Exploring the role of oral microorganisms in the pathogenesis of mucositis by assessing their impact on metabolic activity and reproductive capacity of epithelial cells in vitro.

PURPOSE: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis. METHODS: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells. RESULTS: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used. CONCLUSION: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.

Effect of metalloporphyrins on red autofluorescence from oral bacteria.

The aim of this study was to assess the red autofluorescence from bacterial species related to dental caries and periodontitis in the presence of different nutrients in the growth medium. Bacteria were grown anaerobically on tryptic soy agar (TSA) supplemented with nutrients, including magnesium-porphyrins from spinach and iron-porphyrins from heme. The autofluorescence was then assessed at 405 nm excitation. On the TSA without additives, no autofluorescence was observed from any of the species tested. On the TSA containing sheep blood, red autofluorescence was observed only from Parvimonas micra. When the TSA was supplemented with blood, hemin, and vitamin K, red autofluorescence was observed from Actinomyces naeslundii, Bifidobacterium dentium, and Streptococcus mutans. Finally, on the TSA supplemented with spinach extract, red autofluorescence was observed from Aggregatibacter actinomycetemcomitans, A. naeslundii, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus salivarius, S. mutans, and Veillonella parvula. We conclude that the bacteria related to dental caries and periodontal disease exhibit red autofluorescence. The autofluorescence characteristics of the tested strains depended on the nutrients present, such as metalloporphyrins, suggesting that the metabolic products of the oral biofilm could be responsible for red autofluorescence.

The influence of oral bacteria on epithelial cell migration in vitro.

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.

The consistent application of hydrogen peroxide controls biofilm growth and removes Vermamoeba vermiformis from multi-kingdom in-vitro dental unit water biofilms.

The water systems inside a dental unit are known to be contaminated with a multi-kingdom biofilm encompassing bacteria, fungi, viruses and protozoa. Aerosolization of these micro-organisms can potentially create a health hazard for both dental staff and the patient. Very little is known on the efficacy of dental unit disinfection products against amoeba. In this study we have examined the effect of four different treatment regimens, with the hydrogen peroxide (H2O2) containing product Oxygenal, on an in-vitro multi-kingdom dental unit water system (DUWS) biofilm. The treatment efficacy was assessed in time using heterotrophic plate counts, the bacterial 16S rDNA, fungal 18S rDNA gene load and the number of genomic units for Legionella spp. the amoeba Vermamoeba vermiformis. The results indicated that a daily treatment of the DUWS with a low dose H2O2 (0.02% for 5 h), combined with a weekly shock dose (0.25% H2O2, 30 min) is necessary to reduce the heterotrophic plate count of a severely contaminated DUWS (>106 CFU.mL-1) to below 100 CFU.mL-1. A daily treatment with a low dose hydrogen peroxide alone, is sufficient for the statistically significant reduction of the total amount of bacterial 16S rDNA gene, Legionella spp. and Vermamoeba vermiformis load (p < 0.005). Also shown is that even though hydrogen peroxide does not kill the trophozoite nor the cysts of V. vermiformis, it does however result in the detachment of the trophozoite form of this amoeba from the DUWS biofilm and hereby ultimately removing the amoeba from the system.

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Oral bacteria and yeasts in relationship to oral ulcerations in hematopoietic stem cell transplant recipients.

BACKGROUND: Oral mucositis is a serious and debilitating side effect of conditioning regimens for hematopoietic stem cell transplant (HSCT). Through HSCT, the homeostasis in the oral cavity is disrupted. The contribution of the oral microflora to mucositis remains to be clarified. The aim of our study was to investigate the relationship between yeasts, bacteria associated with periodontitis, and oral ulcerations in HSCT recipients. METHODS: This prospective observational study included 49 adult HSCT recipients. Twice weekly, oral ulcerations were scored, and oral rinsing samples were obtained. Samples were evaluated for the total bacterial load; the Gram-negative bacteria: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Tannerella forsythia, and Treponema denticola; and the yeasts: Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis using real-time polymerase chain reaction with specific primers and probes. Explanatory variables for oral ulcerations were calculated using the multilevel generalized estimated equations (GEE) technique. RESULTS: None of the samples was positive for A. actinomycetemcomitans, while F. nucleatum was found most often (66 % of samples). C. albicans was the most isolated yeast (88 % of samples), whereas C. parapsilosis was found in only 8 % of the samples. Multivariate GEE analyses identified P. gingivalis, P. micra, T. denticola, F. nucleatum, C. glabrata, and C. kefyr as significant explanatory variables of oral ulcerations. CONCLUSIONS: Our data indicate that P. gingivalis in particular, but also P. micra, T. denticola, F. nucleatum, C. glabrata, and C. kefyr may play a role in ulcerative oral mucositis in patients undergoing HSCT.

