The aim of this study was to compare clinical, cytokine and microbiological responses after quadrant-based scaling and root planing (Q-SRP), full-mouth SRP (FM-SRP) and full-mouth disinfection (FMD) in patients with generalized aggressive periodontitis (GAgP), which is currently termed as generalized stage-III and grade-C periodontitis.Forty-two patients with GAgP were randomly assigned into groups as Q-SRP, FM-SRP or FMD with chlorhexidine. Clinical parameters were recorded, and gingival crevicular fluid (GCF) and subgingival plaque samples were collected at baseline, 3 and 6 months after treatment. GCF levels of interleukin (IL)-1β and IL-17 were analysed using ELISA. Quantities of six bacterial species were determined using qPCR.Clinical parameters improved significantly in all groups at 3 and 6 months (p < 0.05). Percentage of sites with probing depth >6 mm was lower in the FMD than Q-SRP group at 3 and 6 months (p < 0.05). FMD showed significantly higher percentage of pocket closure compared with Q-SRP and FM-SRP at both 3 and 6 months after treatment (p < 0.05). The IL-1β levels decreased only in the FMD group (p < 0.05), whereas no changes were found in IL-17 levels in any group. The levels of five out of six bacterial species decreased at 3 and/or 6 months only in the FMD group (p < 0.05).The FMD treatment appears to offer superior outcome than Q-SRP and could be the first choice for patients with GAgP.

Peptidoglycan-associated lipoprotein (PAL) is a conserved pro-inflammatory outer membrane lipoprotein in Gram-negative bacteria. Compared to systemic pathogens, little is known about the virulence properties of PAL in Aggregatibacter actinomycetemcomitans (AaPAL). The aims of this study were to investigate the cytolethality of AaPAL and its ability to induce pro-inflammatory cytokine production in macrophages. Mouse macrophages were stimulated with AaPAL, and the production of IL-1β, IL-6, TNF-α, and MCP-1 was measured after 6, 24, and 48 h. To investigate which receptor AaPAL employs for its interaction with macrophages, anti-toll-like receptor (TLR)2 and anti-TLR4 antibodies were used to block respective TLRs on macrophages. Metabolic activity and apoptosis of the macrophages were investigated after stimulation with AaPAL. AaPAL induced the production of MCP-1, TNF-α, IL-6, and IL-1β from mouse macrophages in order of decreasing abundance. The pre-treatment of macrophages with an anti-TLR2 antibody significantly diminished cytokine production. Under AaPAL stimulation, the metabolic activity of macrophages decreased in a dose- and time-dependent manner. Furthermore, AaPAL induced apoptosis in 56% of macrophages after 48 h of incubation. Our data suggest that AaPAL can kill macrophages by apoptosis. The results also emphasize the role of AaPAL as a potent pro-inflammatory agent in A. actinomycetemcomitans-associated infections.

Phosphorylcholine (ChoP) is covalently incorporated into bacterial surface structures, contributing to host mimicry and promoting adhesion to surfaces. Our aims were to determine the frequency of ChoP display among Aggregatibacter actinomycetemcomitans strains, to clarify which surface structures bear ChoP, and whether ChoP-positivity relates to serum killing. The tested oral (N = 67) and blood isolates (N = 27) represented 6 serotypes. Mab TEPC-15 was used for immunoblotting of cell lysates and fractions and for immunofluorescence microscopy of cell surface-bound ChoP. The lysates were denatured with urea for hidden ChoP or treated with proteinase K to test whether it binds to a protein. Three ChoP-positive and two ChoP-negative strains were subjected to serum killing in the presence/absence of CRP and using Ig-depleted serum as complement source. Cell lysates and the first soluble cellular fraction revealed a < 10 kDa band in immunoblots. Among 94 strains, 27 were ChoP positive. No difference was found in the prevalence of ChoP-positive oral (21/67) and blood (6/27) strains. Immunofluorescence microscopy corresponded to the immunoblot results. Proteinase K abolished ChoP reactivity, whereas urea did not change the negative result. The TEPC-15-reactive protein was undetectable in Δflp1 mutant strain. The survival rate of serotype-b strains in serum was 100% irrespective of ChoP, but that of serotype-a was higher in ChoP-positive (85%) than ChoP-negative (71%) strains. The results suggest that a third of rough-colony strains harbor ChoP and that ChoP is attached to fimbrial subunit protein Flp1. It further seems that ChoP-positivity does not enhance but may reduce A. actinomycetemcomitans susceptibility to serum killing.

