Disruption and bactericidal indices depicted in polygonal graphs to show multiple outcome effects of root canal irrigant supplements on single- and dual-species biofilms.

OBJECTIVES: The aims of this study were to (1) investigate the relative time-dependent disruption and bactericidal effects of detergent-type surfactants on single- or dual-species biofilms of root canal isolates and (2) to examine the utility of polygonal graphs for depiction of biofilm disruption and cell killing. MATERIALS AND METHODS: Single-species biofilms of Streptococcus sanguinis, Enterococcus faecalis, Fusobacterium nucleatum and Porphyromonas gingivalis were grown on nitro-cellulose membranes for 72 h and immersed in Tween®80, cetyltrimethylammoniumbromide (CTAB), and sodium dodecyl sulphate (SDS) for 1-, 5- or 10-min (n = 3 per test). The number of viable and non-viable bacteria "disrupted" from the biofilm and those "remaining-attached" was determined using a viability stain in conjunction with fluorescence microscopy. The data were analysed using non-parametric Kruskal-Wallis test with 5% significance level. RESULTS: Gram-negative obligate anaerobes were more susceptible to cell removal than gram-positive facultative anaerobes. The majority of cells were disrupted after 1-min of exposure; however, the extent varied according to the agent and species. CTAB and SDS were more effective than Tween 80™ at disrupting biofilms and killing cells but all agents failed to achieve 100% disruption/kill. CONCLUSIONS: Biofilm disruption and cell viability were influenced by the species, the test agent and the duration of exposure. CTAB and SDS were more effective in biofilm disruption than Tween 80™. Graphical depiction of biofilm disruption- and viability-outcomes provides an alternative means of simultaneously visualising and analysing relative efficacy in different domains. CLINICAL RELEVANCE: Surfactants were not as effective at biofilm disruption as NaOCl but may be added to other non-disruptive antibacterial agents to enhance this property.

Early Adhesion of Candida albicans onto Dental Acrylic Surfaces.

Denture-associated stomatitis is a common candidal infection that may give rise to painful oral symptoms, as well as be a reservoir for infection at other sites of the body. As poly (methyl methacrylate) (PMMA) remains the main material employed in the fabrication of dentures, the aim of this research was to evaluate the adhesion of Candida albicans cells onto PMMA surfaces by employing an atomic force microscopy (AFM) single-cell force spectroscopy (SCFS) technique. For experiments, tipless AFM cantilevers were functionalized with PMMA microspheres and probed against C. albicans cells immobilized onto biopolymer-coated substrates. Both a laboratory strain and a clinical isolate of C. albicans were used for SCFS experiments. Scanning electron microscopy (SEM) and AFM imaging of C. albicans confirmed the polymorphic behavior of both strains, which was dependent on growth culture conditions. AFM force-spectroscopy results showed that the adhesion of C. albicans to PMMA is morphology dependent, as hyphal tubes had increased adhesion compared with yeast cells ( P < 0.05). C. albicans budding mother cells were found to be nonadherent, which contrasts with the increased adhesion observed in the tube region. Comparison between strains demonstrated increased adhesion forces for a clinical isolate compared with the lab strain. The clinical isolate also had increased survival in blood and reduced sensitivity to complement opsonization, providing additional evidence of strain-dependent differences in Candida-host interactions that may affect virulence. In conclusion, PMMA-modified AFM probes have shown to be a reliable technique to characterize the adhesion of C. albicans to acrylic surfaces.

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.

Physical disruption of oral biofilms by sodium bicarbonate: an in vitro study.

