Interdental and subgingival microbiota may affect the tongue microbial ecology and oral malodour in health, gingivitis and periodontitis.

BACKGROUND AND OBJECTIVE: Oral malodour is often observed in gingivitis and chronic periodontitis patients, and the tongue microbiota is thought to play a major role in malodorous gas production, including volatile sulphur compounds (VSCs) such as hydrogen sulphide (H2 S) and methanethiol (CH3 SH). This study aimed to examine the link between the presence of VSCs in mouth air (as a marker of oral malodour) and the oral bacterial ecology in the tongue and periodontal niches of healthy, gingivitis and periodontitis patients. METHODS: Participants were clinically assessed using plaque index, bleeding on probing (BOP) and periodontal probing depths, and VSC concentrations in their oral cavity measured using a portable gas chromatograph. Tongue scrapings, subgingival and interdental plaque were collected from healthy individuals (n = 22), and those with gingivitis (n = 14) or chronic periodontitis (n = 15). The bacterial 16S rRNA gene region V3-V4 in these samples was sequenced, and the sequences were analysed using the minimum entropy decomposition pipeline. RESULTS: Elevated VSC concentrations and CH3 SH:H2 S were observed in periodontitis compared with health. Significant ecological differences were observed in the tongue microbiota of healthy subjects with high plaque scores compared to low plaque scores, suggesting a possible connection between the microbiota of the tongue and the periodontium and that key dysbiotic changes may be initiated in the clinically healthy individuals who have higher dental plaque accumulation. Greater subgingival bacterial diversity was positively associated with H2 S in mouth air. Periodontopathic bacteria known to be prolific VSC producers increased in abundance on the tongue associated with increased bleeding on probing (BOP) and total percentage of periodontal pockets >6 mm, supporting the suggestion that the tongue may become a reservoir for periodontopathogens. CONCLUSION: This study highlights the importance of the periodontal microbiota in malodour and has detected dysbiotic changes in the tongue microbiota in periodontitis.

Evidence supporting the microbiota-gut-brain axis in a songbird.

Recent research in mammals supports a link between cognitive ability and the gut microbiome, but little is known about this relationship in other taxa. In a captive population of 38 zebra finch(es) (Taeniopygia guttata), we quantified performance on cognitive tasks measuring learning and memory. We sampled the gut microbiome via cloacal swab and quantified bacterial alpha and beta diversity. Performance on cognitive tasks related to beta diversity but not alpha diversity. We then identified differentially abundant genera influential in the beta diversity differences among cognitive performance categories. Though correlational, this study provides some of the first evidence of an avian microbiota-gut-brain axis, building foundations for future microbiome research in wild populations and during host development.

Dental health professional recommendation and consumer habits in denture cleansing.

STATEMENT OF PROBLEM: Regular cleaning of dentures is essential to the oral and general health of denture wearers. Only limited systematic data are available on the recommendations that dental health care professionals (DHCPs) make to patients for denture cleaning. Data on denture wearers' cleaning regimens are also lacking. PURPOSE: The purpose of this study was to provide data on recommendations that DHCPs make to patients for denture cleaning and on the cleaning regimens of denture wearers. MATERIAL AND METHODS: DHCPs (n=613), including dentists and hygienists, were surveyed in developed (Japan, USA, Italy) and developing (Brazil, India) countries. A questionnaire assessing a range of denture cleaning recommendations was used. The questions addressed products, frequency, how to use remedies, the suggested dilution and duration of cleansing treatment, the location of dentures while cleaning, and the reasoning behind the recommendation of particular products or modes of treatment. Denture cleansing methods and the routine of denture wearers in developed and developing countries were also surveyed with a questionnaire (n=2862) and a 1-week diary (n=1462). RESULTS: An average of more than 2 treatments was recommended by DHCPs. Specialist denture cleanser tablets, "regular" toothpaste, mouthwash, soap and water, denture paste, foam or liquid denture cleanser, and dishwashing detergents were most commonly recommended; other product recommendations included baking soda, vinegar, salt water, and bleach. More than 10% of DHCPs made no primary recommendation on cleaning. Denture tablets were more commonly recommended in developed countries, whereas toothpaste was the most common recommendation in developing countries. Denture wearers used products and methods similar to those recommended by DHCPs. Toothpaste, water, and mouthwash were used more frequently than denture tablets. More than 75% of denture wearers reported using denture cleanser tablets for more than 5 minutes, whereas soap and toothpaste were typically used for less than 2 minutes. CONCLUSIONS: DHCP recommendations and denture wearer habits are diverse, with no consensus on the most appropriate denture cleaning methods. This reflects a lack of clear, systematic evidence upon which to base recommendations.

