Oral Treponema species, most notably T. denticola, are implicated in the destructive effects of human periodontal disease. Progress in the molecular analysis of interactions between T. denticola and host proteins is reviewed here, with particular emphasis on the characterization of surface-expressed and secreted proteins of T. denticola involved in interactions with host cells, extracellular matrix components, and components of the innate immune system.

In periodontal disease, host recognition of bacterial constituents, including lipopolysaccharide (LPS), induces p38 MAPK activation and subsequent inflammatory cytokine expression, favoring osteoclastogenesis and increased net bone resorption in the local periodontal environment. In this paper, we discuss evidence that the p38/MAPK-activated protein kinase-2 (MK2) signaling axis is needed for periodontal disease progression: an orally administered p38α inhibitor reduced the progression of experimental periodontal bone loss by reducing inflammation and cytokine expression. Subsequently, the significance of p38 signaling was confirmed with RNA interference to attenuate MK2-reduced cytokine expression and LPS-induced alveolar bone loss. MAPK phosphatase-1 (MKP-1), a negative regulator of MAPK activation, was also critical for periodontal disease progression. In MPK-1-deficient mice, p38-sustained activation increased osteoclast formation and bone loss, whereas MKP-1 overexpression dampened p38 signaling and subsequent cytokine expression. Finally, overexpression of the p38/MK2 target RNA-binding tristetraprolin (TTP) decreased mRNA stability of key inflammatory cytokines at the posttranscriptional level, thereby protecting against periodontal inflammation. Collectively, these studies highlight the importance of p38 MAPK signaling in immune cytokine production and periodontal disease progression.

The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium.

Host defense peptides are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. Deficiency in the human host defense peptide LL-37 has previously been correlated with severe periodontal disease. Treponema denticola is an oral anaerobic spirochete closely associated with the pathogenesis of periodontal disease. The T. denticola major surface protein (MSP), involved in adhesion and cytotoxicity, and the dentilisin serine protease are key virulence factors of this organism. In this study, we examined the interactions between LL-37 and T. denticola. The three T. denticola strains tested were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at the Lys, Phe, Gln, and Val residues. However, dentilisin deletion did not increase the susceptibility of T. denticola to LL-37. Furthermore, dentilisin activity was found to be inhibited by human saliva. In contrast, a deficiency of the T. denticola MSP increased resistance to LL-37. The MSP-deficient mutant bound less fluorescently labeled LL-37 than the wild-type strain. MSP demonstrated specific, dose-dependent LL-37 binding. In conclusion, though capable of LL-37 inactivation, dentilisin does not protect T. denticola from LL-37. Rather, the rapid, MSP-mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. Moreover, in vivo, saliva inhibits dentilisin, thus preventing LL-37 restriction and ensuring its bactericidal and immunoregulatory activities.

The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses. It has been suggested that in the gut microbiome species such as Bacteroides thetaiotaomicron are keystone in maintaining the stability and functional adaptability of the microbial community. In this study, we investigate the interspecies associations in a complex microbial biofilm applying systems biology principles. Using correlation network analysis we identified bacterial modules that represent important microbial associations within the oral community. We used dental plaque as a model community because of its high diversity and the well known species-species interactions that are common in the oral biofilm. We analyzed samples from healthy individuals as well as from patients with periodontitis, a polymicrobial disease. Using results obtained by checkerboard hybridization on cultivable bacteria we identified modules that correlated well with microbial complexes previously described. Furthermore, we extended our analysis using the Human Oral Microbe Identification Microarray (HOMIM), which includes a large number of bacterial species, among them uncultivated organisms present in the mouth. Two distinct microbial communities appeared in healthy individuals while there was one major type in disease. Bacterial modules in all communities did not overlap, indicating that bacteria were able to effectively re-associate with new partners depending on the environmental conditions. We then identified hubs that could act as keystone species in the bacterial modules. Based on those results we then cultured a not-yet-cultivated microorganism, Tannerella sp. OT286 (clone BU063). After two rounds of enrichment by a selected helper (Prevotella oris OT311) we obtained colonies of Tannerella sp. OT286 growing on blood agar plates. This system-level approach would open the possibility of manipulating microbial communities in a targeted fashion as well as associating certain bacterial modules to clinical traits (e.g.: obesity, Crohn's disease, periodontal disease, etc).

Palmer LJ, Chapple ILC, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodont Res 2011; doi: 10.1111/j.1600-0765.2011.01451.x © 2011 John Wiley & Sons A/S Background and Objective:  Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host's immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. Material and Methods:  To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Results:  Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. Conclusion:  The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.

In this study, the association of red complex (RC) bacteria that include Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis with acute, exacerbated or chronic apical periodontitis was evaluated. Seventy-one patients with periapical disease were evaluated by clinical examination and microbiological samples obtained from the root canals were analyzed by a polymerase chain reaction assay. Twenty-one (29.6%) samples were positive for RC bacteria, with T. denticola, T. forsythia and P. gingivalis being detected in 14 (19.7%), 10 (14.1%) and 6 (8.5%) samples, respectively. RC bacteria were mainly associated with acute apical periodontitis (29.2%) and phoenix abscess (63.2%), while they were only sporadically detected (7.1%) in patients with chronic apical periodontitis. Generally, RC bacteria were associated with pain and a higher frequency of intracanalar/intrasulcular pus drainage. Involvement of RC bacteria in symptomatic periapical disease should be suspected in the presence of particularly severe clinical pain and pus drainage.

