Prevotella denticola Lipopolysaccharide from a Cystic Fibrosis Isolate Possesses a Unique Chemical Structure

We report the first complete structural characterization of the lipopolysaccharide (LPS) from a cystic fibrosis (CF) clinical isolate of Prevotella denticola (B003V1S1X). Chemical, spectroscopic, and spectrometric analyses revealed a unique rough-type LPS (LOS) structure. The structure has a highly negatively charged heptasaccharide core region containing hexoses, with the first two sugars, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and mannose, highly phosphorylated. Furthermore, the lipid A moiety has the typical structure for the genus Prevotella, and was also highly phosphorylated.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.

Oral health, smoking and adolescence

The present cross-sectional study examined the effect of smoking on oral health in a birth cohort of 15 to 16-year-old Finnish adolescents. The hypothesis was that oral health parameters were poorer among smoking than non-smoking subjects and that a tobacco intervention program could be effective among the adolescents. The study was conducted in the Kotka Health Center, Kotka, Finland. Altogether 501 out of 545 subjects (15- to 16-year-old boys [n = 258] and girls [n = 243]) were clinically examined in 2004 and 2005. The sample frame was a birth cohort of all subjects in 1989 and 1990, living in Kotka. A structured questionnaire was also filled in by the participants to record their general health and health habits, such as smoking, tooth brushing, and medication used. The participants were classified into nonsmokers, current smokers, and former smokers. Subgingival pooled plaque samples were taken and stimulated salivary samples were also collected. The subjects were asked from which of seven professional groups (doctors, school nurses, dental nurses, general nurses, dentists, teachers and media professionals) they would prefer to receive information about tobacco. The two most popular groups they picked up were dentists and school nurses. Current smokers (n=127) were then randomly assigned into three groups: the dentist group (n =44), the school-nurse group (n =42), and the control group (n =39). The intervention was based on a national recommendation of evidence based guidelines by The Finnish Medical Society Duodecim ( 5A counseling system). Two months after the intervention, a second questionnaire was sent to the smokers in the intervention groups. Smoking cessation, smoking quantity per week, and self-rated addiction for smoking (SRA) were recorded. The results were analyzed using the R-statistical program. The results showed that 15% of the subjects had periodontitis. Smokers (25%) had more periodontitis than non-smokers (66%) (p < 0.001). Smoking boys (24%) also had more caries lesions than non-smokers (69%) (p < 0.001), and they brushed their teeth less frequently than non-smokers. Smoking significantly impaired periodontal health of the subjects, even when the confounding effects of plaque and tooth brushing were adjusted. Smoking pack-years, intensified the effects of smoking. Periodontal bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia and Treponema denticola were more frequently detected among the smokers than non-smokers, especially among smoking girls. Smoking significantly decreased the values of both the salivary periodontal biomarkers MMP-8 (p=0.04) and PMN elastase (p=0.02) in boys. The effect was strengthened by pack years of smoking (MMP-8 p=0.04; elastase p0.01). Of those who participated in the intervention, 19 % quit smoking. The key factors associated with smoking cessation were best friend`s influence, nicotine dependence and diurnal type. When the best friend was not a smoker, the risk ratio (RR) of quit smoking after the intervention was 7.0 (Cl 95% 4.6 10.7). Of the diurnal types, the morning people seemed to be more likely to quit (RR 2.2 [Cl 95% 1.4 3.6]). Nicotine dependence also elicited an opposite effect: those who scored between 3 and 5 dependence scores were less likely to quit. In conclusion, smoking appears to be a major etiological risk factor for oral health. However, the early signs of periodontal disease were mild in the subjects studied. Based on the opinions of the adolescent s, dental professionals may have a key position in their smoking cessation. The harmful effects of smoking on oral health could be used in counselling. Best friend`s influence, nicotine dependence and diurnal type, all factors associated with smoking cessation, should be taken more carefully into account in the prevention programs for adolescents.

