In vitro synergism between berberine and miconazole against planktonic and biofilm Candida cultures

Objectives

To investigate the antimycotic activity of the plant alkaloid berberine (BBR), alone and in combination with antifungal azoles, against planktonic and biofilm Candida cultures.

Design

The minimum inhibitory concentrations (MICs) of BBR, miconazole (MCZ), and fluconazole (FLC) towards Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis were determined by a microdilution method. For C. albicans, the synergistic effects of BBR combined with MCZ or FLC were examined in a paper disc agar diffusion assay and checkerboard microdilution assay. The effect of the BBR/MCZ combination was further investigated in a C. albicans biofilm formation model with a dual-chamber flow cell. The effect on metabolic activity of biofilm cells was established using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)/menadione.

Results

Berberine inhibited the growth of various Candida species (MICs 0.98–31.25 mg/L) in the following order of susceptibility: C. krusei > C. kefyr > C. glabrata > C. tropicalis > C. parapsilosis and C. albicans. Synergism between BBR and MCZ or FLC was observed in the disc diffusion assay as well as in suspension showing an FIC index <0.5 (∑FIC = 0.19). Whilst neither BBR (16 mg/L) nor MCZ (0.8 mg/L) alone significantly inhibited biofilm formation of C. albicans, their combination reduced biofilm formation by >91% after 24 h, as established from the reduction in surface area coverage (P < 0.01). The BBR/MCZ combination also exhibited synergy against the metabolic activity of pre-formed C. albicans biofilms in polystyrene microtiter plates (∑FIC = 0.25).

Conclusion

Berberine exhibits synergistic effects with commonly used antimycotic drugs against C. albicans, either in planktonic or in biofilm growth phases.     

Genetic diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brazilian periodontal patients

Objective

Mucosal surfaces are the primary oral reservoirs of Candida species, but these species can also be found in subgingival biofilm. The present study investigated the genetic diversity and production of exoenzymes of C. albicans and C. dubliniensis isolated from the oral cavity of systemically healthy patients with periodontitis.

Design

Fifty-three patients were analysed. Samples were collected from three oral cavity sites (periodontal pocket, gingival sulci and oral mucosa), plated and, after isolation, suspect strains of C. albicans and C. dubliniensis were identified by PCR. The genetic diversity of the isolates was evaluated by RAPD and the activities of the secreted aspartyl proteinases and phospholipases were evaluated by the agar plate method.

Results

Twenty-one patients showed positive results for Candida spp. There were no statistically significant differences between genders, or between sites. C. albicans was the most frequently found specie, while C. dubliniensis was isolated from the periodontal pocket of only one patient. Sixteen genotypes were detected among the C. albicans isolates, and one among the C. dubliniensis isolates. The similarity coefficient (S_SM) values among the C. albicans genotypes ranged from 0.684 to 1.0 with an average of 0.905 ± 0.074. All isolates produced high levels of Saps and most of them produced high levels of phospholipases. No relationship was found between the genotypes and the pattern of enzymatic production. There was no association between specific genotypes and their site of isolation.

Conclusions

The results of the present study suggest that genetically homogeneous strains of C. albicans are present in the oral cavity of patients with periodontitis and that these strains are capable of producing high levels of exoenzyme.     

Susceptibility of Candida albicans and Candida dubliniensis to erythrosine- and LED-mediated photodynamic therapy

The effect of erythrosine- and LED-mediated photodynamic therapy (PDT) on planktonic cultures and biofilms of Candida albicans and Candida dubliniensis was evaluated. Planktonic cultures of standardized suspensions (10⁶ cells/mL) of C. albicans and C. dubliniensis were treated with erythrosine concentrations of 0.39–200 μM and LEDs in a 96-well microtiter plate. Biofilms formed by C. albicans and C. dubliniensis in the bottom of a 96-well microtiter plate were treated with 400 μM erythrosine and LEDs. After PDT, the biofilms were analysed by scanning electron microscopy (SEM). The antimicrobial effect of PDT against planktonic cultures and biofilms was verified by counting colony-forming units (CFU/mL), and the data were submitted to analysis of variance and the Tukey test (P < 0.05). C. albicans and C. dubliniensis were not detectable after PDT of planktonic cultures with erythrosine concentrations of 3.12 μM or higher. The CFU/mL values obtained from biofilms were reduced 0.74 log₁₀ for C. albicans and 0.21 log₁₀ for C. dubliniensis. SEM revealed a decrease in the quantity of yeasts and hyphae in the biofilm after PDT. In conclusion, C. albicans and C. dubliniensis were susceptible to erythrosine- and LED-mediated PDT, but the biofilms of both Candida species were more resistant than their planktonic counterparts.     

Periodontal conditions and prevalence of putative periodontopathogens and Candida spp. in insulin-dependent type 2 diabetic and non-diabetic patients with chronic periodontitis--a pilot study

Objectives

The aims of this study were to evaluate periodontal conditions and identify the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia, and four different species of Candida (C. albicans, C. dubliniensis, C. glabrata and C. tropicalis) in periodontal pockets and furcation sites of insulin-dependent type 2 diabetic and non-diabetic patients with generalised chronic periodontitis.