Sodium chloride and potassium sorbate: a synergistic combination against Enterococcus faecalis biofilms: an in vitro study.

Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48-h E. faecalis biofilms were evaluated in colony-forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)(2) ] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double-hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections.

Niacin Limitation Promotes Candida glabrata Adhesion to Abiotic Surfaces.

Candida glabrata is a prevalent fungal pathogen in humans, which is able to adhere to host cells and abiotic surfaces. Nicotinic acid (NA) limitation has been shown to promote the adherence of C. glabrata to human epithelial cells. Clinically, the elderly and hospitalized patients who are prone to C. glabrata-related denture stomatitis often suffer from vitamin deficiency. This study aimed to investigate C. glabrata adhesion to abiotic surfaces, including acrylic resin (a denture material) surfaces, cell surface hydrophobicity and adhesion gene expression. C. glabrata CBS138 was grown in media containing decreasing NA concentrations (40, 0.4, 0.04 and 0.004 µM). Adherence of C. glabrata to glass coverslips and acrylic resin was analyzed. C. glabrata adhesion to both surfaces generally increased with decreasing NA concentrations. The highest adhesion was found for the cells grown with 0.004 µM NA. The cell surface hydrophobicity test indicated that NA limitation enhanced hydrophobicity of C. glabrata cells. Quantitative PCR showed that of all adhesion genes tested, EPA1, EPA3 and EPA7 were significantly up-regulated in both 0.004 µM NA and 0.04 µM NA groups compared to those in the 40 µM NA group. No significant up- or down-regulation under NA limitation was observed for the other tested adhesion genes, namely AWP3, AWP4, AWP6 and EPA6. NA limitation resulted in increased expression of some adhesion genes, higher surface hydrophobicity of C. glabrata and enhanced adhesion to abiotic surfaces. NA deficiency is likely a risk factor for C. glabrata-related denture stomatitis in the elderly.

Oral hygiene in patients with motor neuron disease requires attention: A cross-sectional survey study.

AIMS: Motor Neuron Disease (MND) is a progressive neurodegenerative neuromuscular disease, which can progressively impair arm-hand function. Needs and barriers of MND patients and their caregivers in performing oral hygiene were studied. METHODS: An online survey was sent to 706 MND patients. The questions of the survey included self-reliance, self-reported oral health, and oral hygiene. The oral health-related quality of life (GOHAI-NL) and the subjective well-being (ALSAQ-5) were also measured. RESULTS: A total of 259 patients responded (36.7%), of which 71.9% stated not to be informed about the importance of maintaining good oral health by their MND treatment team. Moreover, 40.4% would like to receive help concerning oral hygiene from a dental professional. 19.8% were not satisfied about oral care as conducted by themselves or their caregivers. Patients who do not ask for support with their daily oral care had a significantly worse oral health-related quality of life compared to patients who do ask for support. CONCLUSIONS: The support for daily oral hygiene of MND patients and their barriers to requesting support needs more attention from both MND-treatment teams and general dental professionals.

16S rDNA sequencing and metadata of Dutch dental unit water.

Dental practices were approached to fill out a questionnaire on the infection control protocols in use to control biofilm growth in the dental unit and to send two types of water sample. Sampling of the dental units had to be performed prior to any infection control measures and on the second day of operation, to avoid residual effects of biofilm disinfection protocols performed in the weekend. Instructions were given on how to sample the units. Only samples, accompanied with a completed questionnaire and returned within two days by regular mail, were analysed. Samples were processed for heterotrophic plate counts, 16S (V4) rDNA microbiome sequencing and q-PCR for the concentration of bacterial 16S rDNA, fungal 18S rDNA, Legionella spp. and the presence of amoeba. The files contain the metadata needed to interpret and analyse the microbiome data. This dataset can be used by other scientists, members of infection control units, (trainee) bioinformaticians and policy makers. This dataset can provide leads to further unexplored parameters which could influence the microbial ecology of the dental unit.

The microbiological load and microbiome of the Dutch dental unit; 'please, hold your breath'.