Material compatibility and antimicrobial activity of consumer products commonly used to clean dentures.

STATEMENT OF PROBLEM: Regular denture cleaning is essential to good oral health, but only limited evidence is available regarding the effects of common cleaning routines. PURPOSE: The purpose of this in vitro study was to determine the compatibility of denture materials with and the antimicrobial effects of typical cleaning regimens. MATERIAL AND METHODS: The evaluated treatments were derived from a study of dental professional recommendations and consumer habits, including denture cleanser tablets, toothpaste, mouthwash, isopropyl alcohol (IPA), household bleach, soap, and vinegar. The material integrity of denture materials, including polymethyl methacrylate (PMMA) and metals, was evaluated by scanning electron microscopy and profilometry after treatment with laboratory regimens simulating 2 years of typical consumer use. Treatments were also evaluated in a microbial kill time assay against a range of oral microorganisms with typical treatment regimens. RESULTS: Alcohol-based mouthwash and IPA damaged the surface of PMMA, and brushing with toothpaste caused scratching and surface material loss. Bleach caused limited damage to PMMA, but corroded CoCr alloy (pitting) and solder (layer formation). Denture tablets caused little damage to any materials apart from the layer formation on silver solder. Vinegar and soap were compatible with all materials. In antimicrobial assays, bleach gave excellent results, and IPA and mouthwash required concentrated dilutions to be effective. Cleanser tablets were effective at 5 minutes treatment time against all organisms. Toothpaste was effective against bacteria but not Candida albicans. Vinegar, soaps, salt, and sodium bicarbonate were microbially ineffective. CONCLUSIONS: Bleach was highly antimicrobial but incompatible with metal dental prosthesis components. IPA and mouthwash were antimicrobial but damaged PMMA. Specialist denture cleanser tablets gave a good combination of microbial efficacy and reasonable material compatibility.

Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling.

The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms for diagnostic or anti-infective applications, but that can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerization of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms that produced them. This 'bacteria-instructed synthesis' can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the 'instructing' cell types. We further expand on the bacterial redox chemistries to 'click' fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualization of pathogens.

In vitro growth characteristics and volatile sulfur compound production of Solobacterium moorei.

Solobacterium moorei has recently been implicated as a causative agent of halitosis. In vitro experiments to evaluate the role of S. moorei in halitosis have, however, been complicated by a paucity of information on the ideal conditions for culturing this organism. This work aimed to optimize a liquid culture medium for S. moorei, and to determine the growth-curve of the organism. Further, the ability of S. moorei to generate volatile sulfur compounds was investigated and compared quantitatively to other oral anaerobes by an optimized head-space gas chromatography method. Serum-supplementation of standard liquid growth media gave greater growth of S. moorei than non-supplemented broths, with the best medium found to be serum-supplemented tryptone soya broth. S. moorei was able to metabolize cysteine directly to hydrogen sulfide, but was unable to produce methanethiol from methionine. S. moorei produced 2-3 times more hydrogen sulfide (normalized for colony forming units) than Porphyromonas gingivalis and Veillonella dispar, but considerably less than Fusobacterium nucleatum. The study has identified reliable growth conditions for culture of S. moorei, which were employed to show that S. moorei has the requisite biochemistry consistent with a potential role in halitosis.

Diet and the microbial aetiology of dental caries: new paradigms.

The microbial and dietary factors that drive caries have been studied scientifically for 120 years. Frequent and/or excessive sugar (especially sucrose) consumption has been ascribed a central role in caries causation, while Streptococcus mutans appeared to play the key role in metabolising sucrose to produce lactic acid, which can demineralise enamel. Many authors described caries as a transmissible infectious disease. However, more recent data have shifted these paradigms. Streptococcus mutans does not fulfil Koch's postulates - presence of the organism leading to disease, and absence of the organism precluding disease. Furthermore, molecular microbiological methods have shown that, even with a sugar-rich diet, a much broader spectrum of acidogenic microbes is found in dental plaque. While simple sugars can be cariogenic, cooked starches are also now recognised to be a caries threat, especially because such starches, while not 'sticky in the hand', can be highly retentive in the mouth. Metabolism of starch particles can yield a prolonged acidic challenge, especially at retentive, caries-prone sites. These changes in the paradigms of caries aetiology have important implications for caries control strategies. Preventing the transmission of S. mutans will likely be inadequate to prevent caries if a sufficiently carbohydrate-rich diet continues. Similarly, restriction of sucrose intake, although welcome, would be unlikely to be a panacea for caries, especially if frequent starch intake persisted. Instead, approaches to optimise fluoride delivery, to target plaque acidogenicity or acidogenic microbes, to promote plaque alkali generation, to increase salivary flow or replace fermentable carbohydrates with non-fermentable alternatives may be more promising.