Efeito do tratamento periodontal não cirúrgico em pacientes com periodontite crônica e agressiva: achados microbiológicos e imunológicos

O objetivo do presente estudo foi comparar a expressão de IL-1β, IL-4, IL-8, interferon-γ, atividade de elastase e a composição do perfil microbiano subgengival antes e depois do tratamento periodontal não cirúrgico em pacientes com doença peridontal crônica generalizada (PC) e agressiva generalizada (PA). Vinte pacientes com PC e quatorze com PA foram avaliados. Dados clínicos, fluido gengival e biofilme subgengival foram analisados na visita inicial (VI) e 3 meses (3M) após o tratamento periodontal não cirúrgico. Amostras de fluido gengival (FG) foram coletadas com tiras de papel e os níveis de: IL-1β, IL-4, IL-8 e INF-γ foram medidos, utilizando um tipo de imunoensaio multiplexado (Luminex). Atividade da elastase foi avaliada por um ensaio enzimático. Amostras de placa subgengival foram analisadas através do checkerboard DNA-DNA hybridization. Na avaliação de 3 meses após terapia periodontal foi encontrado melhora significativa para todos os parâmetros clínicos em ambos os grupos. Foram encontradas reduções significativas na atividade de elastase nos sítios rasos e profundos dos pacientes do grupo PA e nos sítios profundos do grupo PC, também foi achado um aumento significativo de INF-γ nos sítios rasos do grupo PA. Os dados microbiológicos, mostraram reduções significativas para os níveis dos membros do complexo vermelho (P. gingivalis, T. forsythia, T.denticola), e para as espécies E.nodatum e P.micra no grupo PC. No grupo PA, ocorreram reduções significativas no níveis de P. gingivalis, T. forsythia, Fusobacterium nucleatum ss polymorphum e Fusobacterium periodonticum. Quando as respostas clínica e imunológica 3M após terapia foram comparadas entre os grupos, apenas diferenças sutis foram observadas. Nenhuma diferença microbiológica foi encontrada entre os grupos após a terapia. Em conclusão, os achados suportam nossa hipótese de que as periodontites cronica e agressiva respondem de forma semelhante ao tratamento periodontal nao cirúrgico.

The association between subgingival periodontal pathogens and systemic inflammation

Aim To investigate associations between periodontal disease pathogens and levels of systemic inflammation measured by C-reactive protein (CRP). Methods A representative sample of dentate 60-70-year-old men in Northern Ireland had a comprehensive periodontal examination. Men taking statins were excluded. Subgingival plaque samples were analysed by quantitative real time PCR to identify the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. High-sensitivity CRP (mg/l) was measured from fasting blood samples. Multiple linear regression analysis was performed using log-transformed CRP concentration as the dependent variable, with the presence of each periodontal pathogen as predictor variables, with adjustment for various potential confounders. Results A total of 518 men (mean age 63.6 SD 3.0 years) were included in the analysis. Multiple regression analysis showed that body mass index (p < 0.001), current smoking (p < 0.01), the detectable presence of P. gingivalis (p < 0.01) and hypertension (p = 0.01), were independently associated with an increased CRP. The detectable presence of P. gingivalis was associated with a 20% (95% confidence interval 4-35%) increase in CRP (mg/l) after adjustment for all other predictor variables. Conclusion In these 60-70-year-old dentate men, the presence of P. gingivalis in subgingival plaque was significantly associated with a raised level of C-reactive protein.

Microbiological Profile of Aggressive Periodontitis in a Sudanese Population

Background: Several bacterial species have been identified as being associated with aggressive periodontitis (AgP) notably Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). There are limited data on bacterial associations with AgP in African populations. Objective: To investigate possible associations between specific bacteria and AgP in a Sudanese population. Methods: Subgingival plaque samples were collected from 93 (20 male, 73 female) Sudanese patients diagnosed with AgP and from 72 (23 male, 48 female) periodontally healthy Sudanese controls. Quantitative PCR was used to identify Aa, Pg, Treponema denticola (Td) and Fusobacterium nucleatum (Fn). The prevalence of these bacterial species was compared using Chi-square analysis. Odds ratios (OR) were calculated using standard methods. Results: The cases with AgP were well matched in age with the controls: 24.8 (SD 5.1) compared with 23.5 (SD 3.7) years, p=0.07. There was a significantly higher prevalence of Pg in AgP (73%) than in the controls (33%), p<0.0001. The OR for Pg to be associated with AgP was 5.44 (95% confidence intervals 2.78-10.64). In 26 (38%) of the AgP cases positive for Pg there were low levels of this bacterium (<100 copies). Both Td and Fn were identified in virtually all (>95%) the plaque samples studied from both AgP and controls. Aa was the least frequently identified species and was present in only 28% of AgP and 18% of controls, p=0.14. The OR for Aa to be associated with AgP was slightly increased at 1.76 (95% CI 0.83-3.74), however, this was not significant (p=0.14). Conclusion: In the Sudanese subjects studied Pg but not Aa was associated with AgP. There were very low levels of Pg in many of the plaque samples from AgP.