In this observational and prospective study, we investigated if microbiological and serological markers of periodontitis associated with conception in 256 non-pregnant women (Mage = 29.2 years; range 19-42 years). Clinical oral and gynecological examinations were performed, major periodontal pathogens in the saliva were detected, and serum and saliva antibodies against major periodontal pathogens were analyzed. The follow-up period for becoming pregnant was 12 months. Porphyromonas gingivalis was significantly (p = 0.032) more frequently detected in the saliva among those who did not become pregnant (8.3%) than among those who became pregnant (2.1%). The median levels of salivary P. gingivalis immunoglobulin A (IgA; p = 0.006) and IgG (p = 0.007) antibodies were higher among those who did not become pregnant compared to those who became pregnant. Hazard ratios (HR) for not becoming pregnant were HR = 3.75 (95% confidence interval [CI] 1.01-13.9; p = 0.048) if the subject was polymerase chain reaction-positive for P. gingivalis with high salivary antibodies against it, and HR = 1.62 (95% CI 1.03-2.54; p = 0.035) if she had high levels of serum P. gingivalis IgA and signs of periodontal infection. P. gingivalis associated with no success in getting pregnant.

Saliva is an attractive source for oral microbial detection and quantification since sampling is non-invasive and rapid.Objectives: To determine whether different saliva preparation methods or preservation time periods affect DNA stability.Methods: Saliva samples from 4 healthy adult volunteers were processed to obtain 3 different preparations: whole saliva, and after centrifugation pellet and supernatant. Purified DNA (MasterPure (TM)) from each sample was divided into 4 aliquots, one for immediate analysis and 3 (stored at -80 degrees C) for later analyses after 1 week and 2 and 6 months. DNA concentrations and qPCR based quantities of Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Filifactor alocis and Streptococcus mutans were determined.Results: DNA concentration did not decrease (P > 0.05) during the 6-month period in any sample. Mean (SE) DNA concentrations (ng/mu l) in whole saliva were 152.2 (51.2) and 147.8 (50) at day 0 and 6 months, respectively. Similarly, the values for pellet were 134.9 (42.5) and 133.6 (42.9), and for supernatant, 11 (1.9) and 8.9 (2.3), the difference being significant (P < 0.001) between supernatant and whole saliva or pellet. The quantities of most bacterial species found at day 0 remained stable over the 6-month period in all saliva preparations. In supernatant, species quantities were lower (P < 0.05) than in whole saliva or pellet.Conclusions: DNA concentrations were comparable between whole saliva and pellet, suggesting that either of them can be used for DNA-based analyses. Our results also demonstrated that DNA extracted from saliva can be preserved at -80 degrees C for at least 6 months without decrease in DNA concentration.

Background: Members of fastidious Granulicatella and Aggregatibacter genera belong to normal oral flora bacteria that can cause serious infections, such as infective endocarditis. Aggregatibacter actinomycetemcomitans has long been implicated in aggressive periodontitis, whereas DNA-based methods only recently showed an association between Granulicatella spp. and dental diseases. As bacterial coaggregation is a key phenomenon in the development of oral and nonoral multispecies bacterial communities it would be of interest knowing coaggregation pattern of Granulicatella species with A. actinomycetemcomitans in comparison with the multipotent coaggregator Fusobacterium nucleatum.The aim was to investigate coaggregation and biofilm formation of Granulicatella elegans and Granulicatella adiacens with A. actinomycetemcomitans and F. nucleatum strains.Results: F. nucleatum exhibited significantly (p < 0.05) higher autoaggregation than all other test species, followed by A. actinomycetemcomitans SA269 and G. elegans. A. actinomycetemcomitans CU1060 and G. adiacens did not autoaggregate. G. elegans with F. nucleatum exhibited significantly (p < 0.05) higher coaggregation than most others, but failed to grow as biofilm together or separately. With F. nucleatum as partner, A. actinomycetemcomitans strains SA269, a rough-colony wild-type strain, and CU1060, a spontaneous smooth-colony laboratory variant, and G. adiacens were the next in coaggregation efficiency. These dual species combinations also were able to grow as biofilms. While both G. elegans and G. adiacens coaggregated with A. actinomycetemcomitans strain SA269, but not with CU1060, they grew as biofilms with both A. actinomycetemcomitans strains.Conclusions: G. elegans failed to form biofilm with F. nucleatum despite the strongest coaggregation with it. The ability of Granulicatella spp. to coaggregate and/or form biofilms with F. nucleatum and A. actinomycetemcomitans strains suggests that Granulicatella spp. have the potential to integrate into dental plaque biofilms.