Development of a new model system to study microbial colonization on dentures.

PURPOSE: Dentures are often colonized with a variety of microorganisms, including Candida albicans, that contribute to denture stomatitis. Several in vitro models have been previously established to study denture-related microbial colonization and evaluate treatment efficacy of denture cleansers; however, those models typically fail to appreciate the complex topology and heterogeneity of denture surfaces and lack effective ways to accurately measure microbial colonization. The purpose of this study was to study microbial colonization with a new model system based on real dentures, to more realistically mimic in vivo conditions. MATERIALS AND METHODS: Scanning electron microscopy was used to observe topological structures among surfaces from different parts of the denture. Employing C. albicans as a model microorganism, we established microbial colonization on different denture surfaces. Moreover, we applied a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) colorimetric assay to quantify C. albicans colonization on dentures without the necessity of biofilm removal and to evaluate treatment efficacy of denture cleansers. RESULTS: There were significant variations in topological structures among surfaces from different parts of the denture, with the unpolished side having the highest amounts of indentations and pores. The distinct denture surfaces support microbial colonization differently, with the unpolished side containing the highest level of microbial colonization and biofilm formation. Furthermore, the modified MTT colorimetric assay proved to be an accurate assay to measure biofilm formation on dentures and evaluate treatment efficacy of denture cleansers. CONCLUSION: This new denture model system in conjunction with the MTT colorimetric assay is a valuable tool to study denture-related microbiology and treatment approaches.

Antimicrobial effects of o-cymen-5-ol and zinc, alone & in combination in simple solutions and toothpaste formulations.

OBJECTIVES: This study aimed to evaluate antimicrobial effects of an o-cymen-5-ol/zinc system. METHODS: o-Cymen-5-ol and zinc gluconate minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined against Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Fusobacterium nucleatum and Candida albicans. Synergy was investigated by checkerboard MIC/MBC; inhibition of P. gingivalis protease activity and S. mutans glycolysis were investigated. Slurried toothpastes containing the system were assessed in kill time assays against S. mutans and E. coli. RESULTS: o-Cymen-5-ol MIC was between 1.7 mM to 3.4 mM; MBC was 3.4 mM to 6.7 mM. Zinc gluconate MIC was 2.8 mM to 11 mM; MBC was between 11 mM and >44 mM. The two agents in solution showed synergy (FICI≤0.50) against P. gingivalis and F. nucleatum, with MIC of 0.42 mM/0.69 mM for o-cymen-5-ol/zinc gluconate, respectively. Zinc inhibited glycolysis and protease to a greater degree than o-cymen-5-ol; glycolysis inhibition by the two agents was additive. o-Cymen-5-ol/zinc chloride in toothpaste showed greater effects than placebo (120s log10 kill=7.35±0.40 and 4.02±0.40, respectively). CONCLUSIONS: The zinc/o-cymen-5-ol system has direct antimicrobial effects and inhibits oral disease-related processes. Synergistic effects were seen against anaerobes. A system combining o-cymen-5-ol and zinc shows properties desirable for incorporation in toothpastes.

In vitro effects of novel toothpaste actives on components of oral malodour.

OBJECTIVES: To evaluate the efficacy of a novel toothpaste containing zinc ions and o-cymen-5-ol to reduce volatile sulfur compounds (VSCs) in in vitro models and to elucidate the mode of action for any activity observed. METHODS: Three models were employed, a chemical neutralisation model to evaluate the chemical reactivity of toothpaste slurries to VSCs, a biofilm perfusion model to measure activity in an orally-relevant biofilm and a planktonic bacterial model to measure antimicrobial effects. RESULTS: The models showed that zinc ions were able to react chemically with hydrogen sulfide to remove this odorous component of halitotic breath. This activity was confirmed within a complex biofilm model, with over 90% of hydrogen sulfide removed from perfusate gas by a slurry of the test toothpaste. CONCLUSIONS: This work provides a mode of action for the clinically observed reduction in VSCs seen for up to 12 hours post brushing with this novel toothpaste.