Dental unit water systems are prone to biofilm formation. During use of the dental unit, clumps of biofilm slough off and can subsequently be aerosolized and inhaled by both patient and staff, potentially causing infections. The aim of this study was to determine the microbial load and microbiome of dental unit water, in the Netherlands, and the factors influencing these parameters. In total, 226 dental units were sampled and heterotrophic plate counts (HPC) were determined on the traditional effluent sample. Of all dental units, 61% exceeded the recommended microbiological guidelines of 100 colony forming units per milliliter. In addition, the microbiome, with additional q-PCR analysis for specific species, was determined on an effluent sample taken immediately after an overnight stagnancy period, in which the biofilm is in its relaxed state. These relaxed biofilm samples showed that each dental unit had a unique microbiome. Legionella spp., amoeba and fungi were found in 71%, 43% and 98% of all units, respectively. The presence of amoeba was positively associated with nine bacterial biomarkers and correlated positively with bacterial and fungal DNA and Legionella spp. concentrations, but not with HPC. Only when adhering to disinfection protocols, statistically significant effects on the microbial load and microbiome were seen. The relaxed biofilm sample, in combination with molecular techniques gives better insight in the presence of opportunistic pathogens when compared to the heterotrophic plate counts. Infection control measures should focus on biofilm analysis and control in order to guarantee patient safety.

The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis.

BACKGROUND: The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss. RESULTS: Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others. CONCLUSION: Analyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized.

An in-vitro dynamic flow model for translational research into dental unit water system biofilms.

Dental unit water systems (DUWS) provide an excellent environment for biofilm formation and can form a potential health risk for patients and staff. To control this biofilm formation, better understanding of the DUWS biofilm ecology is needed. Described is a newly developed in-vitro DUWS model which is easy to build, can be inoculated with different water sources and allows for sampling of both the effluent and biofilm. Unlike most models, a dynamic flow pattern, typical for a dental unit is used to provide water as a nutrient source. Microbial growth and composition were analyzed using heterotrophic plate counts (HPC) and 16S rDNA sequencing. Growth was reproducible in all models, reaching quasi-steady state at day 16 in the effluent (105-106 CFU∙mL-1) and day 23 in the biofilm (108 and 107 CFU∙cm-2) for non-potable and potable water, respectively. Principal component analysis of the microbial composition showed that biofilms originating from either non-potable or potable water were significantly different after 30 days of growth (n = 8, PERMANOVA, F = 35.6, p < .005). Treatment of the biofilms with 1000 ppm active chlorine showed a biological and statistical significant decrease in viable counts in the effluent phase to below the detection limit of 100 CFU∙mL-1. The HPC returned to pre-treatment levels within 14 days. Using this model results in inoculum dependent biofilms with a higher bacterial density compared to previously described models. The relative ease in which samples can be taken allows for the monitoring of antimicrobial disinfection efficacy on the effluent, biofilm and matrix.

Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria.

OBJECTIVE: Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. METHODS: Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. RESULTS: For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. CONCLUSIONS: The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.

Chlorine-based DUWL disinfectant leads to a different microbial composition of water derived biofilms compared to H2O2-based chemical disinfectants in vitro.

BACKGROUND: Biofilm formation in dental unit waterlines (DUWL) may lead to health risks for dental staff and patients. Therefore, dental unit waterlines need to be disinfected, for instance by using chemical disinfectants. However, the application of chemical disinfectants may lead to the selection of specific microorganisms. Therefore, the aim of our study was to assess the microbial composition of water-derived biofilms, after a continuous exposure to maintenance doses of commercially available chemical disinfectants, in vitro. METHODS: The AAA-model was used to grow water derived biofilms. The biofilms were subjected to the maintenance dose of each disinfectant. To determine the microbial composition, the V4 hypervariable region of the 16S rRNA gene was sequenced. The sequences were clustered in operational taxonomic units (OTUs). RESULTS: The bacterial composition of biofilms in all treatment groups differed significantly (PERMANOVA F = 4.441, p = 0.001). Pairwise comparisons revealed Anoxyl treated biofilms were significantly different from all groups (p = 0.0001). In the Anoxyl-treated biofilms, the relative abundance of Comamonadaceae and Sphingopyxis was high compared to the Dentosept, Green and Clean and Oxygenal groups. CONCLUSION: We concluded that exposure to low doses of the chlorine-based chemical disinfectant Anoxyl led to a substantially different composition of water derived biofilms compared to biofilms exposed to H2O2-based chemical disinfectants.

Oral diseases: from detection to diagnostics.

In addition to saliva, other oral components such as gingival crevicular fluid, epithelial cells, bacteria, breath, and dental plaque have diagnostic potential. For oral diseases such as caries and periodontal disease, visual diagnosis is usually adequate, but objective diagnostic tests with predictive value are desired. Therefore, prediction models like the Cariogram have been developed that also include oral aspects such as saliva secretion, buffering capacity, and Streptococcus mutans counts for the prediction of caries. Correlation studies on salivary components and caries have not been conclusive, but correlation studies on functional aspects, such as saliva-induced bacterial aggregation and caries, look promising. Modern proteomic techniques make it possible to study simultaneously the many salivary components involved in these functions.