BACKGROUND:Complexity of oral polymicrobial communities has prompted a need for developing in vitro models to study behavior of coexisting bacteria. Little knowledge is available of in vitro co-growth of several periodontitis-associated species without early colonizers of dental plaque. THE AIM:was to determine temporal changes in the quantities of six periodontal species in an in vitro biofilm model in comparison with parallel planktonic cultures. MATERIAL AND METHODS:Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Parvimonas micra, Campylobacter rectus and Fusobacterium nucleatum were anaerobically grown as multispecies and monospecies biofilms and parallel planktonic cultures using cell culture plates and microfuge tubes, respectively. After incubating 2, 4, 6, 8 days, biofilms and planktonic cultures were harvested, DNA extracted and the target species quantified using qPCR with species-specific 16S rDNA primers. Biofilm growth as monocultures was visualized at day 2 and 8 with confocal microscopy and crystal violet staining. RESULTS:The six species were found throughout the test period in all culture conditions, except that P. gingivalis and F. nucleatum were not detected in multispecies planktonic cultures at day 8. In multispecies biofilm, P. gingivalis qPCR counts (cells/ml) increased (P<0.05) from day 2-8 and were then higher (P<0.05) than those of A. actinomycetemcomitans and C. rectus, whereas in monospecies biofilm, P. gingivalis counts were lower (P<0.05) than those of the other species, except A. actinomycetemcomitans. When multi- and monospecies biofilm cultures were compared, P. gingivalis counts were higher (P<0.05) but those of the other species, except P. intermedia, lower (P<0.05) in multispecies biofilm. Comparison between planktonic and biofilm cultures showed that A. actinomycetemcomitans, P. micra and C. rectus had higher (P<0.05) counts in planktonic cultures no matter whether grown in mono- or multispecies environment. CONCLUSIONS:Six periodontal species were able to form multispecies biofilm up to 8 days in vitro without pioneer plaque bacteria. P. gingivalis seemed to prefer multispecies biofilm environment whereas P. micra and A. actinomycetemcomitans planktonic culture.

The genome of periodontal pathogen Aggregatibacter actinomycetemcomitans exhibits substantial variations in gene content among unrelated strains primarily due to the presence or absence of genomic islands. This study examined the genomic stability of A. actinomycetemcomitans during its persistent infection in the same host. Four pairs of A. actinomycetemcomitans strains, each pair isolated from an individual over time (0-10 years), were examined for their gains/losses of genes by whole genome sequencing, comparative genomic hybridization by microarray and PCR analysis. Possible effects due to genomic changes were further assessed by comparative transcriptome analysis using microarrays. The results showed that each pair of strains was clonally identical based on phylogenetic analysis of 150 core genes. A novel 24.1-Kb plasmid found in strain S23A was apparently lost in the sibling strain I23C. A 353-bp inversion affecting two essential genes of the serotype-specific gene cluster was found in the serotype antigen-nonexpressing strain I23C, while the same gene cluster was intact in the serotype-expressing sibling strain S23A. A 2,293-bp deletion affecting a gene encoding oxaloacetate decarboxylase and its neighbor region was found in strain SCC2302 but not in the sibling strain AAS4a. However, no evidence of gains or losses of genomic islands was found in the paired strains. Transcriptome profiles showed little or no difference in the paired strains. In conclusion, the genome of A. actinomycetemcomitans appears to be relatively stable during short-term infection. Several types of genomic changes were observed in the paired strains of A. actinomycetemcomitans recovered from the same subjects, including a mutation in serotype-specific gene cluster that may allow the bacteria to evade host immune response.

Objective To detect the possible occurrence of the phosphorylcholine(ChoP) epitope in Aggregatibacter actinomycetemcomitans(Aa) serotype b strains.Methods Immunofluorescence microscopy,slot blot,and immunoblot were applied to detect expression of the ChoP in 12 Aa serotype b strains by using mouse anti-ChoP monoclonal antibody TEPC-15 as the primary antiserum and goat anti-mouse IgA as the secondary antiserum.Results Slot blot results revealed positive reactions in 5/12(41.6%) of strains tested.The ChoP could be detected by immunofluorescence microscopy implied that ChoP may be on the surface of Aa.Immunoblot results showed that ChoP on a structure of 9 kDa.The 5 positive strains could be detected by immunofluorescence,slot blot,and immunoblot simultaneously and 7 negative strains were all negative by all 3 methods.Conclusion Phosphorylcholine could be detected in Aa serotype b strains.And the epitope may be on a surface structure of 9 kDa.

ABSTRACT Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.