Benefits of a silica-based fluoride toothpaste containing o-cymen-5-ol, zinc chloride and sodium fluoride.

Fluoride toothpastes in conjunction with tooth brushing are used to clean teeth, control plaque build-up and for anti-caries benefits. Toothpastes are designed with attractive flavours and appearances to encourage regular prolonged use to maximise these benefits. The incorporation of additional ingredients into toothpaste is a convenient way to provide supplementary protection that fits into people's everyday oral care routine. Such ingredients should not compromise the primary health benefits of toothpaste nor discourage its use. o-Cymen-5-ol and zinc chloride have been incorporated into a sodium fluoride (NaF)/silica toothpaste at 0.1%w/w and 0.6%w/w respectively to provide additional benefits. These include improved gingival health maintenance, in terms of the reduction of plaque, gingival index and bleeding, and an immediate and long lasting reduction in volatile sulfur compounds (VSCs) measured on breath. These benefits can be attributed to the antimicrobial and neutralisation actions of the toothpaste. The use of established fluoride models demonstrated no compromise in NaF bioavailability. The toothpaste was formulated without compromising product aesthetics. The combination of o-cymen-5-ol and zinc chloride in toothpaste gave superior maintenance of gingival health and reduction in malodour related VSCs without compromising the primary health benefits of the toothpaste or diminishing attributes preferred for the product's use.

The cariogenic effect of starch on oral microcosm grown within the dual constant depth film fermenter.

Evidence on the link between starch intake and caries incidence is conflicting, therefore the cariogenicity of starch compared with sucrose was explored using a dual Constant Depth Film Fermenter (dCDFF) biotic model system. Bovine enamel discs were used as a substrate and the dCDFF was inoculated using human saliva. CDFF units were supplemented with artificial saliva growth media at a constant rate to mimic resting salivary flow rate over 14 days. The CDFF units were exposed to different conditions, 2% sucrose or 2% starch 8 times daily and either no additional fluoride or 1450 ppm F- twice daily. Bovine enamel discs were removed at intervals (days 3, 7, 10 and 14) for bacterial enumeration and enamel analysis using Quantitative Light Induced Fluorescence (QLF) and Transverse Microradiography (TMR). Results showed that in the absence of fluoride there was generally no difference in mineral loss between enamel exposed to either sucrose or starch when analysed using TMR and QLF (P > 0.05). In the presence of fluoride by day 14 there was significantly more mineral loss under starch than sucrose when analysed with TMR (P < 0.05). It was confirmed that starch and sucrose are similarly cariogenic within the dCDFF in the absence of fluoride. With the aid of salivary amylase, the bacteria utilise starch to produce an acidic environment similar to that of bacteria exposed to sucrose only. In the presence of fluoride, starch was more cariogenic which may be due to the bacteria producing a more hydrophobic intercellular matrix lowering the penetration of fluoride through the biofilm. This is significant as it indicates that the focus on sugars being the primary cause of caries may need re-evaluating and an increase in focus on carbohydrates is needed as they may be similarly cariogenic as sugars if not more so.

Quantifying the demineralisation of enamel using a hyperspectral camera measuring fluorescence loss.

BACKGROUND: The gold standard for quantifying mineral loss of enamel is transverse microradiography (TMR) and is complimented by the non-destructive quantitative light induced fluorescence (QLF) which measures changes in autofluorescence. Fluorescence loss has been shown to correlate with mineral loss. Building upon the established method, the use of hyperspectral fluorescence imaging (HI) allows the capture of a broader range of wavelengths to quantify fluorescence changes more accurately. METHODS: Bovine Enamel was demineralised within the dual constant depth film fermenter over 14 days and analysed using TMR, QLF and HI. The mineral change values were compared using Pearson's Correlation Coefficient. RESULTS: The analysis showed a statistically significant correlation that was equal between TMR and HI (r = 0.844) and TMR and QLF (r = 0.844), but weaker between QLF and HI (r = 0.811). CONCLUSIONS: The correlations indicate that HI is a promising valid non-destructive method for quantifying mineral loss from bovine enamel that is as accurate as QLF and complements TMR.

Dynamics of microcystins and saxitoxin in the Indian River Lagoon, Florida.

