Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Fertility factors affect the vaginal microbiome in women of reproductive age.

PROBLEM: For women of reproductive age, achieving a successful pregnancy requires both the normal functioning of reproductive endocrine and the health of the reproductive tract environment. We aimed to study how these fertility factors, such as female age, baseline sexual hormone levels, tubal patency, and vaginal pH, affect the composition of vaginal microbiome. METHOD OF STUDY: The 16S rRNA sequencing was carried on vaginal microbiome samples from 85 women of reproductive age without vaginal infections or reproductive endocrine diseases. The detailed correlations between fertility factors and vaginal microbiome were quantified by Spearman's rank tests. A linear discriminant analysis was carried out to explore the effects of fertility factors on the relative abundances of vaginal bacterial species. RESULTS: The vaginal pH, levels of basal E2, LH, and FSH all had significant effects on the distribution of vaginal microbiome. The relative abundances of vaginal bacterial species, including Escherichia coli, Streptococcus agalactiae, and Prevotella intermedia, were significantly different due to the host's state of reproductive endocrine and tubal patency. It was worth noting that women with tubal obstruction, or prolonged menstrual cycle, or antral follicle count >15, or vaginal pH > 4.5 all had a higher abundance of Escherichia coli in vagina. CONCLUSION: The fertility factors associated with the reproductive endocrine and the genital tract environment affected vaginal microbiome in women of reproductive age. The species Escherichia coli, Streptococcus agalactiae, Prevotella intermedia, etc could be used as biomarkers to reflect the pathological state of reproductive endocrine and genital tract.

A novel pH-responsive quaternary ammonium chitosan-liposome nanoparticles for periodontal treatment.

The aim of this study was to evaluate the antibacterial activity and cytocompatibility of novel pH-activated nanoparticles (NPs) in vitro and in vivo. The NPs were synthesized from a quaternary ammonium chitosan, i.e., N,N,N-trimethyl chitosan, a liposome, and doxycycline (TMC-Lip-DOX NPs). The cytocompatibility of the NPs was evaluated. The TMC-Lip-DOX NPs achieved superb inhibition of free mixed bacteria and biofilm formation. They also showed excellent biocompatibility with human periodontal ligament fibroblasts. Animal experiments showed that the NPs strongly inhibited biofilm formation and prevented alveolar bone absorption in vivo. All the results indicate that the TMC-Lip-DOX NPs have good potential for use in the treatment of periodontal and other inflammatory diseases.

Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species.

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

Chemical composition, cytotoxic effect and antimicrobial activity of Stachys koelzii Rech.f. essential oil against periodontal pathogen Prevotella intermedia.

Prevotella intermedia is associated with periodontal diseases and endodontic infections. Periodontitis can be suppressed by utilizing the antiseptics, which target the infectious bacteria. The member of Stachys sp. has been used traditionally in the form of decoction or infusion for management of infectious diseases. The subject of this article was to evaluate the chemical composition, antimicrobial and cytotoxic effect of Stachys koelzii essential oil and its main components against Prevotella intermedia. GC-FID and GC-MS analysis were used to determine the chemical composition. The antimicrobial effects of S. koelzii essential oil was evaluated by micro-broth dilution assay. Time kill curve assays, leakage of cytoplasmic materials and anti-biofilm effects were determined. Its cytotoxic effect was evaluated by MTT assay. Essential oil with main components of α-pinene, trans-caryophyllene and 1,8-cineole inhibited P. intermedia with MIC and MBC values of 0.1 and 0.2 mg/mL. Its biofilm formation was higher than α-pinene, followed by trans-caryophyllene and 1,8-cineole. Essential oil and its main components increased the leakage of cytoplasmic components. Essential oil showed cytotoxic effect on HeLa cell lines with IC50 0.06 mg/mL. The cytotoxic effect of α-pinene on healthy cell lines was higher than essential oil. S. koelzii essential oil can be used in mouthwash formulations and its efficacy should be evaluated in large clinical studies.

Antimicrobial Efficacy of Photodynamic Therapy and Light-Activated Disinfection Against Bacterial Species on Titanium Dental Implants.