This chapter addresses the host responses to the microbial biofilm that constitutes the subgingival dental plaque. The host response to infection draws upon the innate, inflammatory and adaptive immune systems, whose role is to provide the appropriate response to the offending microorganisms. In some cases, this will be little or no response when encountering 'commensals', and in other cases a gradated response depending very much on the host's own determination of the pathogenic nature of the microbial insult: and herein lies the root of variation in host responses that govern individual susceptibility. In some individuals and with some bacteria this will be an innate-only response, others will need to invoke the inflammatory response, and yet others will require the adaptive immune response - be it cellular, humoral or both - to reduce or remove the challenge from the microbes. Of course these responses would be somewhat easier to predict with a single pathogen challenge, and become infinitely more complex as the biofilm increases in complexity. Oral infections, in particular gingival inflammation, originate from not just one but many microorganisms. This polymicrobial infection may result in chronic inflammation, which may lead to tissue destruction, as evident in chronic periodontitis. Although many organisms are present in the subgingival biofilm, interestingly, the putative pathogens associated with gingivitis and periodontitis may comprise very small fractions of the total biomass. An understanding of the interaction of structural and defensive host cells with the biofilm is pivotal to understanding periodontal disease etiology and to developing tailored therapeutics. Thus, this chapter addresses the main structural cells, i.e. epithelial cells, exposed to the biofilm.

AIM To identify possible novel biomarkers in gingival crevicular fluid (GCF) samples from chronic periodontitis (CP) and periodontally healthy individuals using high-throughput proteomic analysis. MATERIALS AND METHODS Gingival crevicular fluid samples were collected from 12 CP and 12 periodontally healthy subjects. Samples were trypically digested with trypsin, eluted using high-performance liquid chromatography, and fragmented using tandem mass spectrometry (MS/MS). MS/MS spectra were analysed using PILOT_PROTEIN to identify all unmodified proteins within the samples. RESULTS Using the database derived from Homo sapiens taxonomy and all bacterial taxonomies, 432 human (120 new) and 30 bacterial proteins were identified. The human proteins, angiotensinogen, clusterin and thymidine phosphorylase were identified as biomarker candidates based on their high-scoring only in samples from periodontal health. Similarly, neutrophil defensin-1, carbonic anhydrase-1 and elongation factor-1 gamma were associated with CP. Candidate bacterial biomarkers include 33 kDa chaperonin, iron uptake protein A2 and phosphoenolpyruvate carboxylase (health-associated) and ribulose biphosphate carboxylase, a probable succinyl-CoA:3-ketoacid-coenzyme A transferase, or DNA-directed RNA polymerase subunit beta (CP-associated). Most of these human and bacterial proteins have not been previously evaluated as biomarkers of periodontal conditions and require further investigation. CONCLUSIONS The proposed methods for large-scale comprehensive proteomic analysis may lead to the identification of novel biomarkers of periodontal health or disease.

BACKGROUND Various mouth rinses have been used in the treatment of halitosis, but most of the evidence for the efficacy of mouth rinses is anecdotal. In the present study, 0.2% chlorhexidine rinse and an essential oil mouth rinse are compared for their efficacy in reducing the breath mercaptan levels. MATERIALS AND METHODS Fifteen patients with the chief complaint of oral malodor were randomly divided into 3 groups and were provided with the respective mouth rinses. Pre rinsing measurements were performed on the Day 1 and no other periodontal treatment was instituted. Post rinsing estimation of mercaptan levels was performed after 7 days. RESULTS When comparing chlorhexidine with the essential oil mouth rinse, the reduction in VSCs was highly significant in the chlorhexidine group (P<0.01). However, the reduction in the organoleptic scores was not significant among the two groups. Organoleptic scores showed very highly significant correlations with the VSC concentrations measured by the spectrophotometric method. CONCLUSION The spectrophotometric technique employed in this study appears to be a promising new method for evaluation of oral malodor. Chlorhexidine still appears to be the agent of choice as a short term regimen in cases of oral malodor.

BACKGROUND/AIMS: Previously, we reported that SRP resulted in a decrease in mean pocket depth and attachment level and reduced prevalence and levels of Bacteroidesforsythus, Porphyromonas gingivalis, and Treponema denticola at 3 and 6 months post-SRP in 57 subjects with adult periodontitis. 32 of the 57 subjects were monitored at 9 and 12 months. Thus, the purpose of the present investigation was to evaluate the microbial and clinical effects of SRP in 32 (mean age 48+/-11) subjects over a 12-month period. METHOD: Clinical assessments of plaque, gingival redness, suppuration, bleeding on probing, pocket depth and attachment level were made prior to SRP and at 3, 6, 9, and 12 months post-therapy. Subgingival plaque samples were taken at each visit and analyzed using the checkerboard DNA-DNA hybridization technique for the presence and levels of 40 subgingival species. Each subject also received maintenance scaling at each of the subsequent monitoring visits. Differences in clinical parameters and prevalence and levels of bacterial species were analyzed pre- and post-therapy using the Wilcoxon signed ranks test. The Quade test for related samples was used for analysis of multiple visits. RESULTS: Mean pocket depth (mm+/-SEM) decreased from 3.2+/-0.3 at baseline to 2.9+/-0.3 at 12 months (p<0.01). Mean attachment level showed significant reduction at 6 months, but did not diminish further. Bleeding on probing and plaque were significantly reduced at 12 months (p<0.001, p<0.05, respectively). P. gingivalis, B. forsythus and T. denticola decreased in prevalence and levels up to the 6-month visit and remained at these lower levels at 9 and 12 months. Significant increases in levels and prevalence were noted at 12 months for Actinomyces naeslundii genospecies 2, Actinomyces odontolyticus, Fusobacterium nucleatum ss polymorphum, Streptococcus mitis, Capnocytophaga sp, and Veillonella parvula. CONCLUSIONS: The data suggest that the maintenance phase of therapy may be essential in consolidating clinical and microbiological improvements achieved as a result of initial therapy.