Harmful algal blooms that can produce toxins are common in the Indian River Lagoon (IRL), which covers ~250 km of Florida's east coast. The current study assessed the dynamics of microcystins and saxitoxin in six segments of the IRL: Banana River Lagoon (BRL), Mosquito Lagoon (ML), Northern IRL (NIRL), Central IRL (CIRL), Southern IRL (SIRL), and the St. Lucie Estuary (SLE). Surface water samples (n = 40) collected during the 2018 wet and 2019 dry season were analyzed to determine associations between toxins and temperature, salinity, pH, oxygen saturation, concentrations of dissolved nutrients and chlorophyll-a, presence of biosynthetic genes for toxins, relative abundance of planktonic species, and composition of the microbial community. The potential toxicity of samples was assessed using multiple mammalian cell lines. Enzyme-Linked Immunosorbent Assays were used to determine concentrations of microcystins and saxitoxin. Overall, the microcystins concentration ranged between 0.01-85.70 µg/L, and saxitoxin concentrations ranged between 0.01-2.43 µg/L across the IRL. Microcystins concentrations were 65% below the limit of quantification (0.05 µg/L), and saxitoxin concentrations were 85% below the limit of detection (0.02 µg/L). Microcystins concentrations were higher in the SLE, while saxitoxin was elevated in the NIRL and BRL. Cytotoxicity related to the presence of microcystins was seen in the SLE during the wet season. No significant patterns between cytotoxicity and saxitoxin were identified. Dissolved nutrients were identified as the most highly related parameters, explaining 53% of microcystin and 47% of saxitoxin variability. Multivariate models suggested cyanobacteria, flagellates, ciliates, and diatoms as the subset of microorganisms whose abundances were maximally correlated with saxitoxin and microcystins concentrations. Lastly, biosynthetic genes for microcystins were detected in the SLE and for saxitoxin in the BRL and NIRL. These results highlight the synergistic roles environmental and biological parameters play in influencing the dynamics of toxin production by harmful algae in the IRL.

Defining the sediment prokaryotic communities of the Indian River Lagoon, FL, USA, an Estuary of National Significance.

The Indian River Lagoon, located on the east coast of Florida, USA, is an Estuary of National Significance and an important economic and ecological resource. The Indian River Lagoon faces several environmental pressures, including freshwater discharges through the St. Lucie Estuary; accumulation of anoxic, fine-grained, organic-rich sediment; and metal contamination from agriculture and marinas. Although the Indian River Lagoon has been well-studied, little is known about its microbial communities; thus, a two-year 16S amplicon sequencing study was conducted to assess the spatiotemporal changes of the sediment bacterial and archaeal groups. In general, the Indian River Lagoon exhibited a prokaryotic community that was consistent with other estuarine studies. Statistically different communities were found between the Indian River Lagoon and St. Lucie Estuary due to changes in porewater salinity causing microbes that require salts for growth to be higher in the Indian River Lagoon. The St. Lucie Estuary exhibited more obvious prokaryotic seasonality, such as a higher relative abundance of Betaproteobacteriales in wet season and a higher relative abundance of Flavobacteriales in dry season samples. Distance-based linear models revealed these communities were more affected by changes in total organic matter and copper than changes in temperature. Anaerobic prokaryotes, such as Campylobacterales, were more associated with high total organic matter and copper samples while aerobic prokaryotes, such as Nitrosopumilales, were more associated with low total organic matter and copper samples. This initial study fills the knowledge gap on the Indian River Lagoon bacterial and archaeal communities and serves as important data for future studies to compare to determine possible future changes due to human impacts or environmental changes.

Molecular community profiling of the bacterial microbiota associated with denture-related stomatitis.

Denture-associated stomatitis (DS) affects over two-thirds of denture-wearers. DS presents as erythema of the palatal mucosa in areas where denture-surface associated polymicrobial biofilms containing the fungus Candida albicans exist. The contribution of the oral bacterial microbiota toward the infection is unknown. Therefore, this study characterised the bacterial microbiota of sites within the oral cavity to identify potential associations with occurrence of DS. Denture-wearing patients were recruited (denture stomatitis (DS) n = 8; non-denture stomatitis (NoDS) n = 11) and the oral bacterial microbiota of the tongue, palate and denture-fitting surface was characterised using next-generation sequencing. Operational taxonomic units (OTUs) were identified to bacterial genera and species, and presence/absence and relative abundances were examined. A significant (P = 0.007) decrease in the number of OTUs and thus, diversity of the microbiota was observed in tongue samples of DS patients (vs non-DS). The microbiota of denture-fitting surfaces and palatal mucosae were similar. Large differences in the abundance of bacterial genera and species were observed at each sample site, and unique presence/absence of bacteria was noted. Presence/absence and relative abundance of specific bacteria associated with DS warrants further in vitro and in vivo evaluation, particularly as our previous work has shown C. albicans virulence factor modulation by oral bacteria.