OBJECTIVES: Sodium bicarbonate has been shown clinically to be efficacious at removing dental plaque; however, its effect of mechanism against biofilms has not been evaluated in vitro. Here, we used a well-established in vitro plaque biofilm model to investigate the disruption of dental plaque biofilms. METHODS: Biofilms were grown in a constant depth film fermentor for up to 14 days. The fermentor was inoculated with pooled human saliva and growth maintained with artificial saliva. After various time points, replicate biofilms were removed and subjected to treatment at varying concentrations of sodium bicarbonate. Disruption of the plaque was assessed by viable counts and microscopy. RESULTS: The viable count results showed that younger biofilms were less susceptible to the action of sodium bicarbonate; however, biofilms of 7 days and older were increasingly susceptible to the material with the oldest biofilms being the most susceptible. Sixty-seven percentage of sodium bicarbonate slurry was able to reduce the number of organisms present by approx. 3 log10 . These quantitative data were corroborated qualitatively with both confocal and electron microscopy, which both showed substantial qualitative removal of mature biofilms. CONCLUSIONS: The results from this study have shown that sodium bicarbonate is able to disrupt mature dental plaque grown in vitro and that its reported efficacy in maintaining oral hygiene may be related to this key factor.

Anti-biofilm activity of zinc oxide and hydroxyapatite nanoparticles as dental implant coating materials.

OBJECTIVES: Dental implants are prone to failure as a result of bacterial biofilm accumulation. Such biofilms are often resistant to traditional antimicrobials and the use of nanoparticles as implant coatings may offer a means to control infection over a prolonged period. The objective of this study was to determine the antibiofilm activity of nanoparticulate coated titanium (Ti) discs using a film fermenter based system. METHODS: Metal oxide nanoparticles of zinc oxide (nZnO), hydroxyapatite (nHA) and a combination (nZnO+nHA) were coated using electrohydrodynamic deposition onto Ti discs. Using human saliva as an inoculum, biofilms were grown on coated discs for 96 h in a constant depth film fermenter under aerobic conditions with artificial saliva and peri-implant sulcular fluid. Viability assays and biofilm thickness measurements were used to assess antimicrobial activity. RESULTS: Following 96 h, reduced numbers of facultatively anaerobic and Streptococcus spp. on all three nano-coated surfaces were demonstrated. The proportion of non-viable microorganisms was shown to be higher on nZnO and composite (nZnO+nHA) coated surfaces at 96 h compared with nHA coated and uncoated titanium. Biofilm thickness comparison also demonstrated that nZnO and composite coatings to be the most effective. CONCLUSIONS: The findings support the use of coating Ti dental implant surfaces with nZnO to provide an antimicrobial function. CLINICAL SIGNIFICANCE: Current forms of treatment for implant associated infection are often inadequate and may result in chronic infection requiring implant removal and resective/regenerative procedures to restore and reshape supporting tissue. The use of metal oxide nanoparticles to coat implants could provide osteoconductive and antimicrobial functionalities to prevent failure.

Evaluation of plant and fungal extracts for their potential antigingivitis and anticaries activity.

The link between diet and health has lead to the promotion of functional foods which can enhance health. In this study, the oral health benefits of a number of food homogenates and high molecular mass and low molecular mass fractions were investigated. A comprehensive range of assays were performed to assess the action of these foods on the development of gingivitis and caries using bacterial species associated with these diseases. Both antigingivitis and anticaries effects were investigated by assays examining the prevention of biofilm formation and coaggregation, disruption of preexisting biofilms, and the foods' antibacterial effects. Assays investigating interactions with gingival epithelial cells and cytokine production were carried out to assess the foods' anti- gingivitis properties. Anti-caries properties such as interactions with hydroxyapatite, disruption of signal transduction, and the inhibition of acid production were investigated. The mushroom and chicory homogenates and low molecular mass fractions show promise as anti-caries and anti-gingivitis agents, and further testing and clinical trials will need to be performed to evaluate their true effectiveness in humans.

The prevalence of opportunistic pathogens associated with intraoral implants.