Candida and Porphyromonas gingivalis: the effect on wound closure in vitro.

Microorganisms play a role in oral mucositis after cancer therapy. The current study explored the hypothesis that Candida spp. alone and together with Porphyromonas gingivalis cause delayed healing of oral ulcerations due to the inhibition of wound closure. An in vitro scratch assay model was used to study the influence of viable and heat-killed Candida glabrata, Candida kefyr, and Candida albicans on cell migration of oral epithelial cells. Separately, the effect of conditioned medium of Candida spp. and the effect of a mixed infection of Candida spp. with P. gingivalis on wound closure was studied. In the presence of 10 viable C. glabrata or C. kefyr versus one epithelial cell, with a multiplicity of infection (MOI) of 10, the relative closure of the scratch was 26% and 17%, respectively. At a MOI of 1, this was 60% for C. glabrata and 78% for C. kefyr. The inhibition of oral epithelial cell migration challenged with either C. glabrata or C. kefyr together with P. gingivalis was stronger than the inhibition caused by one of both organisms separately. Candida spp. inhibit cell migration in vitro. A combination of Candida spp. and P. gingivalis inhibited cell migration more than either microorganism separately.

Failed bonded interfaces submitted to microcosm biofilm caries development.

OBJECTIVES: This study aimed to evaluate the dentin wall carious lesion development of different composite-dentin interfaces in the presence of two adhesive bonding materials in the gaps, using a microcosm biofilm model. METHODS: Dentin samples were prepared (10.4mm(2)) and restored with a composite resin using two adhesive systems (etch-and-rinse and self-etch techniques). Different conditions with respect to composite-dentin interfaces were produced with a 200µm gap: failed bonded without ageing or after mechanical ageing, or non-bonded with or without the presence of adhesive material on the dentin wall. For cariogenic challenge, specimens were subjected to a biofilm microcosm model for 14days to create caries-like wall lesions. Before and after caries development, transverse wavelength-independent microradiography images were taken, and lesion depth and mineral loss were measured. Data were analysed with linear regression models (p<0.05). RESULTS: The composite-dentin interface conditions significant influenced the caries development: lesion development was reduced by the presence of the adhesive material on dentin wall, while lesion development was increased by the mechanical ageing (p=0.019). There was no difference between the adhesive materials (p values>0.05). CONCLUSION: Different composite-dentin interfaces influence wall lesion development in gaps, with the interfaces submitted to ageing showing less carious protection than those interfaces with the presence of adhesive covering the dentin. CLINICAL SIGNIFICANCE: The presence of adhesive bonding material in the gaps plays a role on the wall caries lesion development.

Red and Green Fluorescence from Oral Biofilms.

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

The impact of virulence factors of Porphyromonas gingivalis on wound healing in vitro.

BACKGROUND: Porphyromonas gingivalis inhibits oral epithelial wound healing in vitro more strongly than other oral bacteria, but it is unknown why P. gingivalis is such a potent inhibitor of wound healing. OBJECTIVE: Therefore, the aim of this study was to investigate the influence of major virulence factors of P. gingivalis on wound healing in an in vitro wound-healing model. The influence of the capsular polysaccharide, the Arg- and Lys- gingipains, the major fimbriae and lipopolysaccharide (LPS) was investigated. DESIGN: A standardized scratch was made in a confluent layer of human oral epithelial cells HO-1-N-1. The epithelial cells were then challenged with different concentrations of several P. gingivalis wild-type strains and knockout mutants. Closure of the scratch was determined after 17 h and compared to control conditions without bacteria. RESULTS: The P. gingivalis strains ATCC 33277, W83, and W50 significantly inhibited wound healing. The presence of a capsular polysaccharide lowered significantly the inhibition of epithelial cell migration, while gingipain activity significantly increased the inhibition of cell migration. LPS and the major fimbriae did not influence epithelial cell migration. None of the tested P. gingivalis strains completely prevented the inhibition of cell migration, suggesting that other characteristics of P. gingivalis also play a role in the inhibition of wound healing, and that further research is needed. CONCLUSIONS: The capsular polysaccharide and the Arg- and Lys- gingipains of P. gingivalis influenced the capacity of P. gingivalis to hinder wound healing, while LPS and the major fimbriae had no effect.