Modulation of Candida albicans virulence in in vitro biofilms by oral bacteria.

Candida-associated denture stomatitis presents as erythema of the palatal mucosa and is caused by biofilms containing the fungus Candida albicans that co-reside with oral bacteria on the denture-fitting surface. This study aimed to assess the effect of several frequently encountered oral bacteria on the expression of C. albicans virulence factors in in vitro polymicrobial biofilms. Biofilms containing C. albicans and selected bacterial species were grown on denture acrylic, and analysed by microscopy and by qPCR for expression of putative virulence genes. Candida albicans-only biofilms showed limited hyphal production. Hyphal development was significantly (P < 0·001) increased when biofilms also contained four species of oral bacteria (Streptococcus sanguinis, Streptococcus gordonii, Actinomyces odontolyticus and Actinomyces viscosus), as was the expression of virulence genes (P < 0·05). Importantly, inclusion of Porphyromonas gingivalis in the biofilm consortium resulted in significant (P < 0·05) inhibition of virulence gene expression and production of hyphae. The in vitro expression of C. albicans virulence factors was modulated in polymicrobial biofilms. The complexity of this modulation was highlighted by the reversal of effects following introduction of a single bacterial species into a biofilm community. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of individual bacterial species on Candida albicans virulence highlights both the complexity of predicting infection mediated by polymicrobial communities and the potential for management through pro- or prebiotic therapy. The possibility to selectively modulate microbial virulence by addition of, or treatment with pro- or prebiotics avoids the use of conventional antimicrobial compounds, thus reducing the contribution to potential drug resistance. Understanding which bacterial species modulate virulence, and the mechanisms by which this occurs, particularly in biofilms, provides excellent foundations for further research questions, and the potential for novel clinical interventions.

The epithelial cell response to health and disease associated oral biofilm models.

BACKGROUND AND OBJECTIVE: Different bacteria differentially stimulate epithelial cells. Biofilm composition and viability are likely to influence the epithelial response. In vitro model systems are commonly used to investigate periodontitis-associated bacteria and their interactions with the host; therefore, understanding factors that influence biofilm-cell interactions is essential. The present study aimed to develop in vitro monospecies and multispecies biofilms and investigate the epithelial response to these biofilms. MATERIAL AND METHODS: Bacterial biofilms were cultured in vitro and then either live or methanol-fixed biofilms were co-cultured with epithelial cells. Changes in epithelial cell viability, gene expression and cytokine content of culture supernatants were evaluated. RESULTS: Bacterial viability was better preserved within mixed-species biofilm culture than within single-species biofilm culture. Both mixed- and single-species biofilms stimulated increased expression of mRNA for interleukin 8 (IL8), C-X-C motif chemokine ligand 3 (CXCL3), C-X-C motif chemokine ligand 1 (CXCL1), interleukin 1 (IL1), interleukin 6 (IL6), colony-stimulating factor 2 (CSF2) and tumour necrosis factor (TNF), and the response was greatest in response to mixed-species biofilms. Following co-culture, cytokines detected in the supernatants included IL-8, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, with the greatest release of cytokines found following co-culture with methanol-fixed, mixed-species biofilms. CONCLUSIONS: These data show that epithelial cells generate a distinct cytokine gene- and protein-expression signature in response to live or fixed, single- or multispecies biofilms.

Structural modifications of the salivary conditioning film upon exposure to sodium bicarbonate: implications for oral lubrication and mouthfeel.

The salivary conditioning film (SCF) that forms on all surfaces in the mouth plays a key role in lubricating the oral cavity. As this film acts as an interface between tongue, enamel and oral mucosa, it is likely that any perturbations to its structure could potentially lead to a change in mouthfeel perception. This is often experienced after exposure to oral hygiene products. For example, consumers that use dentifrice that contain a high concentration of sodium bicarbonate (SB) often report a clean mouth feel after use; an attribute that is clearly desirable for oral hygiene products. However, the mechanisms by which SB interacts with the SCF to alter lubrication in the mouth is unknown. Therefore, saliva and the SCF was exposed to high ionic strength and alkaline solutions to elucidate whether the interactions observed were a direct result of SB, its high alkalinity or its ionic strength. Characteristics including bulk viscosity of saliva and the viscoelasticity of the interfacial salivary films that form at both the air/saliva and hydroxyapatite/saliva interfaces were tested. It was hypothesised that SB interacts with the SCF in two ways. Firstly, the ionic strength of SB shields electrostatic charges of salivary proteins, thus preventing protein crosslinking within the film and secondly; the alkaline pH (≈8.3) of SB reduces the gel-like structure of mucins present in the pellicle by disrupting disulphide bridging of the mucins via the ionization of their cysteine's thiol group, which has an isoelectric point of ≈8.3.