AIMS: The aim of this study was to determine the prevalence and proportions of opportunistic pathogens harboured on orthodontic retainers. METHODS AND RESULTS: First, Staphylococcus spp. and Candida spp. were isolated from the retainer's inner surface and from other mucosal surfaces of the subject's mouth by routine bacterial culture. The prevalence and proportions of these micro-organisms on retainers was compared in different areas of the mouth within a group of retainer wearers, and mucosal carriage was compared to a group of nonretainer wearers. Staphylococcus spp. were isolated from 50% of the retainers and comprised on average 8·4% of the viable microbiota. Candida spp. comprised 0·13% of the viable microbiota and were recovered from 66·7% of the retainers. Neither genus was isolated from nonretainer wearers. Second, the two most commonly worn retainers manufactured from different materials were sampled; again Staphylococcus spp. and Candida spp. were recovered; however, no statistical differences were observed between the devices. CONCLUSIONS: Opportunistic, nonoral, pathogenic micro-organisms were recovered from orthodontic retainers. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible that an orthodontic retainer could be a reservoir for opportunistic pathogens and act as a source of cross-, self- and re-infection.

Association between interleukin-6 -174 polymorphism and Aggregatibacter actinomycetemcomitans in chronic periodontitis.

BACKGROUND: We previously demonstrated an association between interleukin-6 (IL-6) polymorphisms and the detection of periodontopathogenic bacteria in aggressive and chronic periodontitis on a patient basis (pooled samples). The aim of this study is to comprehensively analyze the relative contribution of IL-6 genetic factors and local (tooth and site) factors on the detection of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis in patients with chronic periodontitis. METHODS: Forty patients diagnosed with severe chronic periodontitis had subgingival samples harvested from four sites (the deepest probing depths in each quadrant). All subjects had a blood sample taken to genotype the single nucleotide polymorphism at position -174 in the IL-6 gene promoter. Nested polymerase chain reaction analysis was performed on subgingival plaque samples for the detection of A. actinomycetemcomitans and P. gingivalis in each of the 160 sampled sites. The association between IL-6 -174 genotypes and bacterial detection was investigated with multilevel analysis accounting for the clustering of multiple sites analyzed within patients. RESULTS: Respectively 60%, 62%, and 40% of subjects had A. actinomycetemcomitans, P. gingivalis, and both bacteria concomitantly detected in ≥1 site. The multilevel analysis confirmed that, among all site and subject factors, IL-6 -174 G homozygosity showed the strongest association with the presence of A. actinomycetemcomitans in all subjects and in the subgroup of whites only. No associations were detected for P. gingivalis. CONCLUSION: This study provides further confirmatory evidence that the detection of A. actinomycetemcomitans is associated with IL-6 genetic factors in chronic periodontitis cases.

Controlled delivery of antimicrobial gallium ions from phosphate-based glasses.

Gallium-doped phosphate-based glasses (PBGs) have been recently shown to have antibacterial activity. However, the delivery of gallium ions from these glasses can be improved by altering the calcium ion concentration to control the degradation rate of the glasses. In the present study, the effect of increasing calcium content in novel gallium (Ga2O3)-doped PBGs on the susceptibility of Pseudomonas aeruginosa is examined. The lack of new antibiotics in development makes gallium-doped PBG potentially a highly promising new therapeutic agent. The results show that an increase in calcium content (14, 15 and 16 mol.% CaO) cause a decrease in degradation rate (17.6, 13.5 and 7.3 microg mm(-2) h(-1)), gallium ion release and antimicrobial activity against planktonic P. aeruginosa. The most potent glass composition (containing 14 mol.% CaO) was then evaluated for its ability to prevent the growth of biofilms of P. aeruginosa. Gallium release was found to reduce biofilm growth of P. aeruginosa with a maximum effect (0.86 log(10) CFU reduction compared to Ga2O3-free glasses) after 48 h. Analysis of the biofilms by confocal microscopy confirmed the anti-biofilm effect of these glasses as it showed both viable and non-viable bacteria on the glass surface. Results of the solubility and ion release studies show that this glass system is suitable for controlled delivery of Ga3+. 71Ga NMR and Ga K-edge XANES measurements indicate that the gallium is octahedrally coordinated by oxygen atoms in all samples. The results presented here suggest that PBGs may be useful in controlled drug delivery applications, to deliver gallium ions in order to prevent infections due to P. aeruginosa biofilms.