This investigation compared the site prevalence of 40 subgingival species in 30 periodontally healthy (mean age 36+/-9 years), 35 elders with a well-maintained periodontium (mean age 77+/-5) and 138 adult periodontitis subjects (mean age 46+/-11). Subgingival plaque samples were taken from the mesial aspect of each tooth (up to 28 samples) in the 203 subjects at baseline. The presence and levels of 40 subgingival taxa were determined in 5003 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The % of sites colonized by each species (prevalence) was computed for each subject. Differences in prevalence and levels among groups were sought using the Kruskal-Wallis test. Commonly detected species, such as Actinomyces naeslundii genospecies 2, Streptococcus sanguis and Streptococcus oralis did not differ significantly among subject groups. After adjusting for multiple comparisons, 4 species were significantly elevated and at greater prevalence in the periodontitis group. Mean % of sites (+/-SEM) colonized by Bacteroides forsythus was 10+/-3, 12+/-2 and 40+/-2 (p<0.001) for healthy, elder and periodontitis groups respectively. The odds ratio was 14.4:1 that a subject had periodontitis when B. forsythus was detected at > or = 5% of sampled sites. Mean prevalence for Porphyromonas gingivalis in healthy, elder and periodontitis subjects was 4+/-2, 5+/-2 and 23+/-2 respectively (p<0.001); for Treponema denticola 12+/-4, 10+/-3 and 30+/-2 (p<0.001) and for Selenomonas noxia 6+/-2, 7+/-2 and 19+/-2 (p<0.01). Similar differences among subject groups were observed when only sites with PD 0-4 mm were analyzed. The data suggest an etiologic role for B. forsythus, P. gingivalis, T. denticola and S. noxia in adult periodontitis.

In a previous report, it was shown that scaling and root planing (SRP) decreased mean pocket depth and attachment level in subjects with adult periodontitis, as well as the levels and prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Treponema denticola. However, a subset of subjects in that study exhibited mean loss of attachment following SRP. The purpose of the present investigation was to seek clinical and microbiological differences between subjects who responded well or poorly to SRP. 57 subjects with adult periodontitis were treated by full-mouth SRP under local anaesthetic. Clinical assessments of plaque, redness, suppuration, BOP, pocket depth and attachment level were made at 6 sites per tooth prior to and 3 months post-SRP. Attachment level measurements were repeated at each visit and differences in means between visits used to assess change. 18 subjects showed mean attachment loss 3 months post-SRP (poor response group), while 39 showed mean attachment level gain (good response group). The prevalence and levels of 40 subgingival taxa in subgingival plaque samples from the mesiobuccal site of each tooth (maximum 28 sites) in each subject prior to and 3 months post-SRP were assessed using checker-board DNA-DNA hybridization. The prevalence of each species was computed for each subject and averaged across subjects in the 2 treatment-response groups at each visit. Differences between groups were sought using the Mann-Whitney test. There were no statistically significant differences between the 2 response groups in any clinical parameter prior to therapy. Subjects in the good response group showed more attachment level gain at sites with baseline pocket depths of < 4 mm, 4-6 and > 6 mm than poor response subjects. Of 40 species evaluated, A. naeslundii genospecies 2 (A. viscosus), T. denticola, C. gracilis and C. rectus were significantly higher and more prevalent pre-therapy in the good response subjects. Mean attachment level change post SRP could be predicted using multiple linear regression with A. naeslundii genospecies 2 (A. viscosus) and T. denticola as the predictor variables (r2 = 0.373, p < 0.00001). Sites that gained > or = 2 mm of attachment post therapy showed a significant decrease in the counts of P. gingivalis (7.5 +/- 3.5 to 0.2 +/- 0.2 x 10(5)), T. denticola (8.2 +/- 3.5 to 1.8 +/- 1.1 x 10(5)) and B. forsythus (11.1 +/- 5.7 to 0.3 +/- 0.2 x 10(5)). The data of the present investigation indicate that SRP is most effective in subjects and sites with high levels of the subgingival species that this therapy affects.