Background: Aggregatibacter actinomycetemcomitans is genetically heterogeneous and comprises distinct clonal lineages that may have different virulence potentials. However, limited information of the strain-to-strain genomic variations is available.Methodology/Principal Findings: The genome sequences of 11 A. actinomycetemcomitans strains (serotypes a-f) were generated de novo, annotated and combined with three previously sequenced genomes (serotypes a-c) for comparative genomic analysis. Two major groups were identified; serotypes a, d, e, and f, and serotypes b and c. A serotype e strain was found to be distinct from both groups. The size of the pangenome was 3,301 genes, which included 2,034 core genes and 1,267 flexible genes. The number of core genes is estimated to stabilize at 2,060, while the size of the pangenome is estimated to increase by 16 genes with every additional strain sequenced in the future. Within each strain 16.7-29.4% of the genome belonged to the flexible gene pool. Between any two strains 0.4-19.5% of the genomes were different. The genomic differences were occasionally greater for strains of the same serotypes than strains of different serotypes. Furthermore, 171 genomic islands were identified. Cumulatively, 777 strain-specific genes were found on these islands and represented 61% of the flexible gene pool.Conclusions/Significance: Substantial genomic differences were detected among A. actinomycetemcomitans strains. Genomic islands account for more than half of the flexible genes. The phenotype and virulence of A. actinomycetemcomitans may not be defined by any single strain. Moreover, the genomic variation within each clonal lineage of A. actinomycetemcomitans (as defined by serotype grouping) may be greater than between clonal lineages. The large genomic data set in this study will be useful to further examine the molecular basis of variable virulence among A. actinomycetemcomitans strains.

Aggregatibacter actinomycetemcomitans is divided into 6 serotypes. Occurrence of non-serotypeable strains is known, but background reasons are unclear. We hypothesized that non-serotypeable strains represent new serotypes or have altered expression of serotype-specific polysaccharide antigen (S-PA). We first characterized 311 strains from 189 individuals using both immunoassay-and PCR-based serotyping. Next, using natural human infection and rabbit immunization approaches, we clarified whether the phenotypically non-serotypeable strains expressed S-PA. Immunoassay identified serotypes a-f among 216 strains from 159 individuals. The remaining 95 strains from 30 individuals were phenotypically non-serotypeable. Yet, all these strains were identified by PCR-typing as serotype a-, b-, c-, or f. Non-serotypeability was confirmed by Western immunoblot with respective rabbit antisera. Patient sera remained non-reactive with autologous non-serotypeable strains at the serotype-specific region. Rabbit immunization with a phenotypically non-serotypeable strain induced no antibody production against S-PA. Thus, phenotypically non-serotypeable strains did not include novel serotypes, but lacked S-PA expression.

Objective To determine serotypes of 29 Aggregatibacter actinomycetemcomitans strains from blood isolates by polymerase chain reaction. Methods ATCC 29523 (serotype a), ATCC 43718 (serotype b), ATCC 33384 (serotype c), IDH 781 (serotype d), IDH 1705 (serotype e) and CU 1000 (serotype f) were used as reference strains. Six pairs of oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype specific polysaccharide antigens were designed. The specificity of the primers were evaluated by the reference strains of Aggregatibacter actinomycetemcomitans. Serotypes of 29 Aggregatibacter actinomycetemcomitans strains from blood iaolates were determined by PCR with the primers. Results Each pair of primers can specifically identify one serotype of Aggregatibacter actinomycetemcomitans. No cross reaction was observed in all strains. The PCR product sizes were: 428 bp (serotype a), 298 bp (serotype b), 559 bp (serotype c), 690 bp (serotype d), 211 bp (serotype e) and 232 bp (serotype f). Of the 29 strains from blood isolates, 16 strains were serotype b (55%), 4 strains were serotype a (14%), 4 strains were serotype c (14%), 2 strains were serotype d (7%), 2 strains were serotype f (7%). One strain could not be serotyped by PCR assay. Conclusion Serotype b was the most frequently detected serotype of the Aggregatibacter actinomycetemcomitans strains from blood isolates. Serotype a and c could be detected more often than the other serotypes.

Background Analysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF. Methodology/Principal Findings We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms.