Assessment of antimicrobial microspheres as a prospective novel treatment targeted towards the repair of perianal fistulae.

BACKGROUND: None of the proposed materials tested for the management of perianal fistulae has proven to be a definitive treatment. AIM: To assess a new repair scaffold and drug delivery device conceived to target perianal fistula repair. METHODS: Poly(D,L-lactide-co-glycolide) porous microspheres containing either antibacterial silver-releasing degradable phosphate glass or metronidazole were prepared using thermally induced phase separation. RESULTS: Ion- and drug-release profiling of the microspheres revealed continued release of silver ions from microspheres filled with silver-doped phosphate glass and high encapsulation efficiency for metronidazole [78% and 82% for microspheres loaded with 2.5% and 1.3% (w/w), respectively]. Microbicidal activity was confirmed by growth inhibition of bacterial species (Staphylococcus aureus, Escherichia coli and Bacteroides fragilis), which characteristically dominate the colonization of perianal fistula tracts. Microspheres containing >3 mol% silver or metronidazole resulted in strong bacterial inhibition/kill against B. fragilis; the presence of one sphere containing >3 mol% silver had a potent inhibitory effect against all the microbes studied. Microspheres became rapidly integrated with host tissue following subcutaneous implantation into a rodent wound model. CONCLUSION: The study demonstrates a novel scaffold for guided tissue regeneration providing local release of antimicrobial agents sufficient to counter bacterial colonization and warrants further investigation.

Fluoroquinolone-resistant mutants of Burkholderia cepacia.

Fluoroquinolone-resistant Burkholderia cepacia mutants were selected on ciprofloxacin. The rate of mutation in gyrA was estimated to be 9.6 x 10(-11) mutations per division. Mutations in gyrA conferred 12- to 64-fold increases in MIC, and an additional parC mutation conferred a large increase in MIC (>256-fold). Growth rate, biofilm formation, and survival in water and during drying were not impaired in strains containing single gyrA mutations. Double mutants were impaired only in growth rate (0.85, relative to the susceptible parent).

Use of quantitative PCR and culture methods to characterize ecological flux in bacterial biofilms.

An in vitro model of supragingival plaque associated with gingivitis was characterized by traditional culture techniques, comparative 16S rRNA gene sequencing of isolates, and quantitative PCR (QPCR). Actinomyces naeslundii, Prevotella spp., and Porphyromonas gingivalis increased under conditions emulating gingivitis. Gram-negative species and total bacteria were dramatically underestimated by culture compared to the estimates obtained by QPCR.

Potential role of Veillonella spp. as a reservoir of transferable tetracycline resistance in the oral cavity.

Twelve out of 96 Veillonella spp. isolated from oral samples harbored tetracycline resistance genes. The most common resistance gene was tet(M). A tet(M)-positive Veillonella dispar strain was shown to transfer a Tn916-like element to four Streptococcus spp. by conjugation at a frequency of 5.2 x 10(-6) to 4.5 x 10(-5) per recipient.

Modeling shifts in microbial populations associated with health or disease.

Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual-species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents.

Characterisation of antibacterial copper releasing degradable phosphate glass fibres.

Phosphate-based glass fibres (PGF) of the general formula Na(2)O-CaO-P(2)O(5) are degradable in an aqueous environment, and therefore can function as antibacterial delivery systems through the inclusion of ions such as copper. In this study, PGF with varying amounts of copper oxide (CuO) were developed for potential uses in wound healing applications. PGF with 0, 1, 5 and 10 mol% CuO were produced with different diameters and characterised in terms of structural and antibacterial properties. The effect of CuO and fibre pulling speed on the glass properties were investigated using rapid differential scanning calorimetry, differential thermal analysis and X-ray diffraction. The effect of two fibre diameters on short-term (3 h) attachment and killing against Staphylococcus epidermidis were investigated and were related to their rate of degradation in deionised water, as well as copper ion release measured using ion chromatography. Thermal analysis showed that there was a significant increase in the PGF glass transition temperature as the CuO content increased. There was a significant decrease in the rate of degradation with increasing CuO content and an increase in fibre diameter. Over 6 h, both the amount and rate of copper ions released increased with CuO content, as well as a reduction in fibre diameter thus increasing the surface area to volume ratio. There was a decrease in the number of viable staphylococci both attached to the CuO-containing fibres and in the surrounding environment.