The purpose of the present investigation was to examine the effect of SRP on clinical and microbiological parameters in 57 subjects with adult periodontitis (mean age 47 +/- 11 years). Subjects were monitored clinically and microbiologically prior to and 3, 6 and 9 months after full-mouth SRP under local anaesthesia. Clinical assessments of plaque, redness, suppuration, BOP, pocket depth and attachment level were made at 6 sites per tooth. The means of duplicate attachment level measurements taken at each visit were used to assess change between visits. Clinical data were averaged within each subject and then averaged across subjects for each visit. Subgingival plaque samples were taken from the mesial aspect of each tooth and the presence and levels of 40 subgingival taxa were determined using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The mean levels and % of sites colonized by each species (prevalence) was computed for each subject at each visit. Differences in clinical and microbiological parameters before and after SRP were sought using the Wilcoxon signed ranks test or the Quade test for more than 2 visits. Overall, there was a mean gain in attachment level of 0.11 +/- 0.23 mm (range -0.53 to 0.64 mm) 3 months post-therapy. There was a significant decrease in the % of sites exhibiting gingival redness (68 to 57%) and BOP (58 to 52%) as well as a mean (+/-SEM) pocket depth (3.3 +/- 0.06 to 3.1 +/- 0.05 mm). Sites with pre-therapy pocket depths of < 4 mm showed a non-significant increase in pocket depth and attachment level, 4.6 mm pockets showed a significant decrease in pocket depth and a non-significant gain in attachment post-therapy, while > 6 mm pockets showed a significant decrease in pocket depth and attachment level measurements post-therapy. Significant clinical improvements were seen in subjects who had never smoked or were past smokers but not in current smokers. Mean prevalences and levels of P. gingivalis, T. denticola and B. forsythus were significantly reduced after SRP, while A. viscosus showed a significant increase in mean levels. The mean decrease in prevalence of P. gingivalis was similar at all pocket depth categories, while B. forsythus decreased more at shallow and intermediate pockets and A. viscosus increased most at deep sites. P. gingivalis. B. forsythus and T. denticola were equally prevalent among current, past and never smokers pre-therapy, decreased significantly post-SRP in never and past smokers but increased in current smokers. Clinical improvement post-SRP was accompanied by a modest change in the subgingival microbiota, primarily a reduction in P. gingivalis, B. forsythus and T. denticola, suggesting potential targets for therapy and indicating that radical alterations in the subgingival microbiota may not be necessary or desirable in many patients.

BACKGROUND Previous studies have shown differences in the mean proportions of subgingival species in samples from periodontitis subjects in different countries, which may relate to differences in diet, genetics, disease susceptibility and manifestation. The purpose of the present investigation was to determine whether there were differences in the subgingival microbiotas of Swedish and American subjects who exhibited periodontal health or minimal periodontal disease. METHOD One hundred and fifty eight periodontally healthy or minimally diseased subjects (N Sweden=79; USA=79) were recruited. Subjects were measured at baseline for plaque, gingivitis, BOP, suppuration, pocket depth and attachment level at 6 sites per tooth. Subgingival plaque samples taken from the mesial aspect of each tooth at baseline were individually analyzed, in one laboratory, for their content of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples=4345).% DNA probe counts comprised by each species was determined for each site and averaged across sites in each subject. Significance of differences in proportions of each species between countries was determined using ancova adjusting for age, mean pocket depth, gender and smoking status. p values were adjusted for multiple comparisons. Cluster analysis was performed to group subjects based on their subgingival microbial profiles using a chord coefficient and an average unweighted linkage sort. RESULTS On average, all species were detected in samples from subjects in both countries. After adjusting for multiple comparisons, 5 species were in significantly higher adjusted mean percentages in Swedish than American subjects: Actinomyces naeslundii genospecies 1 (9.7, 3.3); Streptococcus sanguis (2.5, 1.2); Eikenella corrodens (1.7, 1.0); Tannerella forsythensis (3.5, 2.3) and Prevotella melaninogenica (6.3, 1.8). Leptotrichia buccalis was in significantly higher adjusted mean percentages in American (5.5) than Swedish subjects (3.0). Cluster analysis grouped 121 subjects into 8 microbial profiles. Twenty four of the 40 test species examined differed significantly among cluster groups. Five clusters were dominated by American subjects and 2 clusters by Swedish subjects. Fifty eight of 79 (73%) of the Swedish subjects fell into 1 cluster group dominated by high proportions of A. naeslundii genospecies 1, Prevotella nigrescens, T. forsythensis and P. melaninogenica. Other clusters were characterized by high proportions of Actinomyces gerencseriae, Veillonella parvula, Capnocytophaga gingivalis, Prevotella intermedia, Eubacterium saburreum, L. buccalis and Neisseria mucosa. CONCLUSIONS The microbial profiles of subgingival plaque samples from Swedish and American subjects who exhibited periodontal health or minimal disease differed. The heterogeneity in subgingival microbial profiles was more pronounced in the American subjects, possibly because of greater genetic and microbiologic diversity in the American subjects sampled.

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe-target formats, such as checkerboard DNA-DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin-labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10(4) cells. In all, 93.5% of potential cross-reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross-reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA-DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.

BACKGROUND/AIM The purpose of the present investigation was to determine the effect of self-performed supragingival plaque removal using either manual (Crest Complete) or power (Braun 3D Plaque Remover) toothbrushing on supra and subgingival plaque composition. METHODS 47 periodontal maintenance subjects completed this single-blind 6 month longitudinal study. At baseline, samples of supra and separately subgingival plaque were taken from the mesial aspect of each tooth in each subject using sterile curettes and individually analyzed for their content of 18 bacterial taxa using checkerboard DNA-DNA hybridization. After random assignment to groups receiving either a manual (n=25) or power toothbrush (n=22), subjects received instruction in oral hygiene and used their assigned toothbrush 2x daily for 6 months. Clinical monitoring and microbiological sampling were repeated at 3 and 6 months. Significant differences in microbiological measures over time were sought using the Quade test and between brushing groups at each time point using the Mann-Whitney test. RESULTS Mean total counts were significantly reduced for supra- and subgingival plaque samples in the manual group and subgingival samples in the powered brushing group. Actinomyces naeslundii and Actinomyces israelii/gerencseriae were the most numerous organisms detected at baseline and showed the greatest reductions in counts in both brushing groups. Streptococcus constellatus/intermedius was significantly reduced in both groups, while Streptococcus mitis/oralis/sanguis was significantly reduced in the manual toothbrushing group. Mean counts of species were more markedly altered in subgingival plaque. Major reductions occurred in both groups for A. naeslundii, A. israelii/gerencseriae, Peptostreptococcus micros, Veillonella parvula, Prevotella intermedia/nigrescens, S. mitis/oralis/sanguis and S. constellatus/intermedius. All taxa examined were reduced in prevalence (% of sites colonized) in the subgingival plaque samples for both brushing groups. The reductions in prevalence were greater for A. naeslundii, S. constellatus/intermedius, V. parvula, A. israelii/gerencseriae, S. mitis/oralis/sanguis, P. micros, Streptococcus mutans and P. intermedia/nigrescens. Mean prevalence was decreased more for Porphyromonas gingivalis, Campylobacter rectus/showae, Treponema denticola and Bacteroides forsythus in supragingival plaque than subgingival plaque. CONCLUSIONS The major finding was the effect of supragingival plaque removal on the composition of the subgingival microbiota. Counts and prevalence of most taxa examined were markedly decreased in both toothbrushing groups. This reduction should translate to a decreased risk of periodontal disease initiation or recurrence. Further, the decreased prevalence of periodontal pathogens in supragingival plaque lowers potential reservoirs of these species.