Future MicrobiologyVol. 4, No. 5 EditorialFree AccessPeriodontal bacteria and cardiovascular problemsSirkka E AsikainenSirkka E AsikainenProfessor and Chairman, Section of Oral Microbiology, Umea University, SE-90187 Umea, Sweden. Published Online:4 Jun 2009https://doi.org/10.2217/fmb.09.21AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail The Finnish studies that linked dental infections to cardiovascular diseases [1,2] received widespread interest at the end of the 1980s and kindled great enthusiasm among dental researchers. In addition to the keen scientific interest, a new possibility was also seen to raise the status of dental infections among human infectious diseases and to increase public awareness of dental diseases as infections. The concept that atherosclerosis is an inflammatory disease [3,4] further enhanced research interest in dental infections and particularly in periodontitis as possible inflammatory triggers. After two decades, the controversial results from epidemiological studies [5] have gradually raised doubts of the validity of the suggested association between periodontitis and cardiovascular diseases. By contrast, the fact remains that in untreated or undertreated periodontitis the persistent bacterial growth on tooth surfaces and chronic inflammation in surrounding periodontal tissues provide a biologically feasible basis for inducing systemic effects that are considered to increase the risk of cardiovascular pathology.The systemic inflammatory effect of periodontitis has been studied by measuring levels of surrogate biomarkers for inflammation, such as proinflammatory cytokines and acute phase proteins, especially C-reactive protein [6]. These levels seem to be elevated in periodontitis patients in comparison to periodontally healthy subjects and the levels tend to decrease after treatment of periodontitis [7]. However, the inflammatory response is nonspecific by nature and can be induced by various stimuli. For example, coexisting but undiagnosed silent or acute infections and other pathological conditions and/or their treatment have their effects on patient's inflammatory status. Thus, the relative contribution of periodontitis, or of any other specified infection, to systemic inflammation remains difficult to determine.Antibody reaction may serve as a more specific approach than inflammatory biomarkers for determining systemic host response to periodontitis infection. Along this line of thinking, serum antibody levels were measured against selected periodontitis-associated species and a positive association was found between periodontitis and cardiovascular disease [8] for the first time on a biological basis. However, periodontitis is a multibacterial infection and even though the two bacterial species were selected according to culture-based knowledge and after validation in patients with and without periodontitis [9], it can today be discussed whether relevant bacterial species specific to periodontitis were/are used in corresponding immunoassays.Culture-independent nucleic acid-based techniques, particularly 16S rRNA gene sequencing approaches, have recently made it possible to detect and identify a variety of previously unknown bacteria from subgingival samples. At present, approximately 700 phylotypes are known [10], of the more than 1000 estimated phylotypes. Currently available applications that utilize the acquired sequence data include DNA microarrays that provide simultaneous and rapid detection of several hundred cultivable and not-yet-cultured bacterial species/phylotypes. Quantitative real-time PCR allows prompt discovery of the quantity and proportions of selected species in complex subgingival samples. In addition, the rapid development of sequencing technology makes faster and faster sequence data collection possible. Although handling and interpretation of the huge amount of data are key issues that first need to be resolved, thereafter, new models for periodontal microbiota may be constructed on, for example, the basis of bacterial community profiling.Besides the technological advances in molecular biology, another driving force revolutionizing our knowledge of periodontal infections is the biofilm science. Despite being pioneered in environmental microbiology several decades ago, it was only relatively recently launched to medicine [11,12]. It is obvious that the biofilm concept is changing the view of chronic infections that were previously strongly influenced by concepts on acute infections. Compared with other biofilm-associated infections the complexity of subgingival microbial biofilm represents a challenge. Taking on this challenge seems necessary, since the current knowledge of the health impact of subgingival microbiota is mainly based on studies comparing clinical periodontal status to bacterial culture or PCR detection of target bacteria from subgingival samples or on studies using pure cultures of selected bacterial species in a variety of experimental models. By contrast, little is understood of the community life style of subgingival microbiota. The good news today is that the biofilm science connects dental infections to a wider biological context provided by environmental and medical microbiology.For the time being, culture results still retain their historical position as the conceptual basis in periodontal microbiology. Extensive efforts have revealed that no single bacterium can be regarded as the particular periodontal pathogen for all patients. It appears that different species belonging to the cultured indigenous oral microbiota can overgrow in the complex subgingival bacterial community in different patients and even at different subgingival sites or time points in the same patient. Despite this, culture-dependent studies provide evidence for shared characteristics among the microbiota in periodontitis when compared with the microbiota in periodontally healthy individuals. They also pinpoint certain species as possible periodontal pathogens [13]. These species include mainly Gram-negative bacteria, such as Aggregatibacter (previously Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Campylobacter rectus. Consistent with the specific-pathogen concept, periodontal microbiology research during the last decades has mainly focused on culture and PCR detection of these individual species from clinical samples in a variety of study set-ups. In mechanistic studies, bacteria such as P. gingivalis and A. actinomycetemcomitans have been commonly used as model species to investigate, for example, how periodontal bacteria could contribute to the development of atherosclerosis and its complications.The new methods to detect and identify subgingival bacteria independent of culture methods have already revealed results that shake the traditional grounds in this field. For example, the predominant bacteria or bacterial groups found in periodontitis [14] are not the same periodontitis-associated bacteria identified in hundreds of culture-based studies during the past 30 years. Questions are raised as to whether culture-dependent methods bias our understanding of periodontal microbiology and whether they have directed our attention to irrelevant bacteria. If so, there may be