Interkingdom interactions on the denture surface: Implications for oral hygiene.

BACKGROUND: Evidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices. MATERIALS AND METHODS: In vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition. RESULTS: It was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium). CONCLUSIONS: Overall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises.

Denture-associated biofilm infection in three-dimensional oral mucosal tissue models.

PURPOSE: In vitro analyses of virulence, pathogenicity and associated host cell responses are important components in the study of biofilm infections. The Candida-related infection, denture-associated oral candidosis, affects up to 60 % of denture wearers and manifests as inflammation of palatal tissues contacting the denture-fitting surface. Commercially available three-dimensional tissue models can be used to study infection, but their use is limited for many academic research institutions, primarily because of the substantial purchase costs. The aim of this study was to develop and evaluate the use of in vitro tissue models to assess infections by biofilms on acrylic surfaces through tissue damage and Candida albicans virulence gene expression. METHODOLOGY: In vitro models were compared against commercially available tissue equivalents (keratinocyte-only, SkinEthic; full-thickness, MatTek Corporation). An in vitro keratinocyte-only tissue was produced using a cancer-derived cell line, TR146, and a full-thickness model incorporating primary fibroblasts and immortalised normal oral keratinocytes was also generated. The in vitro full-thickness tissues incorporated keratinocytes and fibroblasts, and have potential for future further development and analysis. RESULTS: Following polymicrobial infection with biofilms on acrylic surfaces, both in-house developed models were shown to provide equivalent results to the SkinEthic and MatTek models in terms of tissue damage: a significant (P<0.05) increase in LDH activity for mixed species biofilms compared to uninfected control, and no significant difference (P>0.05) in the expression of most C. albicans virulence genes when comparing tissue models of the same type. CONCLUSION: Our results confirm the feasibility and suitability of using these alternative in vitro tissue models for such analyses.

Viable Compositional Analysis of an Eleven Species Oral Polymicrobial Biofilm.

PURPOSE: Polymicrobial biofilms are abundant in clinical disease, particularly within the oral cavity. Creating complex biofilm models that recapitulate the polymicrobiality of oral disease are important in the development of new chemotherapeutic agents. In order to do this accurately we require the ability to undertake compositional analysis, in addition to determine individual cell viability, which is difficult using conventional microbiology. The aim of this study was to develop a defined multispecies denture biofilm model in vitro, and to assess viable compositional analysis following defined oral hygiene regimens. METHODS: An in vitro multispecies denture biofilm containing various oral commensal and pathogenic bacteria and yeast was created on poly (methyl methacrylate) (PMMA). Denture hygiene regimens tested against the biofilm model included brushing only, denture cleansing only and combinational brushing and denture cleansing. Biofilm composition and viability were assessed by culture (CFU) and molecular (qPCR) methodologies. Scanning electron microscopy and confocal laser scanning microscopy were also employed to visualize changes in denture biofilms following treatment. RESULTS: Combinational treatment of brushing and denture cleansing had the greatest impact on multispecies denture biofilms, reducing the number of live cells by more than 2 logs, and altering the overall composition in favor of streptococci. This was even more evident during the sequential testing, whereby daily sequential treatment reduced the total and live number of bacteria and yeast more than those treated intermittently. Bacteria and yeast remaining following treatment tended to aggregate in the pores of the PMMA, proving more difficult to fully eradicate the biofilm. CONCLUSIONS: Overall, we are the first to develop a method to enable viable compositional analysis of an 11 species denture biofilm following chemotherapeutic challenge. We were able to demonstrate viable cell reduction and changes in population dynamics following evaluation of various denture cleansing regimens. Specifically, it was demonstrated that daily combinational treatment of brushing and cleansing proved to be the most advantageous denture hygiene regimen, however, residual organisms still remained within the pores of PMMA surface, which could act as a reservoir for further biofilm regrowth. We have identified an industry need for denture cleansing agents with the capacity to penetrate these pores and disaggregate these complex biofilm consortia.