BACKGROUND, AIMS Polymorphisms in the cluster of IL-1 genes have been significantly associated with the severity of adult periodontitis. The purpose of this study was to compare microbiological parameters in IL-1 genotype negative and positive adult subjects with a range of periodontitis severities. METHOD The study included 108 subjects in good general health. Clinical parameters were recorded at 6 sites/tooth excluding 3rd molars and included: plaque accumulation, gingival erythema, bleeding on probing, suppuration, pocket depth and attachment level. Subgingival plaque samples were collected from the mesiobuccal surface of up to 28 teeth in each subject (mean 25.3) providing a total of 2736 samples. The levels of 40 subgingival taxa were determined in each sample using checkerboard DNA-DNA hybridization. Fingerstick blood samples were collected for IL-1A (+4845) and IL-1B (+3954) genotyping using PCR-based methods. RESULTS The proportion of IL-1 genotype positive subjects that exhibited mean counts of specific subgingival species above selected thresholds was significantly higher than the proportion of genotype negative subjects. Prominent among species that were detected at higher levels in genotype positive subjects were members of the "red" and "orange" complexes and included: Bacteroides forsythus, Treponema denticola, the Fusobacterium nucleatum subspecies, Fusobacterium periodonticum, Campylobacter gracilis, Campylobacter showae and Streptococcus constellatus. Streptococcus intermedius, Streptococcus gordonii and 3 Capnocytophaga species were also detected more frequently at high numbers in genotype positive subjects. Significantly higher mean counts of B. forsythus, Porphyromonas gingivalis, T. denticola, the F. nucleatum subspecies, F. periodonticum, Campylobacter rectus, C. showae, Eubacterium nodatum, S. constellatus, S. gordonii, and S. intermedius were detected at periodontal pockets >6 mm in subjects who were genotype positive when compared with genotype negative subjects. The increase was due to increased numbers of cells of these species rather than a major shift in proportion. CONCLUSION The data suggest that genotype positive subjects more frequently had higher levels of "red" and "orange" complex species that are known to be strongly associated with measures of periodontal inflammation.

An association has been reported between polymorphisms in the genes encoding IL-1alpha (-889) and IL-1beta (+3953)(periodontitis susceptibility trait, PST), and an increased severity of periodontitis (18). The IL-1beta polymorphism was reported to correlate with increased IL-1beta expression by monocytes in response to bacterial stimulants. In the present study, we determined if PST positive subjects with periodontitis exhibit elevated production of IL-1beta, compared to PST negative periodontitis patients. Peripheral blood monocytes were obtained from 10 PST+ and 10 PST- age- and disease-balanced subjects with adult forms of periodontitis. Monocytes were cultured with a panel of bacterial stimulants, including Escherichia coli and Porphyromonas gingivalis LPS, and whole formalinized periodontal pathogens P. gingivalis, Bacteroides forsythus and Prevotella intermedia, and health-associated organisms Veillonella parvula and Streptococcus sanguis. Our results demonstrate that monocytes from PST+ and PST- patients showed no significant differences in IL-1beta production in response to any stimulant tested. In addition, the periodontal pathogens P. gingivalis, B. forsythus and P. intermedia failed to stimulate higher IL-1beta responses compared to health-associated species V. parvula and S. sanguis. A marked interindividual variation in production of IL-1beta was seen, with high, low and intermediate responders present in both PST+ and PST- groups. We conclude that genetic loci other than the PST polymorphisms are also important regulators of monocyte IL-1 responses.

The purpose of this investigation was to examine the short-term effect of apically repositioned flap surgery on clinical and microbiologic parameters in patients with adult periodontitis. A total of 11 patients with moderate to advanced periodontitis received apically repositioned flap surgery. Subjects were monitored during a 3-month pretreatment phase, the baseline surgical phase, and for 3 months post-surgery. Clinical assessments including plaque accumulation, gingival redness, suppuration, bleeding on probing, pocket depth, and attachment level were made at 6 sites per tooth. Subgingival plaque samples were taken from the mesial aspect of each tooth and the presence and levels of 29 subgingival taxa were determined using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The mean levels and percentage of sites colonized by each species (prevalence) were computed for each subject at each visit. After surgery, there was a significant decrease in mean pocket depth and percentage of sites exhibiting gingival redness. Significant decreases were seen in the percentage of sites that had attachment levels < 4 mm, with a significant increase in the percentage of sites with attachment levels of 4 to 6 mm after therapy. The mean total DNA probe count for all bacterial species was significantly decreased by both scaling and root planing and surgical therapy. P gingivalis and B forsythus, 2 bacteria previously shown to be susceptible to mechanical therapy, exhibited statistically significant decreases in mean total DNA probe count. Because surgical therapy decreased levels of the suspected periodontal pathogens C rectus, P nigrescens, and C gracilis, it may be speculated that there was a potential added beneficial effect of surgery on the periodontal microbiota.