consequences for the prevailing concept of the specific role of the aforementioned bacterial species in the etiology and progression of periodontitis, as well as for the validity of using these bacteria as model species of periodontal pathogens.Subgingivally colonizing bacteria live on tooth surfaces outside the epithelial lining of the body. How could these bacteria contribute to a systemic inflammatory state in periodontitis patients? The traditional explanation has been that bacteremic episodes occur during their everyday activities, such as mastication and tooth brushing. Interestingly, bacteria can only be cultured from blood samples for a brief time period after the oral stimulation is discontinued, indicating that bacteria are efficiently cleared from circulation. That the translocated bacteria are capable of colonizing distant body sites is supported by the occasional occurrence of severe nonoral infections, such as endocarditis, brain abscesses and other types of infections, caused by these bacteria [15].The main route for subgingival bacteria to enter the parenteral space is probably through microulcerations in nonkeratinized crevicular epithelium facing the tooth surface. Ulcerations already appear during the early phases of periodontal inflammation and they continuously break the integrity of crevicular epithelium in the course of periodontitis progression. After the bacteria have gained access to the abundant blood and lymph vessel networks in the inflamed periodontal tissues the route to larger neck vessels and via the superior vena cava to the heart is straightforward. With every heartbeat periodontal bacteria can then be spread to the pulmonary and systemic circulatory systems. Still, it is surprising that nonoral infections caused by periodontitis-associated bacteria are infrequent, even despite the high prevalence of periodontitis in adult populations.Viable oral or nonoral bacteria have hardly ever been found in atheroma plaques, whereas the recovery of bacterial DNA is common. Since this DNA originates from a variety of nonoral and oral bacteria [16], it seems unlikely that only certain bacterial species or groups would be associated with atherogenesis. Rather it may mean, if the concept is accepted, that many species have the potential to induce host responses that predispose to atheroma development. Thus, instead of restricting our attention to bacteremia or certain bacterial species or specified infections, an approach considering bacteria-associated molecular patterns (BAMPs) [17] as a basic unit opens new possibilities for mechanistic studies on atherogenesis in chronic infections. The BAMPs are independent of bacterial viability or species and their significance as inflammatory stimulators is well known. They are established ligands for Toll-like receptors [18] and ligand binding initiates signaling that leads to the upregulation of gene expression in the key cells of atherogenesis, for example adhesion molecules in endothelial cells and proinflammatory cytokines and chemokines in monocytes/macrophages and lymphocytes.Even though the pathogenic potential of BAMPs as individual bacterial cell components is widely discussed in the literature, little is known of the mechanisms by which they are delivered in biofilm-associated chronic infections. Aside from the fact that lysed bacteria liberate bacterial material, the release of BAMPs from live biofilms provides an interesting scenario for periodontitis-associated systemic effects. While releasing outer membrane vesicles that serve as delivery packages for membrane-bound BAMPs, live Gram-negative periodontal bacteria can also release free-soluble BAMPs independently of vesicles [19]. The released free-soluble BAMPs from both biofilm and planktonic life-forms of Gram-negative bacteria have been shown to induce an upregulation of gene and protein expression of a variety of proinflammatory cytokines in the human whole blood [20]. Based on this early experimental data it is tempting to speculate that the release of BAMPs from subgingival biofilms could provide an important mechanism by which chronic periodontal inflammation is perpetuated and systemic dissemination of bacterial material sustained. In free-soluble form, the BAMPs may readily gain access from the subgingival space into blood/lymph microvasculature in adjacent periodontal tissues, which opens the route for their systemic spread. In blood circulation and arterial intima they may induce proinflammatory and proatherogenic host responses that promote endothelial dysfunction and atherothrombogenesis.In periodontitis, the environment for bacterial growth is unique. Nowhere else in the body is the epithelial lining penetrated by calcified tissue that provides a solid, nonregenerating surface for bacterial colonization and biofilm formation. An evoked host defense cannot remove bacterial biofilms from the subgingival tooth surfaces. It can, however, gradually segregate teeth from alveolar bone and thus get rid of the tenacious biofilms. From the whole-body perspective, the final outcome, loss of affected teeth, can be regarded as a successful intrinsic anti-infection strategy. Most commonly, periodontal tissue destruction is, however, slow and the affected teeth may only finally loosen after decades. That for most of its progression time periodontitis is silent and painless and, therefore, easily unnoticed, does not diminish the significance of the concurrent immunoinflammatory host reactions.When considering that periodontitis in its early phase is easily diagnosed and inexpensively treated, it is surprising that periodontitis is still widespread in middle-aged and older populations [21,22]. This situation is unexpected particularly in the USA and Europe, where a massive number of periodontists and dental hygienists have been educated and information of periodontal disease has been actively spread to the general public during the past 30 years. A treatment strategy that involves replacement of periodontitis-affected teeth with dental implants has, however, exponentially increased in dentate patients during recent decades. The dilemma is that these patients have proven their susceptibility to periodontal tissue destruction. In addition, similar to the natural teeth, dental implants also penetrate the oral epithelium and provide solid surfaces for bacterial biofilm growth. As expected, the composition of bacterial biofilms [23] and the inflammatory response in implant-surrounding tissues [24] closely resembles those around natural teeth. An analysis of the literature showed that exposure of dental implants to the oral environment for at least five years led to peri-implantitis more frequently than in half of the treated subjects [25]. Furthermore, like periodontitis, peri-implantitis also provides subgingival biofilms and inflammatory response in surrounding tissues, which can induce systemic effects.The controversial results on the association of periodontitis and atherosclerosis-related cardiovascular diseases have gradually raised doubts about the validity of the suggested association. Still, in periodontitis the enduring pathology involving persistent bacterial infection and chronic inflammation forms a biological basis for systemic effects related to the development of atherosclerosis and its complications. But, obtaining proof for a causal relationship between periodontitis and cardiovascular diseases can be expected to be difficult. One of the main reasons is that both diseases are chronic and progress gradually and asymptomatically over decades. Expectations for clear-cut results may be a remnant of the concepts and knowledge concerning diseases caused by acute single-pathogen infections. Other approaches may be needed for chronic infections associated with complex bacterial communities.New concepts, such as the community lifestyle of subgingival microbiota, and new molecular biological tools, such as metagenomics, are available for a fresh start in periodontal research. Analysis of bacterial genes (gene-centric approach) instead of bacteria in subgingival bacterial communities and a better understanding of the pathogenic mechanisms of these communities will provide exciting local and systemic corollaries.