Presence of negative charge on the basal planes of New York talc.

Potentiometric titration measurements as well as rheological measurements of talc aqueous suspensions indicate that the behavior of the New York talc particles is consistent with the presence of a negative charge on their basal planes. The possibility of the presence of a negative electrical charge on the basal planes of talc particles is analyzed in this paper. Samples of New York talc were studied using electron microprobe analysis and dehydration techniques and the exact chemical formula of New York talc was determined. It was found that there exists a deficiency of protons in the tetrahedral layers of talc, resulting from substitution of Si(4+) ions with Al(3+) and Ti(3+) ions. The comparison of the level of substitution of Si(4+) ions with ions of a lower valency was found to be of a similar order of magnitude as that found in other talc deposits. This strongly points to the presence of a negative charge on the talc basal planes.

Attitudes of general dental practitioners in Europe to the microbial risk associated with dental unit water systems.

UNLABELLED: Dental Unit Water Systems (DUWS) are used in dental practices to provide water for cooling of dental equipment and irrigation of the oral cavity. However, they have been demonstrated to be contaminated with micro-organisms. There are currently no European Union (EU) Commission guidelines for the microbial quality of water discharged by DUWS. This study was part of an EU research programme to investigate the microbial contamination of DUWS in general dental practice (GDP) in the UK, Denmark, Germany, The Netherlands, Ireland, Greece and Spain. OBJECTIVE: To undertake a questionnaire survey on the type of DUWS in use and determine the attitude of GDPs to the risk of microbial infection from DUWS. MATERIALS AND METHODS: The questionnaire was written and translated into the language of each country before being posted to each participating dentist. Dentists were asked to complete the questionnaire survey and return it by post. RESULTS AND CONCLUSIONS: The major findings were that the majority of dentists did not clean, disinfect or determine the microbial load of their DUWS, and that dentists would welcome regular monitoring and advice on maintaining their DUWS; the introduction of guidelines; and recommendations on controlling the microbial load of DUWS.

Comparison of the efficacies of disinfectants to control microbial contamination in dental unit water systems in general dental practices across the European Union.

Water delivered by dental unit water systems (DUWS) in general dental practices can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWS provide a reservoir for microorganisms and should be controlled. This study compared disinfection products for their ability to meet the American Dental Association's guideline of <200 CFU x ml(-1) for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal, Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS (n = 134) in Denmark, Germany, Greece, Ireland, The Netherlands, Spain, and the United Kingdom. Weekly water samples were tested for total viable counts (TVCs) on yeast extract agar, and, where possible, the effects of products on established biofilm (TVCs) were measured. A 4- to 5-week baseline measurement period was followed by 6 to 8 weeks of disinfection (intermittent or continuous product application). DUWS water TVCs before disinfection ranged from 0 to 5.41 log CFU x ml(-1). Disinfectants achieved reductions in the median water TVC ranging from 0.69 (Ster4Spray) to 3.11 (Dentosept) log CFU x ml(-1), although occasional high values (up to 4.88 log CFU x ml(-1)) occurred with all products. Before treatment, 64% of all baseline samples exceeded American Dental Association guidelines, compared to only 17% following commencement of treatment; where tested, biofilm TVCs were reduced to below detectable levels. The antimicrobial efficacies of products varied (e.g., 91% of water samples from DUWS treated with Dentosept or Oxygenal met American Dental Association guidelines, compared to 60% of those treated with Ster4Spray). Overall, the continuously applied products performed better than those applied intermittently. The most effective products were Dentosept and Oxygenal, although Dentosept gave the most consistent and sustained antimicrobial effect over time.

Creation of oral care flavours to deliver breath-freshening benefits.