Caractérisation du produit du gène sty4221, unique à Salmonella enterica sérovar Typhi

Salmonella enterica sérovar Typhi (Typhi) est une bactérie pathogène spécifique à l’homme. Typhi est l’agent étiologique de la fièvre typhoïde chez l’humain, causant plus de 16 millions de nouveaux cas par année et plus de 600 000 morts. Il a été démontré que pour causer une infection systémique, Salmonella doit nécessairement survivre dans les macrophages de l'hôte. Paradoxalement, S. enterica sérovar Typhimurium, très apparenté à Typhi (près de 90 % d’homologie), n’a pas la capacité de se disséminer dans l’organisme humain et peut infecter plusieurs espèces animales. Nous avons antérieurement identifié 36 gènes uniques à Typhi (absents chez Typhimurium) situés sur 15 régions différentes et exprimés sélectivement lors de l’infection de macrophages humains. Ainsi, l’une de ces régions a suscité notre attention, soit la région sty4217-4222 et plus particulièrement le produit du gène sty4221, une aminotransférase hypothétique. Ce dernier gène est d’intérêt dû à l’homologie qu’il détient avec une hémolysine connue (Hly) produite par Treponema denticola, possédant elle-même une activité d’aminotransférase. Chez T. denticola, Hly dégrade la cystéine et produit du H2S qui est toxique pour l’hôte. Notre hypothèse est que la spécificité d’hôte et la capacité de produire une infection systémique de Typhi sont dues à l’expression de gènes qui ne se retrouvent pas chez d’autres salmonelles. Le but de cette étude était donc de caractériser le gène sty4221 quant à son activité hémolytique, cytotoxique et tenter de déterminer son rôle dans la virulence de cette bactérie. Le gène sty4221 a été cloné sous le contrôle d’un promoteur inductible à l’arabinose et exprimé par E. coli. L’activité hémolytique du clone a été déterminée par simple observation sur gélose sang. Ce clone a également permis d’observer l’effet cytotoxique du surnageant de culture sur différentes lignées cellulaires, par quantification de la relâche de LDH. Le gène sty4221 a été muté chez la souche sauvage de Typhi, ISP1820, l’implication pathogénique du gène a ainsi pu être étudiée. Des tests de phagocytose, d’invasion et de survie dans des macrophages humains ont été effectués, ainsi que des tests d’adhésion et d’invasion sur des cellules HeLa. Par ailleurs, une première tentative de purification de la protéine a été entreprise. En somme, nous savons maintenant que STY4221 a des propriétés hémolytiques, augmentées par la présence de cystéine. De plus, STY4221 a un effet cytotoxique sur les macrophages THP-I, mais aucun effet sur les HeLa. Or, sty4221 ne semble pas impliqué dans les étapes d’adhésion, d’invasion, de phagocytose ou de survie. La caractérisation de sty4221 permettra sans doute d’approfondir nos connaissances sur les toxines trouvées uniquement chez Typhi.