PURPOSE: The aim of this study was to evaluate the efficacy of photodynamic therapy (PDT) and light-activated disinfection (LAD) against a 3-day-old bacterial suspension prepared from three different bacterial species present on titanium dental implants, and to analyze the possible alterations of the implant surfaces as a result of the PDT and LAD. MATERIALS AND METHODS: The study was conducted on 72 titanium dental implants contaminated with a bacterial suspension prepared from three bacterial species: Prevotella intermedia, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. The contaminated implants were incubated under anaerobic conditions for 72 hours and then were randomly divided into four experimental groups and two control groups (n = 12 each), according to the following treatment protocols: group 1 (PDT1): PDT (660 nm, 100 mW, 60 seconds) with toluidine blue; group 2 (PDT2): PDT (660 nm, 100 mW, 60 seconds) with phenothiazine chloride dye; group 3 (LAD): light-emitting diode (LED) with toluidine blue; group 4 (toluidine blue): treatment with only toluidine blue for 60 seconds. In the positive control group, the implants were treated with a 0.2% chlorhexidine-based solution for 60 seconds, and in the negative control group, no treatment was used. RESULTS: The highest bacterial reduction was recorded in the PDT1 (98.3%) and PDT2 (97.8%) groups. The results of this study showed that there was a statistically significant reduction of bacteria in the PDT1 and PDT2 groups compared with the negative control group (P < .05), individually for each bacterial species as well as for all three species together. LAD was less effective than PDT1 and PDT2, and did not show a statistically significant difference compared with the negative control or any other treatment group. Toluidine blue was the least effective treatment in terms of both the total bacterial count and the individual count for each bacterial species. CONCLUSION: Both PDT1 and PDT2 protocols showed a high efficacy against a 3-day-old bacterial biofilm on dental implants and were more effective compared with LAD.

Components in Lentinus edodes mushroom with anti-biofilm activity directed against bacteria involved in caries and gingivitis.

The present study investigated the compounds present in the low molecular mass fraction of Lentinus edodes mushroom (shiitake) extract and their anti-virulence activity against oral pathogens (reference and clinical Streptococcus mutans, Actinomyces naeslundii, and Prevotella intermedia strains). Oxalic, succinic, and quinic acids, and adenine, inosine, and uridine were identified by HPLC-DAD-ESI-MS/MS. Their anti-biofilm production and preformed biofilm disaggregation activities were studied using commercial standard compounds at different concentrations. As regards S. mutans, the highest activity was shown by adenine at 5 mg mL-1 both in the biofilm inhibition (BI 50%) and biofilm disaggregation tests (BD 20%). Considering A. naeslundii, BI values close to 80% were registered for oxalic acid at 1 mg mL-1 and 2 mg mL-1 and BD 50% for quinic acid at 3 mg mL-1. A weaker activity was found against P. intermedia. Furthermore, different mixtures of the commercial standards were tested showing that the activity of a compound can be strongly and sometimes negatively affected by the presence of the other compounds.

Polymorphism IL-1RN rs419598 reduces the susceptibility to generalized periodontitis in a population of European descent.

Interleukin (IL) 1-ra is a potent endogenous competitive inhibitor of IL-α and ß and has an anti-inflammatory role. Study objectives were: 1) to assess the associations of IL-1RN genetic single nucleotide polymorphism (SNP) (rs419598) with generalized chronic periodontitis (GCP), generalized aggressive periodontitis (GAgP), and absence of periodontitis and 2) to assess its association with the load of five periodontopathogenic bacteria and periodontal clinical variables. A cross-sectional analytic study was conducted in 123 patients with GCP, 60 patients with GAgP, and 20 controls. Reverse hybridization PCR was used for genotyping analysis to detect SNPs in IL-1A (rs1800587), IL-1B (rs1143634), and IL-1RN (rs419598) genes and for determination of the load of five periodontopathogenic bacteria. The severity and extension of periodontitis were assessed. Multinomial logistic regression and mediated regression analyses were performed. Considering results for GCP and GAgP patients together, the presence of polymorphism in IL-1A and/or IL-1B gene was associated with a higher likelihood of periodontitis, (OR = 8.11; 95%CI [1.85-35.48]), but this likelihood was reduced when IL-1RN polymorphism was also present, (OR = 5.91; 95%CI [1.08-32.27]). IL-1RN polymorphism was significantly associated with lower counts of red complex bacteria, specifically Porphyromona gingivalis, Tannerella forsythia, and Prevotella intermedia, which were associated with improved clinical outcomes. The polymorphic expression of IL-1RN (rs419598) gene may be associated with a reduced susceptibility to GAgP and GCP in populations of European descent. This effect may be mediated by a decreased load of Porphyromona gingivalis, Tannerella forsythia, and Prevotella intermedia.

