Wander José da Silva

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.

Improvement of XTT assay performance for studies involving Candida albicans biofilms

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

Efficacy of denture cleansers on Candida spp. biofilm formed on polyamide and polymethyl methacrylate resins

STATEMENT OF PROBLEM Although new materials have emerged as options to fabricate removable dental prostheses, the development of Candida biofilms on these materials and the effectiveness of methods to control these pathogenic biofilms are poorly understood. PURPOSE The purpose of this study was to evaluate the efficacy of denture cleansers on Candida single- and dual-species biofilms formed on polyamide resin. MATERIAL AND METHODS Polymethyl methacrylate (PMMA) resin (Acron MC) and polyamide resin (Flexite M.P.) specimens (n=116) were prepared, and their surface roughness was standardized (0.34 ±0.02 μm). Surface free energy (SFE) was measured for some specimens (n=20 per resin), while the remainder were randomly divided by lottery into 24 groups (n=8) for biofilm assay. C. albicans and/or C. glabrata biofilm was formed for 72 hours, and then specimens were treated with an enzymatic cleanser solution (Polident 3 Minutes), a cleanser solution (Corega Tabs), or 0.5% sodium hypochlorite (NaOCl) solution. Water served as the negative control. Remaining adherent microorganisms were removed from the treated specimens by ultrasonic waves, and colony-forming units (CFU) of each microorganism were calculated. SFE data were analyzed by 1-way ANOVA, and Candida species data were analyzed by 3-way ANOVA followed by the Tukey-Kramer test (P=.05). RESULTS All tested biofilms displayed significantly higher growth on polyamide resin (P<.001), which presented the lowest SFE. Denture cleansers significantly decreased Candida levels; however, the 0.5% NaOCl solution was the only effective cleanser. C. glabrata revealed significantly higher CFU counts under all experimental conditions (P<.001). CONCLUSIONS The highest Candida spp. biofilm growth was shown to occur on polyamide resin when compared with PMMA. Denture cleansers were able to remove Candida spp. biofilm formed on both denture base resins.

Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria

This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.

Preload loss and bacterial penetration on different implant-abutment connection systems

Preload loss can favor the occurrence of implant-abutment interface misfit, and bacterial colonization at this interface may lead to implant failure. The aim of this study was to evaluate the preload loss and bacterial penetration through the implant-abutment interface of conical and external hexagon connection systems subjected to thermal cycling and mechanical fatigue (TM). Four different implant-abutment connection systems were evaluated (n=6): external hexagon with universal post, Morse taper with universal post, Morse taper with universal post through bolt, and locking taper with standard abutment. The assemblies (implant-abutment) were subjected to a thermal cycling regimen (1,000 cycles of 5 degrees C and 55 degrees C) and to mechanical fatigue (1.0 million cycles, 1.0 Hz, 120 N). The assemblies were immersed in Tryptic Soy + Yeast Extract broth containing Streptococcus sanguinis and incubated at 37 degrees C and 10% CO(2) for 72 h. Detorque values were recorded. The bacterial penetration was assessed and the abutments were observed by scanning electron microscopy. The preload data were analyzed statistically by two-way ANOVA and Tukeys test at 5% significance level. All screw abutment systems showed significantly higher (p<0.05) detorque values when subjected to TM and all conical systems presented bacterial penetration. The results show no relationship between the preload loss and the bacterial penetration.

Long-term efficacy of denture cleansers in preventing Candida spp. biofilm recolonization on liner surface

This study evaluated the long-term efficacy of denture cleansers against Candida spp. biofilm recolonization on liner surface. Specimens were fabricated of a poly(methyl methacrylate)-based denture liner and had their surface roughness evaluated at baseline and after cleansing treatments. C. albicans or C. glabrata biofilms were formed on liner surface for 48 h, and then the specimens were randomly assigned to one of cleaning treatments: two alkaline peroxides (soaking for 3 or 15 min), 0.5% sodium hypochlorite (10 min) or distilled water (control; 15 min). After the treatments, the specimens were sonicated to disrupt the biofilm, and residual cells were counted (cell/mL). Long-term effectiveness of the cleaning processes was determined by submitting a set of cleaned specimens to biofilm growth conditions for 48 h followed by estimation of cell counts. The topography of specimens after cleaning treatments was analyzed by SEM. Data were analyzed by ANOVA and Tukeys test (α; = 0.05). Results of cell count estimation showed significant differences in cleanliness among the treatments (p < 0.001), and it could be observed by SEM. However, no significant difference (p > 0.05) was observed among the Candida species regarding the recolonization condition. Alkaline denture cleansers showed similar cleaning performance and both differed from the control (p < 0.001). Sodium hypochlorite was the only treatment that removed biofilm efficiently, since no viable cells were found after its use. In conclusion, alkaline peroxide denture cleansers were not effective in removing Candida spp. biofilm from denture liner surfaces and preventing biofilm recolonization.