The objective of this study was to characterize the subgingival microbiota of African-American children with Localized Aggressive Periodontitis (LAP). Fifty-one children were included. Subgingival plaque samples were taken from diseased (DD) and healthy sites (DH) in LAP and from healthy sites in HS and HC and analyzed by 16S rRNA-based microarrays. Aggregatibacter actinomycetemcomitans (Aa) was the only species found to be both more prevalent (OR = 8.3, p = 0.0025) and abundant (p < 0.01) in DD. Filifactor alocis (Fa) was also found to be more prevalent in DD (OR 2.31, CI 1.06-5.01, p = 0.03). Most prevalent species in healthy sites were Selenomonas spp, Veillonella spp, Streptococcus spp, Bergeyella sp, and Kingella oralis. Overall, Streptococcus spp, Campylobacter gracilis, Capnocytophaga granulosa, Haemophilus parainfluenzae, and Lautropia mirabilis were most abundant in healthy children, while Aa, Fa, Tannerella sp, Solobacterium moorei, Parvimonas micra, and Capnocytophaga sp were most abundant in LAP. Based on a comprehensive analysis with 16S rRNA-based microarrays, Aa was strongly associated and site-specific in LAP. In contrast, other species were found to be associated with healthy sites and individuals (ClinicalTrials.gov number CT01330719).Abbreviations: healthy site in healthy sibling (HS); healthy site in healthy control child (HC).

Background: Surfaces and fluids can affect oral bacterial colonization. The aim of this study is to compare redeveloping biofilms on natural teeth and dentures. Methods: Supragingival plaque samples were taken from 55 dentate individuals and the denture teeth of 62 edentulous individuals before and after professional cleaning. Also, samples from seven "teeth"(samples included dentures) in randomly selected quadrants were collected after 1, 2, 4, and 7 days of no oral hygiene. Samples were analyzed using checkerboard DNA-DNA hybridization. Counts and proportions of 41 bacterial taxa were determined at each time point, and significant differences were determined using the Mann-Whitney U test. Ecological succession was determined using a modified moving window analysis. Results: Mean total DNA probe counts were similar precleaning but were higher in dentate individuals at all post-cleaning visits (P <0.01). Precleaning edentate biofilms had higher counts and proportions of Streptococcus mitis, Streptococcus oralis, and Streptococcus mutans, whereas dentate individuals had higher proportions of Tannerella forsythia, Selenomonas noxia, and Neisseria mucosa. By day 2, mean counts of all taxa were higher in natural teeth, and most remained higher at day 7 (P <0.01). Succession was more rapid and complex in dentate individuals. Both groups demonstrated increased proportions of S. mitis and S. oralis by day 1. N. mucosa, Veillonella parvula, and Eikenella corrodens increased in both groups, but later in samples from edentate individuals. Conclusions:"Mature" natural and denture teeth biofilms have similar total numbers of bacteria but different species proportions. Post-cleaning biofilm redevelopment is more rapid and more complex on natural teeth than on denture teeth.

AIM Although it is established that peri-implantitis is a bacterially induced disease, little is known about the bacterial profile of peri-implant communities in health and disease. The purpose of the present investigation was to examine the microbial signatures of the peri-implant microbiome in health and disease. MATERIALS AND METHODS Subgingival and submucosal plaque samples were collected from forty subjects with periodontitis, peri-implantitis, periodontal and peri-implant health and analysed using 16S pyrosequencing. RESULTS Peri-implant biofilms demonstrated significantly lower diversity than subgingival biofilms in both health and disease, however, several species, including previously unsuspected and unknown organisms, were unique to this niche. The predominant species in peri-implant communities belonged to the genera Butyrivibrio, Campylobacter, Eubacterium, Prevotella, Selenomonas, Streptococcus, Actinomyces, Leptotrichia, Propionibacterium, Peptococcus, Lactococcus and Treponema. Peri-implant disease was associated with lower levels of Prevotella and Leptotrichia and higher levels of Actinomyces, Peptococcus, Campylobacter, non-mutans Streptococcus, Butyrivibrio and Streptococcus mutans than healthy implants. These communities also demonstrated lower levels of Prevotella, non-mutans Streptococcus, Lactobacillus, Selenomonas, Leptotrichia, Actinomyces and higher levels of Peptococcus, Mycoplasma, Eubacterium, Campylobacter, Butyrivibrio, S. mutans and Treponema when compared to periodontitis-associated biofilms. CONCLUSION The peri-implant microbiome differs significantly from the periodontal community in both health and disease. Peri-implantitis is a microbially heterogeneous infection with predominantly gram-negative species, and is less complex than periodontitis.