Pathogenesis and Treatment of Bovine Foot Rot.

Bovine foot rot (BFR) is an infectious disease of the interdigital skin and subcutaneous tissues of beef and dairy cattle that occurs under a variety of management and environmental settings. The anaerobic, gram-negative bacteria Fusobacterium necrophorum, Porphyromonas levii, and Prevotella intermedia are commonly isolated from lesions. A multitude of host, agent, and environmental factors contribute to the development of BFR. Initiation of systemic antimicrobial therapy early in the course of disease commonly leads to resolution. Delays in treatment may result in extension of infection into deeper bone, synovial structures, or ligamentous structures, and the prognosis for recovery is reduced.

Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms.

As a member of subgingival multispecies biofilms, Tannerella forsythia is commonly associated with periodontitis. The bacterium has a characteristic cell surface (S-) layer modified with a unique O-glycan. Both the S-layer and the O-glycan were analyzed in this study for their role in biofilm formation by employing an in vitro multispecies biofilm model mimicking the situation in the oral cavity. Different T. forsythia strains and mutants with characterized defects in cell surface composition were incorporated into the model, together with nine species of select oral bacteria. The influence of the T. forsythia S-layer and attached glycan on the bacterial composition of the biofilms was analyzed quantitatively using colony-forming unit counts and quantitative real-time polymerase chain reaction, as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. This revealed that changes in the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers. The localization of T. forsythia within the bacterial agglomeration varied depending on changes in the S-layer glycan, and this also affected its aggregation with Porphyromonas gingivalis. This suggests a selective role for the glycosylated T. forsythia S-layer in the positioning of this species within the biofilm, its co-localization with P. gingivalis, and the prevalence of C. rectus. These findings might translate into a potential role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and the immune system, from within or beyond the biofilm.

Genome sequence of Prevotella intermedia SUNY aB G8-9K-3, a biofilm forming strain with drug-resistance

Abstract Prevotella intermedia has long been known to be as the principal etiologic agent of periodontal diseases and associated with various systemic diseases. Previous studies showed that the intra-species difference exists in capacity of biofilm formation, antibiotic resistance, and serological reaction among P. intermedia strains. Here we report the genome sequence of P. intermedia SUNY aB G8-9K-3 (designated ATCC49046) that displays a relatively high antimicrobial resistant and biofilm-forming capacity. Genome sequencing information provides important clues in understanding the genetic bases of phenotypic differences among P. intermedia strains.

Genome sequence of Prevotella intermedia SUNY aB G8-9K-3, a biofilm forming strain with drug-resistance.

Prevotella intermedia has long been known to be as the principal etiologic agent of periodontal diseases and associated with various systemic diseases. Previous studies showed that the intra-species difference exists in capacity of biofilm formation, antibiotic resistance, and serological reaction among P. intermedia strains. Here we report the genome sequence of P. intermedia SUNY aB G8-9K-3 (designated ATCC49046) that displays a relatively high antimicrobial resistant and biofilm-forming capacity. Genome sequencing information provides important clues in understanding the genetic bases of phenotypic differences among P. intermedia strains.

Effect of Helicobacter pylori infection on chronic periodontitis by the change of microecology and inflammation.

