronic periodontitis, and for whom "checkerboard" DNA-DNA hybridization quantification of 8 periodontal pathogens was performed. We examined 3 traits: "high red" and "high orange" bacterial complexes, and "high" Aggregatibacter actinomycetemcomitans (Aa) colonization. Genotyping was performed on the Affymetrix 6.0 platform. Imputation to 2.5 million markers was based on HapMap II-CEU, and a multiple-test correction was applied (genome-wide threshold of p < 5 × 10(-8)). We detected no genome-wide significant signals. However, 13 loci, including KCNK1, FBXO38, UHRF2, IL33, RUNX2, TRPS1, CAMTA1, and VAMP3, provided suggestive evidence (p < 5 × 10(-6)) of association. All associations reported for "red" and "orange" complex microbiota, but not for Aa, had the same effect direction in a second sample of 123 African-American participants. None of these polymorphisms was associated with periodontitis diagnosis. Investigations replicating these findings may lead to an improved understanding of the complex nature of host-microbiome interactions that characterizes states of health and disease.

Predominant bacterial species in subgingival plaque in dogs.

BACKGROUND AND OBJECTIVE: The dog has been used extensively for experimental and microbiological studies on periodontitis and peri-implantitis without detailed knowledge about the predominant flora of the subgingival plaque. This study was designed to evaluate the predominant cultivable bacterial species in dogs and compare them phenotypically and genotypically with corresponding human species. MATERIAL AND METHODS: Four subgingival samples were taken from two upper premolars in each of six Labrador retrievers. The samples from each dog were processed for anaerobic culture. From the samples of each dog, the five or six predominating bacteria based on colony morphology were selected and pure cultured. Each of the strains was characterized by Gram stain, anaerobic/aerobic growth and API-ZYM test. Eighteen strains showing clear-cut phenotypic differences were further classified based on DNA sequencing technology. Cross-reactions of DNA probes from human and dog strains were also tested against a panel of both human and dog bacterial species. RESULTS: Thirty-one strains in the dogs were isolated and characterized. They represented 21 different species, of which six belonged to the genus Porphyromonas. No species was found consistently in the predominant flora of all six dogs. Porphyromonas crevioricanis and Fusobacterium canifelinum were the two most prevalent species in predominant flora in dogs. DNA probes from human and dog species cross-reacted to some extent with related strains from humans and dogs; however, distinct exceptions were found. CONCLUSION: The predominant cultural subgingival flora in dogs shows great similarities with the subgingival bacteria from humans at the genus level, but distinct differences at the species level; however, a genetic relatedness could be disclosed for most strains investigated.

Rapid identification of oral anaerobic bacteria cultivated from subgingival biofilm by MALDI-TOF-MS.

INTRODUCTION: To facilitate the identification of anaerobes cultivated from periodontal disease, whole cell bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was evaluated. METHODS: A total of 84 strains (nine reference strains and 75 recent clinical isolates from 33 patients with aggressive periodontitis) previously identified with phenotypic methods were used. All the references and 10 clinical isolates belonging to the same species as the reference strains were genotypically identified by sequence analysis of the 16S ribosomal RNA gene. All the strains were then analyzed using MALDI-TOF-MS. RESULTS: The reference strains of anaerobic bacteria used showed characteristic MALDI-TOF-MS spectra with peaks between m/z 2000 and up to about m/z 13,000. On visual inspection, the similarity of spectra produced by strains of a single genus could be recognized. Obvious differences between spectra produced by strains of different species were also easily noticed. The reproducibility of the method was proved by the similarity of spectra belonging to the same species. The spectra of the Prevotella intermedia strains identified with MALDI clustered together and clustered separately from the spectra of Prevotella nigrescens, proving that MALDI-TOF-MS is an accurate method that is capable of separating these two species. The quality of clustering was characterized by calculating an inconsistency coefficient (Mathworks:/Matlab Reference Manual v2007a/, Statistical toolbox). CONCLUSION: Our results suggest that MALDI-TOF-MS might become a useful method for the identification of anaerobic bacteria, especially for those that cannot be readily identified by biochemical analysis. It may become an attractive system even for the routine identification of clinical isolates.

Treatment outcomes of dental flossing in twins: molecular analysis of the interproximal microflora.

BACKGROUND: The purpose of this study was to assess the effects of dental flossing on the microbial composition of interproximal plaque samples in matched twins. METHODS: The study was a two-treatment, examiner-masked, randomized, parallel-group, controlled study. Fifty-one twin pairs between 12 and 21 years of age were randomized to a 2-week supervised and unsupervised treatment regimen consisting of tongue brushing and toothbrushing or tongue brushing and toothbrushing plus flossing. The reverse-capture checkerboard hybridization assay was used to assess levels (abundance) of 26 microbial species in interproximal plaque samples collected from six sites per subject. An integrative computational predictive model estimated average changes in microbial abundance patterns of selected bacterial species from baseline to 2 weeks by comparing treatment groups. RESULTS: After the 2-week study period, putative periodontal pathogens and cariogenic bacteria were overabundant in the group that did not floss compared to the group that performed flossing. Those included Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), Prevotella intermedia, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), and Streptococcus mutans. Microbial species that are not consistent with the development of periodontal disease or dental caries were overabundant in the group that did floss compared to the non-flossing group. CONCLUSION: In a well-matched twin cohort, tooth and tongue brushing plus flossing significantly decreased the abundance of microbial species associated with periodontal disease and dental caries after a 2-week program.