Bioactivity and architecture of Candida albicans biofilms developed on poly(methyl methacrylate) resin surface

The aim of this study was to evaluate the bioactivity and architecture of Candida albicans biofilms developed on the surface of poly(methyl methacrylate) (PMMA) resin. To do this, surface roughness (SR) and surface free energy of PMMA specimens were measured. Next, the biofilms of two different C. albicans strains (ATCC 90028 and SC5314) were allowed to develop on the PMMA surface and evaluated at 24, 48, and 72 h after adhesion. The bioactivity of the biofilms was measured by the XTT reduction assay. Biofilm topography was evaluated by scanning electron microscopy. Confocal microscopy was used to evaluate the architectural properties of bio-volume, average thickness, biofilm roughness, surface area/volume ratio and the proportion of live/dead cells in the different biofilm development stages. SR and SFE had no influence on biofilm development. Each strain exhibited a different biofilm activity (P < 0.001). Confocal images showed different architectures for the different biofilm development stages. We conclude that the main differences detected in biofilm bioactivity and architecture were related to the characteristics of each C. albicans strain and to biofilm development time.

Effects of Undecylenic Acid Released from Denture Liner on Candida Biofilms

Denture liners (DL) are easily colonized by Candida spp. In an attempt to prevent biofilm colonization, manufacturers have incorporated undecylenic acid (UDA) into DL. In this in vitro study, the effects of UDA released from DL on Candida biofilms were investigated. The concentrations of UDA released from commercial DL were determined by gas chromatography–mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) and minimum fungistatic concentration (MFC) tests were performed for C. albicans or C. glabrata, with UDA for comparison with the concentrations released from DL. Specimens of DL with (experimental group) and without UDA (control group) were fabricated, and Candida biofilms were developed on DL surfaces. Biofilms were evaluated by cell counts, metabolic activity, structure, and secretion of proteinase or phospholipase. The concentrations of UDA released were within the MIC and MFC ranges. In the presence of UDA, C. albicans biofilms were thinner and had lower numbers of viable and active cells, although no significant enzymatic changes were observed relative to the control group (p > 0.05). In contrast, C. glabrata biofilms exhibited higher cell counts and greater metabolic activity and also increased proteinase activity in the presence of UDA relative to the control group (p < 0.05). Overall, UDA did not prevent Candida biofilm formation.

Evaluation of sodium hypochlorite as a denture cleanser: a clinical study.

OBJECTIVES To evaluate the effect of sodium hypochlorite (NaOCl) on biofilms, colour stability (ΔE) and surface roughness (Ra) of complete dentures and patient acceptability. BACKGROUND Denture cleansers should be able to reduce the accumulation of biofilms without affecting the acrylic resin properties. Patient satisfaction is important to maintaining their daily use. MATERIALS AND METHODS Fifteen participants were instructed to keep their dentures immersed daily in a 0.5% NaOCl solution for 3 min over 90 days. Swabs were taken from dentures and inoculated on CHROMagar and blood agar. The number of colony-forming units (cfu) was counted after a 48-h incubation period. ΔE was assessed using the CIE L*a*b* system. Ra was measured using a profilometer. Patient acceptability was checked based on their degree of satisfaction. Cell counts, ΔE and Ra were analysed using anova, Friedmans and Kruskal-Wallis tests, respectively (α = 0.05). RESULTS A significant reduction in the total number of microorganisms (p = 0.001) and Candida spp. was noticed. No significant differences were found for ΔE (p = 0.68) and for Ra (p = 0.47). The level of the patient satisfaction increased throughout the follow-up period. CONCLUSION The 0.5% NaOCl solution was effective in reducing microorganisms without significant changes in colour or roughness of denture resin. The participants reported satisfaction with the cleaning results.

Dietary Carbohydrates Modulate Candida albicans Biofilm Development on the Denture Surface

The purpose of this study was to investigate whether dietary carbohydrates can modulate the development of Candida albicans biofilms on the denture material surface. Poly (methyl methacrylate) acrylic resin discs were fabricated and had their surface roughness measured. Biofilms of C. albicans ATCC 90028 were developed on saliva-coated specimens in culture medium without (control) or with carbohydrate supplementation by starch, starch+sucrose, glucose, or sucrose for 72 h. The cell count, metabolic activity, biovolume, average thickness, and roughness coefficient were evaluated at the adhesion phase (1.5 h) and after 24, 48, and 72 h. The secretion of proteinases and phospholipases, cell surface energy, and production of extra/intracellular polysaccharides were analyzed after 72 h of biofilm development. Data were analyzed by one- and two-way ANOVA followed by Tukey’s test at 5% significance level. In the early stages of colonization (adhesion and 24 h), the glucose group showed the highest cell counts and metabolic activity among the groups (p<0.05). After maturation (48 and 72 h), biofilms exposed to glucose, sucrose, or starch+sucrose showed higher cell counts and metabolic activity than the control and starch groups (p<0.001). Compared to the control group, biofilms developed on starch or starch+sucrose had more proteinase activity (p<0.001), whereas biofilms developed on glucose or sucrose had more phospholipase activity (p<0.05). Exposure to starch+sucrose increased the production of extracellular and intracellular polysaccharides (p<0.05). Biofilms developed on starch or without carbohydrate supplementation presented cells with more hydrophobic behavior compared to the other groups. Confocal images showed hyphae forms on biofilms exposed to starch or starch+sucrose. Within the conditions studied, it can be concluded that dietary carbohydrates can modulate biofilm development on the denture surface by affecting virulence factors and structural features.