OBJECTIVE The aim of this study was to distinguish between generalized aggressive (GAgP) and chronic periodontitis (CP) based on the subgingival microbial profiles predominant in these diseases. METHODS Two-hundred and sixty subjects, 75 with GAgP and 185 with CP were recruited. Full-mouth clinical measurements were recorded. Individual subgingival plaque samples were taken from 7 sites per subject and analyzed for the prevalence and levels of 51 species by chequerboard. Differences between groups were examined by the Mann-Whitney test. Associations between bacterial species and GAgP were examined by logistic regression analysis. RESULTS Actinomyces gerensceriae, Actinomyces israelli, Eubacterium nodatum and Propionibacterium acnes were detected in significantly greater counts in GAgP, whereas Capnocytophaga ochracea, Fusobacterium periodonticum, Staphylococcus aureus and Veillonella parvula were more predominant in CP patients (adjusted p < 0.001). E. nodatum (at mean levels ≥4 × 10(5)) increased significantly the probability of a subject being diagnosed with GAgP than CP (OR 2.44 [0.96-6.20]), whereas P. gingivalis (OR 0.34 [0.11-0.93]) and T. denticola (OR 0.35 [0.11-0.94]) were associated with CP. CONCLUSIONS Very few subgingival species differed in prevalence and/or levels between GAgP and CP in this sample population. In particular, E. nodatum was strongly related to GAgP, whereas P. gingivalis and T. denticola were associated with CP.

Aim: This study compared the changes on the subgingival microbiota of subjects with "refractory" periodontitis (RP) or treatable periodontitis (GR) before and after periodontal therapy by using the Human Oral Microbe Identification Microarray (HOMIM). Methods: Individuals with chronic periodontitis were classified as RP (n=17) based on mean attachment loss (AL) and/or >3 sites with AL ≥2.5 mm after scaling and root planing, surgery and systemically administered amoxicillin and metronidazole or as GR (n=30) based on mean attachment gain and no sites with AL ≥2.5 mm after treatment. Subgingival plaque samples were taken at baseline and 15 months after treatment and analyzed for the presence of 300 species by HOMIM analysis. Significant differences in taxa before and after therapy were sought using the Wilcoxon test. Results: The majority of species evaluated decreased in prevalence in both groups after treatment; however, only a small subset of organisms was significantly affected. Species that increased or persisted in high frequency in RP but were significantly reduced in GR included Bacteroidetes sp., Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella spp., Tannerella forsythia, Dialister spp., Selenomonas spp., Catonella morbi, Eubacterium spp., Filifactor alocis, Parvimonas micra, Peptostreptococcus sp. OT113, Fusobacterium sp. OT203, Pseudoramibacter alactolyticus, Streptococcus intermedius or Streptococcus constellatus and Shuttlesworthia satelles. In contrast, Capnocytophaga sputigena, Cardiobacterium hominis, Gemella haemolysans, Haemophilus parainfluenzae, Kingella oralis, Lautropia mirabilis, Neisseria elongata, Rothia dentocariosa, Streptococcus australis and Veillonella spp. were more associated with therapeutic success. Conclusion: Persistence of putative and novel periodontal pathogens, as well as low prevalence of beneficial species was associated with chronic "refractory" periodontitis.

OBJECTIVE Nowadays, necrotizing periodontal diseases have a low prevalence; however, a better understanding of the etiopathogenesis of these diseases is necessary for determining more adequate preventive and therapeutic strategies. METHOD AND MATERIALS From a pool of 1,232 HIV-infected patients, 15 presented with necrotizing periodontal diseases, which were evaluated by full-mouth periodontal clinical measurements. Subgingival biofilm samples were collected from necrotizing lesions of six of these individuals. The presence and levels of 47 bacterial species were determined by checkerboard DNA-DNA hybridization. RESULTS All 15 patients (10 had severe immunodeficiency) had been infected sexually. Thirteen patients were taking antiretroviral medication (66.7% undergoing highly active antiretroviral therapy). Regarding necrotizing periodontal diseases, necrotizing ulcerative gingivitis (60%) was more prevalent than necrotizing ulcerative periodontitis (40%). The frequency of supragingival biofilm and bleeding on probing ranged from 11.5% to 59.2% and 3.0% to 54.0%, respectively, whereas the mean probing depth and clinical attachment level were between 1.48 and 2.61 mm and 1.30 and 2.62 mm, respectively. Species detected in high prevalence and/or counts in necrotizing lesions included Treponema denticola, Eikenella corrodens, Dialister pneumosintes, Enterococcus faecalis, Streptococcus intermedius, Aggregatibacter actinomycetemcomitans, and Campylobacter rectus. In contrast, Parvimonas micra, Prevotella melaninogenica, Fusobacterium nucleatum, Eubacterium nodatum, and Helicobacter pylori were observed in the lowest mean prevalence and/or counts. CONCLUSION Necrotizing periodontal disease lesions in HIV-infected patients present a microbiota with high prevalence and/or counts of classical periodontal pathogens, in particular T denticola, as well as species not commonly considered as periodontal pathogens, such as E faecalis and D pneumosintes. In addition, these individuals with necrotizing periodontal disease frequently display severe immunodeficiency and AIDS-defining diseases such as tuberculosis.