Interferon-gamma and interleukin-12 gene polymorphisms and their relation to aggressive and chronic periodontitis and key periodontal pathogens.

BACKGROUND: The gene polymorphisms interferon-gamma (IFN-gamma) 874 T/A and interleukin (IL)-12 1188 A/C have been associated with the altered production of cytokines. Therefore, they might be indicative of the occurrence of chronic periodontitis (CP) or aggressive periodontitis (AgP) and the prevalence of key periodontal pathogens. For this purpose, we analyzed these polymorphisms in subjects with generalized AgP or generalized CP. Moreover, we assessed the relationship between these polymorphisms and five periodontopathic bacteria. METHODS: A total of 124 unrelated German white subjects with periodontitis (AgP=72 and CP=52) and 74 periodontitis-free subjects were studied. Gene polymorphisms were determined by polymerase chain reaction with sequence-specific primers. Subgingival bacteria were molecular biologically analyzed using multiplex polymerase chain reaction and reverse hybridization. The distributions of alleles and genotypes were calculated by the chi(2) test with Yates correction. Risk factor analyses were carried out by logistic regression considering established confounders for periodontitis. RESULTS: Allele and genotype frequencies of both investigated polymorphisms were not significantly different between subjects with periodontitis and periodontitis-free controls. However, in the total study group, IL-12 AA-positive subjects had a significantly higher bleeding index than individuals who expressed IL-12 CC (68.2% versus 50.0%, P=0.025). Moreover, IFN-gamma AA carriers had a decreased odds ratio (OR) for the individual presence of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) (OR=0.39, P=0.012) after adjustment for age, gender, smoking, and probing depth. IFN-gamma TA predisposed an individual to infection with Prevotella intermedia (OR=2.15, P=0.019). CONCLUSION: Although a relationship between the bleeding index and the presence of bacteria was shown, IFN-gamma and IL-12 polymorphisms are not suitable diagnostic features for AgP and CP.

The reproducibility of curet sampling of subgingival biofilms.

BACKGROUND: Because few studies have examined the critical issue of sampling reproducibility, the purpose of the present study was to examine the reproducibility of curet sampling of subgingival biofilms. METHODS: Seven subgingival biofilm samples were taken successively, using a curet, from each of 80 sites and individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. One healthy site was sampled in each of 20 periodontally healthy subjects, and one sulcus/pocket of < or =3, 4 to 5, and > or =6 mm was sampled in each of 20 subjects with chronic periodontitis. The significance of differences in counts and proportions of individual species at the seven successive samplings for each probing depth (PD) category was determined using the Kruskal-Wallis test. The reproducibility of species proportions at each PD category was measured using the coefficient of variation (CV), and the consistency of microbial profiles across samples was examined using the minimum similarity coefficient. RESULTS: There was no significant difference in the mean proportions of the 40 test species in the seven successive samples in each of the four PD categories. The median CV for individual species in the same site was 0.79 (95% confidence interval [CI]: 0.76 to 0.82) compared to 1.76 (95% CI: 1.69 to 1.82) in samples from different sites. The within-site mean minimum similarity coefficient (+/- SEM) was 51.2% +/- 2.2%, and it was 27.9% +/- 0.3% between sites. CONCLUSION: The proportions of species remained consistent in successive curet samples, indicating that the use of curets provided a reliable and reproducible method to obtain subgingival samples.

[Dissertations 25 years after date 12. Classification and virulence of black-pigmented bacteria in relation to periodontitis]. / Proefschriften 25 jaar na dato 12. Classificatie en virulentie van zwart-gepigmenteerde bacteriën in relatie tot parodontitis.

Bacteria in dental plaque play an essential role in the origin and development of periodontitis. In the seventies of the last century it became clear that black-pigmented bacteria of the genus Bacteroides play a vital role in this process. These bacteria are currently known as Porphyromonas gingivalis and Prevotella intermedia. In a PhD dissertation 25 years ago it was shown by DNA analysis that this group of bacteria is very heterogeneous, and that different species exist, which are associated with different oral infections. Because Porphyromonas gingivalis plays an important role in periodontitis, this bacterium has been investigated extensively during the last decades. The entire genome is now known at the DNA level. In addition, transmission between spouses has been shown to be possible, although it does not always cause periodontal disease. It is not yet possible to conclude if for patients with Porphyromonas gingivalis a different antibiotic policy should be used compared to patients without this bacterium.

