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.

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.

The effect of fluoride on the stability of oral bacterial communities in vitro.

An in vitro model system has been used to study the combined influences of fermentable carbohydrate, pH, and fluoride on the stability of complex oral microbial communities. The pH generated from carbohydrate pulses rather than the availability of substrate per se was responsible for the enrichment of the cariogenic species S. mutans and L. casei. The addition of sub-MIC levels of sodium fluoride (1 mmol/L; 19 ppm) reduced both the rate of acid production and the fall in terminal pH from glucose pulses, thereby enabling pH-sensitive bacteria, including many Gram-negative species, to persist. Furthermore, the combination of even a moderately-low environmental pH (ca. pH 5.0) with a low level (1 mmol/L) of fluoride was able to prevent S. mutans from out-competing other species and resulted in its proportions within the bacterial community remaining low. By this mechanism, fluoride could make a significant contribution to preventing dental caries.

The effects of triclosan and zinc citrate, alone and in combination, on a community of oral bacteria grown in vitro.

A mixed-culture chemostat system has been used as a more stringent laboratory system for evaluation of the antimicrobial effects of Triclosan and zinc citrate. The inhibitors were added alone, and in combination, as a pulse (a high initial inhibitor concentration which decreased with time) or as a dose (concentration of inhibitor increased with time) to give maximum concentrations of 34.5 mumol/L Triclosan and 39.8 mumol/L zinc citrate. When dosed, Triclosan inhibited A. viscosus and all five Gram-negative species, whereas zinc citrate had less effect, probably due to complexation by media components. Similar effects were seen when Triclosan was pulsed, except that S. mutans was the most sensitive Gram-positive species and V. dispar was unaffected. However, when the inhibitors were dosed or pulsed in combination, marked complementary and additive inhibitory effects were observed, particularly against Gram-negative species, although S. gordonii and S. oralis were relatively unaffected. The data confirm that increased effects can be obtained with suitable combinations of antimicrobial agents and suggest that, under certain conditions, apparently broad-spectrum antimicrobial agents may be acting more selectively than hitherto suspected.

Prevention of population shifts in oral microbial communities in vitro by low fluoride concentrations.

A continuous culture system has been used to study the effects of low (sub-MIC) levels of sodium fluoride on the stability and metabolism of a defined oral microbial community. The microflora was also subjected to glucose pulses at pH 7.0, with and without subsequent pH control. At pH 7.0, a continuous supply of 1 mmol/L NaF reduced slightly the viable counts of the oral microflora, although their proportions were relatively unaffected. At pH 7.0, during glucose pulsing, 1 mmol/L NaF prevented the rise in proportions of A. viscosus and reduced the levels of B. intermedius. Glucose pulsing without pH control and in the absence of fluoride markedly inhibited the growth of many species, and L. casei, V. dispar, and S. mutans predominated in the culture. Fluoride (1 mmol/L), either pulsed with the glucose or provided continuously, reduced both the rate of change and the degree of fall in pH, and in doing so prevented the enrichment of S. mutans in the culture. Fluoride also reduced the pH-mediated inhibition of other members of the oral community, although S. sanguis was inhibited even further. Thus, even sub-MIC levels of fluoride may have a beneficial anti-bacterial effect on dental plaque by interfering with acid production. This would reduce the pH-mediated disruption to the balance of the microflora and suppress the selection of S. mutans.

Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro.

A mixed culture chemostat system was used to distinguish between the effects of carbohydrate availability per se and the low pH generated from carbohydrate metabolism on the proportions of bacteria within microbial communities. Nine oral bacteria were grown at pH 7 and pulsed with glucose on ten consecutive days. In one chemostat, the pH was maintained automatically at 7 throughout the experimental period, while in the other, pH control was discontinued for six hours after each pulse. Glucose pulses at neutral pH had little effect on the composition of the microflora. Only the proportions of A. viscosus and V. dispar increased; L. casei and S. mutans remained at low levels (0.2% and 1.0%, respectively). Acetate and propionate were low. In contrast, when pH was allowed to fall after each glucose pulse, the composition of the microflora altered dramatically. The amounts of L. casei and S. mutans increased both as a proportion of the total count and in absolute numbers, as did V. dispar, whereas the amounts of the other Gram-negative organisms (B. intermedius, F. nucleatum, and N. subflava) and S. sanguis were considerably reduced. Lactate formed a major portion of the metabolic end-products. Successive glucose pulses resulted in both amplified changes in the microflora and a steadily greater rate and final extent of acid production. This is in agreement with the reported shifts in the oral microflora in vivo in response to frequent carbohydrate intake. Analysis of the data strongly suggests that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, is responsible for the widely reported shifts in composition and metabolism of the oral microflora in vivo.

gamma-Aminobutyric acidA receptor function is modulated by cyclic GMP.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate nervous system. Modulatory effects of intracellular ATP on the GABA response in isolated bullfrog dorsal root ganglion neurons were examined using whole-cell voltage clamp. Investigation of the plausible mechanisms ATP might utilize to regulate the GABA response led to the discovery that intracellular cyclic GMP may play an important role in modulating inhibitory neurotransmission. This modulatory effect of cyclic GMP is likely to be mediated via a cyclic GMP-dependent protein kinase.

Microbiological effects of new agents in dentifrices for plaque control.

The incorporation of antimicrobial agents in dental products has been proposed for many years as a possible valuable adjunct to mechanical plaque control. Out of the diverse collection of potential agents, relatively few can be formulated into dentifrices. Several products have been developed based on Triclosan, a broad spectrum antimicrobial agent. By itself, Triclosan has only moderate antiplaque activities; consequently, it has been combined with other molecules in attempts to boost its clinical efficacy. The combination of Triclosan with a copolymer (polyvinyl methyl ether maleic acid) has been shown to increase the retention of Triclosan to surfaces, and to raise its antiplaque and antimicrobial activity in a range of laboratory tests. Other studies, however, have found greater inhibitory effects on both liquid and biofilm mixed cultures when Triclosan was combined with either pyrophosphate or zinc citrate. Both of these combinations were selectively inhibitory to those species implicated in gingivitis and advanced periodontal diseases. There is little microbiological data on the clinical efficacy of a combination of Triclosan with pyrophosphate, but as pyrophosphate has only a bacteriostatic mode of action and is lost rapidly from the mouth, this may limit the significance of any of its potential antimicrobial effects in vivo. In contrast, zinc citrate is a proven inhibitor of bacterial metabolism and growth, and is retained in the mouth for prolonged periods. Several clinical studies have confirmed an additive, complementary and selective inhibitory antimicrobial effect on dental plaque of a dentifrice containing Triclosan and zinc citrate, without any adverse effects on the natural microbial ecology of the mouth.

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.

Orbital inflammatory syndrome and systemic lupus erythematosus.

Orbital inflammatory syndrome (OIS) is a rare condition characterized by inflammation of orbital structures in the absence of underlying infection, neoplasm or endocrinopathy. The pathogenesis of OIS has not been fully described, but histopathology reveals diffuse lymphocytic infiltration of the involved structures. This disorder presents clinically as acute pain and swelling in the orbit and usually responds rapidly to high dose corticosteroids. This entity has been reported previously in seven patients with systemic lupus erythematosus (SLE). Herein, we describe a patient with SLE who developed OIS, review the previously reported cases that have occurred in association with SLE, and describe the diagnostic evaluation and therapy for OIS. This rare entity can pose both a diagnostic and therapeutic dilemma in SLE patients given immunoppressive therapy.

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.

Dental plaque as a biofilm.

Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin. Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface. Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle). To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface. Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions. These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession. As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogenous environment. Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases). Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.