OBJECTIVE: Oral care products deliver breath freshening primarily via mechano-chemical cleaning or by antimicrobial active systems. Dental flavours provide taste benefits, and freshen breath mainly by sensorial masking. We aimed to determine whether flavours could deliver breath freshening in products by inhibiting bacterial volatile sulphide compound (VSC) production. SUBJECTS AND METHODS: Flavour materials were screened for inhibition of hydrogen sulphide formation by Klebsiella pneumoniae in vitro, grouped by efficacy, and data provided to flavourists. Flavours were formulated to maximize the content of VSC-effective ingredients and re-screened to confirm performance. Extensive, iterative testing of flavours identified reliable creative rules to deliver efficient inhibition of H2S generation. Breath-freshening flavours in whole products were then tested in-house in a 'breath freshness panel'. MAIN OUTCOME MEASURES: Malodour of panellists (not preselected for malodour score) was scored before and after product use, on the 'Rosenberg' 0-5 scale, together with residual flavour score, by extensively trained judges. Products were tested in double-blind, crossover studies, and results analysed using ANOVA. RESULTS AND CONCLUSIONS: Products flavoured using these rules delivered significantly greater breath freshening at 2 h than control products, and equivalent benefits to products containing 0.1% (w/w) triclosan or 0.2% (w/w) zinc sulphate.

Microbiological evaluation of dental unit water systems in general dental practice in Europe.

A range of opportunistic pathogens have been associated with dental unit water systems (DUWS), particularly in the biofilms that can line the tubing. This study therefore aimed to assess the microbiology of DUWS and biofilms in general dental practices across seven European countries, including the United Kingdom (UK), Ireland (IRL), Greece (GR), Spain (ES), Germany (D), Denmark (DK) and the Netherlands (NL). Water supplied by 51% of 237 dental unit water lines exceeded current American Dental Association recommendations of < or = 200 colony-forming units (CFU) ml(-1). Microbiological loading of the source waters was between 0 (Denmark, the Netherlands and Spain) and 4.67 (IRL) log CFU ml(-1); water line samples from the DUWS ranged from 1.52 (ES) to 2.79 (GR) log CFU ml(-1); and biofilm counts ranged from 1.49 (GR) to 3.22 (DK) log CFU.cm(-2). Opportunistic pathogens such as legionellae (DK and ES), including Legionella pneumophila SG1 (DK and GR), and Mycobacterium spp. (DK, NL, GR, D and ES) were recovered occasionally. Presumptive oral streptococci (ES and NL), oral anaerobes (GR), Candida spp. (UK, NL and ES) and blood (GR and IRL) were detected at relatively low frequencies, but their presence indicated a failure of the 3-in-1 antiretraction valve, leading to back siphonage of oral fluids into the water and biofilm phase. These findings confirm that a substantial proportion of DUWS have high levels of microbial contamination, irrespective of country, type of equipment and source water. The study emphasizes the need for effective mechanisms to reduce the microbial burden within DUWS, and highlights the risk of occupational exposure and cross-infection in general dental practice.

Microbiological evaluation of a range of disinfectant products to control mixed-species biofilm contamination in a laboratory model of a dental unit water system.

Dental unit water system (DUWS) tubing harbors complex multispecies biofilms that are responsible for high microbial levels at the distal outlet. The aim of this study was to use an established biofilm laboratory model to simulate biofouling of DUWS to evaluate practical, cost-effective, and evidence-based methods of microbial decontamination. Reproducible biofilms were developed in the model over 14 days; decontamination was assessed using total viable counts (TVC) and microscopic-image analysis techniques to view the inner surface of tubing. Flushing did not reduce the biofilm coverage or TVC. Combizyme and ozone did not completely eliminate the viable bacteria (70 and 65% reduction in biofilm TVC, respectively), nor did they remove the biofilm (45 and 57% reduction in biofilm coverage, respectively). Chlorhexidine and Bio2000 (active agent: ethanol and chlorhexidine), Tegodor and Gigasept Rapid (aldehyde based), and Grotanol (hydroxide based) completely eliminated the TVC but did not completely remove biofilm (31, 53 33, 34, and 64.9% reduction of biofilm coverage, respectively). Other products including Grotanol Flussig (phenol based), Betadine (povidone-iodine based), Alpron (chlorite based), and the hydroxide-containing products Sporklenz, Sterilex Ultra, Dialox, Sterilox, Sanosil, Oxigenal, and Grotanat Bohrerbad resulted in a 100% reduction in the biofilm TVC and a >95% reduction in biofilm coverage. The study demonstrated that while many disinfectants achieve a sufficient reduction in TVC they may not necessarily remove unwanted biofilm from the tubing surfaces as tested in this laboratory-controlled biofilm model.