Helicobacter pylori (H. pylori), a pathogen inducing peptic disease, is recently found to be binding to the progress of periodontitis. Most previous studies are case-controlled, and they investigate the risk of H. pylori infection in disease the development of while few studies evaluate the correlation between H. pylori and periodontal pathogens. Therefore, we investigated the correlation between H. pylori infection with periodontal parameters, periodontal pathogens and inflammation. The results indicated that patients with H. pylori showed significantly higher probing depth and attachment loss than those without (p < 0.05). Among 28 subgingival plaque samples from 14 patients, the frequencies of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Treponema denticola were significantly higher with H. pylori infection than those without H. pylori infection (p < 0.05). However, the frequency of Aggregatibacter actinomycetemcomitans was lower (p < 0.05). Furthermore, after human acute monocytic leukemia cell line (THP-1) was stimulated with cagA-positive standard strains (cagA+ H. pylori 26695), the expression of periodontitis-related molecules Wnt5a, interleukin 8 (IL-8), interleukin 6 (IL-6) and interferon gamma (IFN-γ) significantly increased (p < 0.05). Conversely, the expression of tumor necrosis factor alpha (TNF-α) was almost stable. Meanwhile, cagA+ H. pylori promoted significantly higher expression of IL-8 and Wnt5a than isogenic cagA mutants strains (cagA- H. pylori 26695) did. Taken together, our data suggested that H. pylori might promote the growth of some periodontal pathogens and aggravate the progress of chronic periodontitis.

Evaluating the effect of local pH on fluorescence emissions from oral bacteria of the genus Prevotella.

A number of anaerobic oral bacteria, notably Prevotellaceae, exhibit red fluorescence when excited by short-wavelength visible light due to their accumulation of porphyrins, particularly protoporphyrin IX. pH affects the fluorescence of abiotic preparations of porphyrins due to transformations in speciation between monomers, higher aggregates, and dimers. To elucidate whether the porphyrin speciation phenomenon could be manifested within a microbiological system, suspensions of Prevotella intermedia and Prevotella nigrescens were examined by fluorescence spectrophotometry while being titrated against NaOH. The initial pH of the samples was <6, which was then raised toward the maximum found within a diseased periodontal pocket, being ∼pH 8.7. The intensity of the fluorescence emissions increased between 600 and 650 nm with increasing pH. Peak fluorescence emissions occurred at 635±1 nm with a second emission peak developing with increasing pH at 622 nm. A linear relationship was demonstrated between pH and the log10 ratio of 635:622 nm excitation fluorescence intensities. These findings suggest that the pH range found within the oral cavity could affect the fluorescence of oral bacteria in vivo, which may in turn have connotations for any clinical diagnoses that may be inferred from dental plaque fluorescence.

Osteoblast integration of dental implant materials after challenge by sub-gingival pathogens: a co-culture study in vitro.

Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium (Ti), titanium-zirconium (TiZr) alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.

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

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

Does estradiol have an impact on the dipeptidyl peptidase IV enzyme activity of the Prevotella intermedia group bacteria?

Initiation and development of pregnancy-associated gingivitis is seemingly related to the microbial shift towards specific gram-negative anaerobes in subgingival biofilms. It is known that Prevotella intermedia sensu lato is able to use estradiol as an alternative source of growth instead of vitamin K. The aim of the present study was to investigate the impact of estradiol on the bacterial dipeptidyl peptidase IV (DPPIV) enzyme activity in vitro as a virulent factor of the Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca. In all experiments, 2 strains of each Prevotella species were used. Bacteria were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol and were allowed to build biofilms at an air-solid interface. DPPIV activities of biofilms were measured kinetically during 20 min using a fluorometric assay. The enzyme activity was later related to the amount of protein produced by the same biofilm, reflecting the biofilm mass. Estradiol significantly increased DPPIV activities of the 8 Prevotella strains in a strain- and dose-dependent manner. In conclusion, our in vitro experiments indicate that estradiol regulates the DPPIV enzyme activity of P. intermedia, P. nigrescens, P. pallens, and P. aurantiaca strains differently. Our results may, at least partly, explain the role of estradiol to elicit a virulent state which contributes to the pathogenesis of pregnancy-related gingivitis.

Quantitation of biofilm and planktonic life forms of coexisting periodontal species.

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.

Relationship between methanogenic archaea and subgingival microbial complexes in human periodontitis.

We compared the amounts of methanogenic archaea with ten of the most important periodontal pathogens in 125 clinical samples. Correlation analysis suggests that the support of the periodontitis-associated bacterial consortium by methanogenic archaea may be driven through direct or indirect interactions with Prevotella intermedia.

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.