Khocht A, Yaskell T, Janal M, Turner BF, Rams TE, Haffajee AD, Socransky SS. Subgingival microbiota in adult Down syndrome periodontitis. J Periodont Res 2012; 47: 500-507. © 2012 John Wiley & Sons A/S Background and Objective:  The subgingival microbiota in Down syndrome and non-Down syndrome adults receiving periodic dental care was examined for 40 bacterial species using checkerboard DNA-DNA hybridization and the results were related to clinical periodontal attachment loss. Material and Methods:  A total of 44 Down syndrome, 66 non-Down syndrome mentally retarded and 83 mentally normal adults were clinically evaluated. This involved, for each subject, the removal of subgingival specimens from three interproximal sites on different teeth; all subgingival samples per subject were then pooled and assessed for the presence and levels of 40 bacterial species using species-specific whole-genomic DNA probes and checkerboard DNA-DNA hybridization. Significant group differences in species proportions averaged across subjects were evaluated using the Kruskal-Wallis test, and associations between subgingival species and mean subject attachment loss within Down syndrome and non-Down syndrome subject groups were quantified using Pearson correlation and multiple linear regression analysis. Results:  Down syndrome subjects exhibited greater attachment loss than non-Down syndrome subjects (p = 0.05). Most microbial species were present in Down syndrome subjects at levels similar to non-Down syndrome subjects, except for higher proportions of Selenomonas noxia, Propionibacterium acnes, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis in Down syndrome subjects compared with non-Down syndrome study subjects, higher proportions of Treponema socranskii in Down syndrome subjects compared with non-Down syndrome mentally retarded subjects, and higher proportions of Streptococcus constellatus in Down syndrome subjects compared with mentally normal subjects. Down syndrome adults classified with periodontitis revealed higher subgingival levels of T. socranskii than Down syndrome subjects with no periodontitis (p = 0.02). Higher subgingival proportions of S. constellatus, Fusobacterium nucleatum ssp. nucleatum, S. noxia and Prevotella nigrescens showed significant positive correlations (r = 0.35-0.42) and higher proportions of Actinomyces naeslundii II and Actinomyces odontolyticus showed negative correlations (r = -0.36 to -0.40), with increasing mean subject attachment loss in Down syndrome adults. Conclusion:  Individuals with Down syndrome show higher levels of some subgingival bacterial species and specific associations between certain subgingival bacterial species and loss of periodontal attachment. These findings are consistent with the notion that certain subgingival bacteria may contribute to the increased level of periodontal disease seen in Down syndrome individuals and raise the question as to the reason for increased colonization in Down syndrome.

INTRODUCTION Knowing the microbiota that colonizes orthodontic appliances is important for planning strategies and implementing specific preventive measures during treatment. The purpose of this clinical trial was to evaluate in vivo the contamination of metallic orthodontic brackets with 40 DNA probes for different bacterial species by using the checkerboard DNA-DNA hybridization (CDDH) technique. METHODS Eighteen patients, 11 to 29 years of age having fixed orthodontic treatment, were enrolled in the study. Each subject had 2 new metallic brackets bonded to different premolars in a randomized manner. After 30 days, the brackets were removed and processed for analysis by CDDH. Data on bacterial contamination were analyzed descriptively and with the Kruskal-Wallis and Dunn post tests (α = 0.05). Forty microbial species (cariogenic microorganisms, bacteria of the purple, yellow, green, orange complexes,"red complex +Treponema socranskii," and the cluster of Actinomyces) were assessed. RESULTS Most bacterial species were present in all subjects, except for Streptococcus constellatus, Campylobacter rectus, Tannerella forsythia, T socranskii, and Lactobacillus acidophillus (94.4%), Propionibacterium acnes I and Eubacterium nodatum (88.9%), and Treponema denticola (77.8%). Among the cariogenic microorganisms, Streptococcus mutans and Streptococcus sobrinus were found in larger numbers than L acidophillus and Lactobacillus casei (P <0.001). The periodontal pathogens of the orange complex were detected in larger numbers than those of the "red complex +T socranskii"(P <0.0001). Among the bacteria not associated with specific pathologies, Veillonella parvula (purple complex) was the most frequently detected strain (P <0.0001). The numbers of yellow and green complex bacteria and the cluster of Actinomyces were similar (P >0.05). CONCLUSIONS Metallic brackets in use for 1 month were multi-colonized by several bacterial species, including cariogenic microorganisms and periodontal pathogens, reinforcing the need for meticulous oral hygiene and additional preventive measures to maintain oral health in orthodontic patients.

BACKGROUND AND OBJECTIVE The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease. MATERIAL AND METHODS Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time-point. Ecological succession was determined using a modified moving-window analysis. RESULTS Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1-4 d. At 4-7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations. CONCLUSION There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning.

OBJECTIVE To evaluate changes in subgingival microbiota before and during the leveling and alignment orthodontic stage using the polymerase chain reaction (PCR) method. MATERIALS AND METHODS Thirty orthodontic patients (17 females and 13 males; aged 16.7 ± 6.5 y) were included in this study. Subgingival microbial samples were taken from the disto-buccal gingival crevice of the left upper central incisors, the left lower central incisors, the mesio-buccal gingival crevice of the left upper first molars, and the left lower first molars, at four different times: at baseline, before placement of orthodontic appliances (T1), and 1 week (T2), 3 months (T3), and 6 months after placement of orthodontic appliances (T4). DNA was extracted from the samples, and the 16S rRNA-based PCR detection method was used to determine the prevalence of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobactor rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, and Treponema denticola, which are considered as putative periodontopathogens. RESULTS The frequency of T forsythia, C rectus, and P nigrescens significantly increased after placement of orthodontic appliances. For the other species, the frequency tended to increase but no statistically significant difference was noted. The frequency of the change, representing microorganisms not existing at T1 but newly developing at T2, T3, and T4, was higher at the molars than at the incisors. CONCLUSION The placement of orthodontic appliances affects the subgingival microbial composition even during the early period of orthodontic treatment, increasing the prevalence of periodontopathogens, especially in the molar region.