Effects of glucose and fluoride on competition and metabolism within in vitro dental bacterial communities and biofilms.

Antimicrobial effects of fluoride in vivo remain contentious. Previous studies suggested that 1 mM NaF reduced acid production from glucose, and prevented the enrichment of bacteria associated with caries in a chemostat model. The present study examines the effects of a lower fluoride concentration (0.53 mM, 10 ppm NaF) in both biofilm and planktonic microbial communities. Nine oral species were grown at pH 7.0 and pulsed on 10 successive days with glucose; bacterial metabolism was allowed to reduce the pH for 6 h before being returned to neutrality, either in the presence or absence of NaF. In addition, 10-day-old mixed culture biofilms were overlaid with glucose, with or without NaF, and the pH change followed by microelectrode. After 10 days, chemostat pH dropped to ca. pH 4.5 following glucose pulses, and the community was dominated by Streptococcus mutans (rising from 4 to 23% of total CFU) and Veillonella dispar (16 to 73%). In comparison, after 10 days pulsing with glucose + fluoride, the final pH was significantly higher (ca. pH 4.9) (paired t test, p < 0.0001). The culture was predominated by V. dispar (70%) and Actinomyces naeslundii (13%), whereas S. mutans proportions were significantly lower (t test, p = 0.04), remaining <3% of the total flora, compared to the culture without fluoride. Biofilm pH fell to only pH 5.55 1 h after glucose/fluoride overlay, compared to 4.55 with glucose alone (paired t test, p < 0.000001). Analysis of the data suggests that fluoride exerts dual antimicrobial modes of action. Fluoride prevents enrichment of S. mutans by inhibiting critical metabolic processes (direct effect) and, in an inter-related way, by reducing environmental acidification (indirect effect) in biofilms.

Microbial biofilm formation and contamination of dental-unit water systems in general dental practice.

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10(5) CFU. ml(-1); in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this "viable by BacLight" fraction produced colonies on agar plates. Legionella pneumophila, Mycobacterium spp., Candida spp., and Pseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and Fusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered "cleaner" than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.

Microbial aerosols in general dental practice.

OBJECTIVE: To measure the concentration of microbial aerosols in general dental practices and to use this information to carry out quantitative microbiological risk assessments. METHODOLOGY: Microbial air sampling was carried out continuously during 12 treatment sessions in 6 general dental practices in the South West of England. RESULTS: The microbial aerosol concentration in treatment rooms was generally less than 10(3) colony forming units per cubic metre of air (cfu x m(-3)). However, in 6 out of the 12 visits, at least one peak concentration with much higher numbers of bacteria was detected. The peak concentrations were associated with increased recoveries of presumptive oral streptococci suggesting these aerosols originated from the mouths of patients. These aerosol peaks dissipated within 30 minutes and no dissemination into waiting areas was detected. The peak concentrations were associated with mechanical scaling procedures (47% of procedures giving rise to a peak) and to a lesser extent by cavity preparation (11%). No aerosolised blood was detected. CONCLUSIONS: The data have been used to generate a framework for quantifying risk of exposure of staff to aerosolised microbial pathogens in general dental practice. For example, dentists and their assistants may have a slightly higher risk of exposure to Mycobacterium tuberculosis than the general public. The use of face seal masks that have been shown to protect against aerosolised micro-organisms may reduce this exposure.

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.

Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration.

Coaggregation is a well-characterized phenomenon by which specific pairs of oral bacteria interact physically. The aim of this study was to examine the patterns of coaggregation between obligately anaerobic and oxygen-tolerant species that coexist in a model oral microbial community. Obligate anaerobes other than Fusobacterium nucleatum coaggregated only poorly with oxygen-tolerant species. In contrast, F. nucleatum was able to coaggregate not only with both oxygen-tolerant and other obligately anaerobic species but also with otherwise-noncoaggregating obligate anaerobe-oxygen-tolerant species pairs. The effects of the presence or absence of F. nucleatum on anaerobe survival in both the biofilm and planktonic phases of a complex community of oral bacteria grown in an aerated (gas phase, 200 ml of 5% CO2 in air x min-1) chemostat system were then investigated. In the presence of F. nucleatum, anaerobes persisted in high numbers (>10(7) x ml-1 in the planktonic phase and >10(7) x cm-2 in 4-day biofilms). In an equivalent culture in the absence of F. nucleatum, the numbers of black-pigmented anaerobes (Porphyromonas gingivalis and Prevotella nigrescens) were significantly reduced (P

Analysis of pH-driven disruption of oral microbial communities in vitro.