Isolation of bacteriophages from the oral cavity.

AIMS: To isolate bacteriophages lytic for oral pathogens from human saliva, dental plaque and mature biofilms constituted from saliva-derived bacteria. METHODS AND RESULTS: Saliva and dental plaque samples from healthy volunteers and from patients with gingivitis and periodontitis were examined for the presence of lytic bacteriophage using a panel of oral pathogens and bacteria isolated from the samples. Samples were also enriched for bacteriophage using static culture techniques and mature biofilms. A limited number of samples contained bacteriophage particles that were visualized using electron microscopy. Cultures yielded phage infecting non-oral bacteria (Proteus mirabilis) but no bacteriophage specific for recognized oral pathogens were found. Some micro-organisms from the oral microflora elaborated antibacterial substances that inhibited growth of other residents of the oral cavity. CONCLUSIONS: Unlike other ecosystems, the composition of the oral cavity does not appear to be heavily influenced by interactions between bacteriophages and their hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage for control of oral infections may need to be obtained from other sources. Antibacterial substances derived from some members of the oral microflora warrant investigation as potential antibiotics.

Modelling oral malodour in a longitudinal study.

The aim of this study was to develop an in vitro model of the tongue microflora in order to assess anti-malodour compounds before clinical evaluation. Biofilms, derived from the tongue microflora, were grown in a constant depth film fermentor (CDFF) with nutrients supplied in the form of mucin- and serum-containing artificial saliva. Differential agars and a halimeter were used to determine the bacterial microflora and production of volatile sulphur compounds (VSCs), respectively. The resulting biofilms had a bacterial population which contained, on average, 29% streptococci, 48% Gram-negative anaerobes and 2.5% with an H2S-producing phenotype. When the biofilms were pulsed with either chlorhexidine or zinc acetate there was a reduction in the number of H2S-producing bacteria, however these counts subsequently recovered as pulsing continued. The generation of VSCs was correlated to the viable counts of the H2S-producing bacteria. By pulsing with anti-malodour compounds over time we observed a reduction in the quantity of VSCs produced and a change in the composition of the plaque to one which contained fewer H2S-producing bacteria.

The effect of orthodontic bonding materials on dental plaque accumulation and composition in vitro.

The aim of this study was to investigate the accumulation and composition of microcosm dental plaque on different orthodontic bonding materials using an in vitro model. Microcosm plaques were grown on discs of a range of bonding materials in a constant depth film fermentor. The biofilms were derived from human saliva and supplied with artificial saliva as a source of nutrients. The number of viable bacteria in the biofilms was determined and the streptococci present were identified to species level. The results showed that there was no significant difference in bacterial accumulation between different bonding materials, however, biofilms grown on materials which were fluoride releasing, did not contain Streptococcus mutans. This in vitro study has shown that the use of fluoride-releasing bonding materials may support the growth of supragingival plaque, which does not contain S. mutans.

Characterization of in vitro oral bacterial biofilms by traditional and molecular methods.

The aim of this study was to compare culture-based bacterial isolation methods with direct amplification and cloning of 16S rRNA genes from oral biofilms grown in an in vitro model. The model used was a constant depth film fermentor which was inoculated with pooled human saliva. The use of culture techniques and cloning resulted in the identification of 36 different bacterial species from the saliva inoculum and from the biofilms. Of these, only five were detected solely by molecular methods. Three taxa were detected which, according to the databases, were unidentified. Using the molecular methods of detection, differences in the number of species observed were found using different 16S rRNA gene primers and numbers of PCR cycles. We have shown that microcosm supragingival plaque biofilms grown in a fermentor consisted of a community most of the members of which could be cultivated on laboratory media.