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Bibliography1 Mattila KJ, Nieminen MS, Valtonen VV et al.: Association between dental health and acute myocardial infarction. BMJ298,779–781 (1989).Crossref, Medline, CAS, Google Scholar2 Syrjanen J, Peltola J, Valtonen V et al.: Dental infections in association with cerebral infarction in young and middle-aged men. J. Intern. Med.225,179–184 (1989).Crossref, Medline, CAS, Google Scholar3 Ross R: Atherosclerosis is an inflammatory disease. Am. Heart J.138,S419–S420 (1999).Crossref, Medline, CAS, Google Scholar4 Libby P: Inflammation in atherosclerosis. Nature420,868–874 (2002).Crossref, Medline, CAS, Google Scholar5 Beck JD, Offenbacher S: Systemic effects of periodontitis: epidemiology of periodontal disease and cardiovascular disease. J. Periodontol.76,2089–2100 (2005).Crossref, Google Scholar6 Willerson JT, Ridker PM: Inflammation as a cardiovascular risk factor. Circulation109,2–10 (2004).Crossref, Medline, Google Scholar7 Paraskevas S, Huizinga JD, Loos BG: A systematic review and meta-analyses on C-reactive protein in relation to periodontitis. J. Clin. Periodontol.35,277–290 (2008).Crossref, Medline, CAS, Google Scholar8 Pussinen PJ, Jousilahti P, Alfthan G et al.: Antibodies to periodontal pathogens are associated with coronary heart disease. Arterioscler. Thromb. Vasc. Biol.23,1250–1254 (2003).Crossref, Medline, CAS, Google Scholar9 Pussinen PJ, Vilkuna-Rautiainen T, Alfthan G, Mattila K, Asikainen S: Multiserotype enzyme-linked immunosorbent assay as a diagnostic aid for periodontitis in large-scale studies. J. Clin. Microbiol.40,512–518 (2002).Crossref, Medline, CAS, Google Scholar10 Paster BJ, Olsen I, Aas JA, Dewhirst FE: The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol. 200042,80–87 (2006).Crossref, Medline, Google Scholar11 Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science284,1318–1322 (1999).Crossref, Medline, CAS, Google Scholar12 del Pozo JL, Patel R: The challenge of treating biofilm-associated bacterial infections. Clin. Pharmacol. Ther.82,204–209 (2007).Crossref, Medline, CAS, Google Scholar13 Socransky SS, Haffajee AD: The bacterial etiology of destructive periodontal disease: current concepts. J. Periodontol.63,322–331 (1992).Crossref, CAS, Google Scholar14 Kumar PS, Griffen AL, Moeschberger ML, Leys EJ: Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J. Clin. Microbiol.43,3944–3955 (2005).Crossref, Medline, CAS, Google Scholar15 Gendron R, Grenier D, Maheu-Robert L: The oral cavity as a reservoir of bacterial pathogens for focal infections. Microbes Infect.2,897–906 (2000).Crossref, Medline, CAS, Google Scholar16 Ott SJ, El Mokhtari NE, Musfeldt M et al.: Detection of diverse bacterial signatures in atherosclerotic lesions of patients with coronary heart disease. Circulation113,929–937 (2006).Crossref, Medline, Google Scholar17 Erridge C: The roles of pathogen-associated molecular patterns in atherosclerosis. Trends Cardiovasc. Med.18,52–56 (2008).Crossref, Medline, CAS, Google Scholar18 Akira S, Takeda K, Kaisho T: Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol.2,675–680 (2001).Crossref, Medline, CAS, Google Scholar19 Karched M, Ihalin R, Eneslatt K et al.: Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form. BMC Microbiol.8,18 (2008).Crossref, Medline, Google Scholar20 Oscarsson J, Karched M, Thay B, Chen C, Asikainen S: Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells. BMC Microbiol.8,206 (2008).Crossref, Medline, Google Scholar21 Baelum V, van Palenstein Helderman W, Hugoson A, Yee R, Fejerskov O: A global perspective on changes in the burden of caries and periodontitis: implications for dentistry. J. Oral Rehabil.34,872–906 (2007).Crossref, Medline, CAS, Google Scholar22 Boehm TK, Scannapieco FA: The epidemiology, consequences and management of periodontal disease in older adults. J. Am. Dent. Assoc.138(Suppl. 1),S26–S33 (2007).Crossref, Medline, Google Scholar23 Mombelli A: Microbiology and antimicrobial therapy of peri-implantitis. Periodontol. 200028,177–189 (2002).Crossref, Medline, Google Scholar24 Lindhe J, Berglundh T: The interface between the mucosa and the implant. Periodontol. 200017,47–54 (1998).Crossref, Medline, CAS, Google Scholar25 Zitzmann NU, Berglundh T: Definition and prevalence of peri-implant diseases. J. Clin. Periodontol.35,286–291 (2008).Crossref, Medline, Google ScholarFiguresReferencesRelatedDetailsCited ByBAX Gene Overexpression in the Tongue Could Warn of Infection Risk due to Periodontal PathogensThe Open Dentistry Journal, Vol. 12, No. 1Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles18 September 2015 | PLOS ONE, Vol. 10, No. 9Periodontal Proteomics: Wonders Never Cease!International Journal of Proteomics, Vol. 2013Clinically Classified Periodontitis and Its Association in Patients with Preexisting Coronary Heart DiseaseJournal of Oral Diseases, Vol. 2013Atherosclerosis and infection: is the jury still not in?Dimitrios Chatzidimitriou, Dimitrios Kirmizis, Eleni Gavriilaki, Maria Chatzidimitriou & Nikolaos Malisiovas3 October 2012 | Future Microbiology, Vol. 7, No. 10Proteomics of Protein Secretion by Aggregatibacter actinomycetemcomitans25 July 2012 | PLoS ONE, Vol. 7, No. 7New insights into Prevotella diversity and medical microbiologyCorentine Alauzet, Hélène Marchandin & Alain Lozniewski6 December 2010 | Future Microbiology, Vol. 5, No. 11 Vol. 4, No. 5 STAY CONNECTED Metrics History Published online 4 June 2009 Published in print June 2009 Information© Future Medicine LtdFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p<0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p<0.05, ECC), Veillonella parvula (p<0.01), Veillonella atypica (p<0.05), and Veillonella sp. HOT-780 (p<0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p <0.05), and Porphyromonas gingivalis (p<0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p<0.05), and visible dental plaque (p<0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p<0.01) by oligonucleotide DNA probes, and P. gingivalis (p<0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p<0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p<0.05). P. gingivalis (p<0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.