Previously, a mixed culture chemostat system was used to demonstrate that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, was responsible for the shifts observed in the oral microflora which are associated with high carbohydrate diets and the development of dental caries. The aim of this study was to determine more accurately the microbially generated pH at which such shifts occurred. Nine oral bacteria were grown in three independent chemostats, and pulsed with glucose on 10 consecutive days. In one chemostat, pH control was discontinued for 6 h, and the pH fall was restricted to a minimum value of pH 5.5; the pH fall was arrested in the other two chemostats at either pH 5. 0, or at pH 4.5. When the pH was allowed to fall, the numbers and proportions of Streptococcus mutans and Lactobacillus rhamnosus increased; this increase was directly related to the magnitude of the pH fall. Veillonella dispar was the most numerous organism following all glucose pulsing regimes, especially at low pH. The increase in proportions of acidogenic bacteria was accompanied by a fall in the proportions of acid-sensitive species (Fusobacterium nucleatum, Prevotella nigrescens, Streptococcus gordonii and Streptococcus oralis). The counts of these species were relatively stable between pH 5.5 and 4.5, but were markedly reduced when the pH fell below pH 4.5; Neisseria subflava could not persist in the culture at pH 4.5 or below. The data suggest that the disruption of communities associated with glucose metabolism and low pH can be explained in terms of a two-stage process. A fall in pH to a value between pH 5.5 and 4.5 may allow the enrichment of potentially cariogenic species, whilst permitting species associated with health to remain relatively unaffected. A further reduction in pH (

Physiological approaches to the control of oral biofilms.

Evidence that physiological strategies may be potential routes for oral biofilm control has come from (i) observations of the variations in the intra-oral distribution of members of the resident oral microflora, (ii) changes in plaque composition in health and disease, and (iii) data from laboratory model systems. Key physiological factors that were identified as significant in modulating the microflora included the local pH, redox potential (Eh), and nutrient availability. Increases in mutans streptococci and lactobacilli occur at sites with caries; growth of these species is selectively enhanced at low pH. In contrast, periodontal diseases are associated with plaque accumulation, followed by an inflammatory host response. The increases in Gram-negative, proteolytic, and obligately anaerobic bacteria reflect a low redox potential and a change in nutrient status due to the increased flow of gingival crevicular fluid (GCF). Consequently, physiological strategies for oral biofilm control should focus on reducing the frequency of low pH in plaque by (i) inhibiting acid production, (ii) using sugar substitutes, and (iii) promoting alkali generation from arginine or urea supplements. Similarly, strategies to make the pocket environment less favorable to periodonto-pathogens include (i) anti-inflammatory agents to reduce the flow of (and hence nutrient supply by) GCF, (ii) bacterial protease inhibitors, and (iii) redox agents to raise the Eh locally. Most laboratory and clinical findings support the concept of physiological control. However, some data suggest that the ordered structure and metabolically interactive organization of mature dental plaque could generate a community with a high level of homeostasis that is relatively resistant to deliberate external manipulation.

A modified chemostat system to study the ecology of oral biofilms.

Previously, we developed a chemostat system to study the behaviour and properties of a community of up to 10 species of oral bacteria. The present study describes modification of this system to incorporate removable and replaceable hydroxyapatite (the major mineral in human dental enamel) disks on which biofilms could develop. Hydroxyapatite disks were immersed in the chemostat for known time periods, and the bacterial content of biofilms determined by viable counting. Initial deposition rates were rapid, with all 10 species detected after 1 h, and the numbers of bacteria in biofilms continued to increase for 21 d. The species composition of biofilms reflected that of the surrounding fluid phase, and showed only limited signs of the type of 'species succession' which is observed in developing dental plaque in vivo, although anaerobic species increased in proportion in older biofilms. Four-day biofilms showed the least variability and were chosen as the 'standard biofilm' for more detailed study. Variability in the bacterial composition of 4-d biofilms was comparable both within a single chemostat run and between independent chemostat runs. Glucose pulsing in the absence of pH control resulted in the selection of cariogenic species; the disruption of the biofilm community was less marked than that of the equivalent planktonic culture. The model system has considerable potential in studying the effects of a variety of factors on biofilm development, as well as in comparing the efficacy of antimicrobial systems against biofilms.