The association between caries and periodontal diseases

Objectifs: Le but de cette étude clinique était de comparer un groupe d’adultes ayant un parodonte sain avec un groupe d’adultes atteints de parodontite chronique en terme de risque carieux et mesures cliniques et microbiologiques de la carie. Méthodes: Quatre-vingt-seize individus ont été divisés en deux groupes en fonction de leur état de santé parodontal et ont été appariés pour l'âge, le sexe et l'origine ethnique. Trente-huit sujets étaient atteints de parodontite chronique définie comme ayant au moins quatre dents avec ≥ 1 site avec une profondeur de sondage ≥ 4 mm et une perte d'attache clinique ≥ 2 mm, et 58 sujets présentaient un parodonte sain. Par la suite, les groupes ont été subdivisés en deux groupes en fonction de leur statut carieux : les participants ayant au moins une lésion carieuse non traitée sur une surface dentaire et ceux n’ayant pas de lésion carieuse non traitée. Les données ont été recueillies par le biais d’un questionnaire, un examen clinique et des échantillons de plaque supra- et sous-gingivale. L’évaluation de la charge buccale de Streptococcus mutans et de six agents pathogènes parodontaux a été réalisée par la technique d'amplification de la réaction en chaine de la polymérase (PCR). Les données ont été analysées à l'aide d’analyses statistiques descriptives et bivariées. Résultats: Les individus atteints de parodontite chronique étaient 3,5 fois plus susceptibles d'avoir des caries que les individus en bonne santé (OR 3,5 ; IC: 1,5 - 8,3 ; P = 0,006). Les sujets à la fois atteints de parodontite chronique et de caries dentaires ont eu un niveau d’éducation significativement plus faible que les sujets ayant un parodonte sain et sans caries dentaires (OR 6,0 ; IC: 1,7 à 21,7 ; P = 0,04). La proportion de sujets ayant une charge buccale élevée de Porphyromonas gingivalis (P. g.) et Treponema denticola (T. d.) était significativement plus élevée chez les patients atteints de parodontite chronique et de carie que chez les patients sains présentant des caries (P. g.: OR 8,6 ; IC: 2,4 - 30,3 ; P = 0,004 et T. d.: OR 10,0 ; CI: 2,6 - 38.1 ; P = 0,003). Conclusions: Les résultats de cette étude suggèrent que, chez les sujets adultes atteints de la parodontite chronique, la fréquence des caries est plus élevée que chez les sujets ayant un parodonte sain. De plus, le faible niveau d'éducation influence négativement le statut parodontal des individus.

Spirochaetes and other bacterial species associated with bovine digital dermatitis

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis. (C) 1998 Elsevier Science B.V.

Genetic relatedness and phenotypic characteristics of Treponema associated with human periodontal tissues and ruminant foot disease

Treponema have been implicated recently in the pathogenesis of digital dermatitis (DID) and contagious ovine digital dermatitis (CODD) that are infectious diseases of bovine and ovine foot tissues, respectively. Previous analyses of treponemal 16S rDNA sequences, PCR-amplified directly from DID or CODD lesions, have suggested relatedness of animal Treponema to some human oral Treponema species isolated from periodontal tissues. In this study a range of adhesion and virulence-related properties of three animal Treponema isolates have been compared with representative human oral strains of Treponema denticola and Treponema vincentii. In adhesion assays using biotinylated treponemal cells, T denticola cells bound in consistently higher numbers to fibronectin, laminin, collagen type 1, gelatin, keratin and lactoferrin than did T. vincentii or animal Treponema isolates. However, animal DID strains adhered to fibrinogen at equivalent or greater levels than T denticola. All Treponema strains bound to the amino-terminal heparin l/fibrin I domain of fibronectin. 16S rDNA sequence analyses placed ovine strain UB1090 and bovine strain UB1467 within a cluster that was phylogenetically related to T vincentii, while ovine strain UB1466 appeared more closely related to T denticola. These observations correlated with phenotypic properties. Thus, T denticola ATCC 35405, GM-1, and Treponema UB1466 had similar outer-membrane protein profiles, produced chymotrypsin-like protease (CTLP), trypsin-like protease and high levels of proline iminopeptidase, and co-aggregated with human oral bacteria Porphyromonas gingivalis and Streptococcus crista. Conversely, T vincentii ATCC 35580, D2A-2, and animal strains UB1090 and UB1467 did not express CTLP or trypsin-like protease and did not co-aggregate with P. gingivalis or S. crista. Taken collectively, these results suggest that human oral-related Treponema have broad host specificity and that similar control or preventive strategies might be developed for human and animal Treponema-associated infections.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

An interleukin-1 beta (IL-1 beta) single-nucleotide polymorphism at position 3954 and red complex periodontopathogens independently and additively modulate the levels of IL-1 beta in diseased periodontal tissues

Inflammatory cytokines such as interieukin-1 beta (IL-1 beta) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-10 mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-10 promoter single-nucleotide polymorphism at position 3954 [IL-1 beta(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1 beta levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects in = 175) and the possible correlations with the clinical parameters of the disease. IL-1 beta(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1 beta levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1 beta(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1 beta (3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1 beta levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1 beta(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1 beta in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1 beta(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1 beta in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.