Periodontitis and coronary artery disease (CAD) are inflammatory diseases and associated with each other. The major histocompatibility complex (MHC) region carries genes involved in immune response and inflammation. We investigated whether the MHC genes correlate with the presence of periodontitis or with the occurrence of periodontal pathogens in patients with CAD. Blood and saliva samples from CAD patients (n = 106) were collected at the time of hospitalization. Nine MHC genetic markers [human leukocyte antigen (HLA)-A, HLA-B, HLA-DRB1, lymphotoxin alpha (LTA) +253(a/g), +496(C/T), +633(c/g), +724(C/A), C4A and C4B)] were typed. Based on panoramic tomography, patients were categorized into nonperiodontitis and periodontitis groups. Two major periodontal pathogens, Aggregatibacter (Actinobacillus) actinomycetemcomitans and Porphyromonas gingivalis, were cultivated and polymerase chain reaction-amplified from salivary samples. Serum immunoglobulin (Ig)A and IgG antibody levels to these pathogens were measured. In the univariate analysis, LTA+496C allele (OR = 5.29; 95% CI = 2.07-13.51, P = 0.00027), and the occurrence of P. gingivalis in saliva (OR = 4.74; 95% CI = 1.64-13.70; P = 0.002) were more frequent in periodontitis when compared with nonperiodontitis. Similarly, serum IgA antibody level against the pathogen was increased in periodontitis (P = 0.048). In the multiple logistic regression analysis, when a wide range of covariates was included, the LTA+496C allele (OR = 10.87; 95% CI = 3.23-36.60; P = 0.00012) and the elevated serum IgA antibody level against P. gingivalis (OR = 1.56; 95% CI = 1.05-2.30; P = 0.026) remained as significant risk factors for periodontitis. In conclusion, the major finding of this study is that the LTA+496C allele is associated with periodontitis in patients with CAD.

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fat ty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specif ic oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 2561IT and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including f ive mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. infermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.