Increase in probing depth is correlated with a higher number of Prevotella intermedia genotypes.

BACKGROUND: The aims of this study were to determine the genotypic diversity of Prevotella intermedia in subgingival plaque samples by using two techniques, arbitrarily primed polymerase chain reaction (AP-PCR) and heteroduplex analysis, and to assess the relationship of this diversity with increase in probing depth. METHODS: The subgingival plaque samples were obtained from 12 patients using paper points inserted into periodontal pockets (diseased sites) and healthy gingival sulci (healthy sites) of the same subjects. After isolation and identification, AP-PCR was performed for genotypic characterization of P. intermedia (80 isolates). The clinical samples with a positive result for P. intermedia were amplified by 16S rRNA-based PCR method, and the amplicons were subjected to heteroduplex analysis. RESULTS: The agreement between the two methods was very high; the AP-PCR and heteroduplex analysis showed that subjects harbored between one and five distinct genotypes of P. intermedia, with a positive association between numbers of genotypes by AP-PCR (P = 0.0042) or heteroduplex (P = 0.0099) and increase in probing depth. No matching of P. intermedia genotypes was observed between healthy and diseased sites of the same individual. Interindividual analyses demonstrated absence of identical clones and indicated a high level of genetic diversity in the species. CONCLUSION: A clear relationship was observed between a higher number of genotypes and increase in probing depth; these results suggest that environmental challenges in the periodontal pockets may modulate the microbiota by selecting genotypes best able to exploit the environment.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level.

Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.

Distribution of periodontal pathogens in Korean aggressive periodontitis.

BACKGROUND: Microbial associations in aggressive periodontitis versus different ethnic origins are substantially unknown. We undertook this study to determine the prevalence of seven putative periodontopathogens in Korean patients and to evaluate microbial differences in localized and generalized aggressive periodontitis patients. METHODS: Thirty-nine aggressive periodontitis patients between 20 and 35 years old (24 males and 15 females; mean age 29.6 years) were selected according to clinical criteria. The patients were subclassified into 17 localized and 22 generalized aggressive periodontitis patients. In each of the 39 individuals, subgingival plaque samples were collected from four diseased teeth (> or = 6 mm probing depth, 156 sites) and one healthy site (< or = 3 mm probing depth, 39 sites). Polymerase chain reaction (PCR) of the 16S ribosomal RNA gene fragments (about 530 bp) of plaque bacteria and their subsequent detection by dot-blot hybridization using specific oligonucleotide probes were performed to determine the presence of seven periodontopathogens. RESULTS: The prevalences were 75% for Actinobacillus actinomycetemcomitans, 94.2% for Tannerella forsythensis (formerly Bacteroides forsythus), 99.4% for Fusobacterium sp., 85.9% for Micromonas micros (formerly Peptostreptococcus micros), 96.8% for Porphyromonas gingivalis, 78.8% for Prevotella intermedia, and 96.8% for Treponema sp. The prevalences of these bacteria were significantly higher in diseased sites than in healthy sites. Logistic regression analysis showed that P. intermedia was more significantly associated with generalized aggressive periodontitis than the localized form, with an odds ratio of 3.28 (95% confidence interval 1.26-8.56, P = 0.015). CONCLUSIONS: Our results demonstrate that the seven periodontal pathogens analyzed are strongly associated with Korean aggressive periodontitis. In particular, P. intermedia are more significantly associated with generalized aggressive periodontitis, a more severe and progressive form, than with localized aggressive periodontitis.

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque.

OBJECTIVES: Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. MATERIALS AND METHODS: The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. RESULTS: Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. CONCLUSION: Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.

Comparison of microbial cultivation and a commercial PCR based method for detection of periodontopathogenic species in subgingival plaque samples.

OBJECTIVES: Microbiological laboratory procedures are involved in diagnosis and therapy control of progressive and refractory forms of periodontitis. In recent years techniques have been developed based on the detection of nucleic acids. The purpose of this study was to validate the commercially available micro-Dent(R) test which employs probes for A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus and T. denticola. METHODS: 122 plaque samples obtained from periodontal pockets with various depths from 33 early onset periodontitis (EOP) patients and 15 periodontally healthy subjects were analysed by cultivation and the microDent(R) kit. RESULTS: Both cultivation and the nucleic acid based assay showed a positive correlation of pocket depth with the frequency and quantity of periodontopathogenic species. T. denticola was found only in pockets > 4 mm in EOP patients. Comparison of the two methods revealed that the microDent(R) kit identified both P. gingivalis and B. forsythus more often than did the cultivation method. CONCLUSIONS: Nucleic acid techniques should replace cultivation methods as gold standard in microbiological diagnosis of progressive periodontitis. The micro-Dent(R) kit can be recommended for microbiological laboratories analysing subgingival plaque samples.