Design

Clinical parameters, including oral status assessed using plaque index, gingival index, probing depth, gingival recession and clinical attachment level and systemic conditions with fasting glucose level or glycosylated haemoglobin were measured in diabetic and non-diabetic patients with chronic periodontitis. Samples of subgingival biofilm were obtained from the periodontal pockets and furcation sites and submitted to phenol-chloroform DNA extraction and PCR analysis using specific primers.

Results

Clinical conditions of diabetic and non-diabetic patients were similar, without statistical differences in both periodontal indexes and glucose levels (p > 0.05). Diabetics had a higher prevalence of Candida spp., mainly C. albicans and C. dubliniensis, and a lower frequency of T. forsythia, when compared to non-diabetic patients, for both periodontal sites. C. glabrata and C. tropicalis were not found in periodontal pockets and furcation sites of non-diabetic patients.

Conclusion

The results demonstrated a strong colonisation of Candida spp. in the periodontal sites of diabetic patients that have generalised chronic periodontitis with a higher prevalence of C. dubliniensis followed by C. albicans.     

Isolation of Candida dubliniensis in denture stomatitis

Objective

To describe the isolation of Candida dubliniensis from a patient with denture stomatitis and to compare with the presence of yeasts in the oral cavities of denture wearers.

Design

One hundred and fifty-two Candida isolates were recovered through oral swabs from denture as well as the underlying mucosa from 100 patients wearing denture. For detection and identification of fungal isolates, standard phenotypic and genotypic methods were used.

Results

Forty-five of 100 denture wearers suffered from denture stomatitis. Seventy-three Candida isolates were recovered from 38 denture wearers without denture stomatitis. In this group, Candida albicans was the predominant species (58.9%), followed by Candida tropicalis (15.1%), Candida guilliermondii (13.7%), Candida glabrata (9.6%), and Candida parapsilosis (2.7%).Seventy-nine isolates were yielded from 40 patients suffering from denture stomatitis. C. albicans was also the most frequently isolated species (58 isolates, 73.4%), followed by C. glabrata and C. tropicalis (7 isolates each, 8.9%), and Saccharomyces cerevisiae (2 isolates, 2.5%). One isolate was yielded of the following species: Candida famata, Candida krusei, C. parapsilosis and C. guilliermondii. Moreover 1 isolate was phenotypic and genotypic identified as C. dubliniensis genotype 1.

Conclusions

C. albicans is the predominant fungal species isolated from denture wearers. C. dubliniensis could be isolated from adults with denture stomatitis.     

Microbicidal efficacy of thiocyanate hydrogen peroxide after adding lactoperoxidase under saliva loading in the quantitative suspension test

Objective

As shown in the quantitative suspension test adding lactoperoxidase to a thiocyanate (SCN⁻) hydrogen peroxide (H₂O₂) combination over the physiological saliva level has significant positive antimicrobial effects to a level of totally killing Streptococcus mutans, Streptococcus sanguinis, and Candida albicans. The aim of this study was to evaluate this positive effect under human saliva loading.

Methods

The bactericidal and fungicidal effect of lactoperoxidase was evaluated in a quantitative suspension test by using two test mixtures of a 2.0% thiocyanate and 1.2% hydrogen peroxide solution, one without (Group A) and one with (Group B) lactoperoxidase under saliva loading. Following the quantitative suspension tests (EN-13727/EN-13624), the growth of surviving bacteria and fungi in a nutrient broth was measured. The exposure times were restricted to 1, 3, 5, and 15 min. All statistical analyses were carried out with SPSS 11.5.

Results

In the quantitative suspension test, the combination of thiocyanate and hydrogen peroxide showed relatively low antimicrobial effectiveness on S. mutans, S. sanguinis, and C. albicans in the presence of human saliva at measured time points in comparison to the mixture with lactoperoxidase, which showed a high bactericidal activity within 15 min (S. mutans and S. sanguinis) and fungicidal activity within 3 min (C. albicans).

Conclusion

The antimicrobial effectiveness of the tested thiocyanate hydrogen peroxide combination was increased significantly by adding lactoperoxidase in the quantitative suspension test under human saliva loading.     

Efficacy of denture cleansers on denture liners contaminated with Candida species

As poor denture hygiene is related to Candida colonisation, disinfectant solutions have been proposed as an effective method of preventing denture stomatitis. This study assessed the efficacy of denture cleansers on Candida albicans and Candida glabrata adherence on denture liners. Another aim was to correlate materials’ surface roughness (Ra) to Candida adherence. Specimens of three denture liners (soft and hard polymethyl methacrylate (PMMA)-based and soft silicone-based) were prepared and had their Ra measured. Specimens were randomly divided to adherence assays with C. albicans or C. glabrata. After contamination with the fungi, specimens were treated with an enzymatic cleanser solution, a cleanser solution or a 0.5% NaOCl solution by soaking for 3, 15 or 10 min, respectively. Control group specimens were soaked in distilled water for 15 min. Number of remaining Candida cells after treatment was determined by light microscopy (×400). Analysis of variance (α = 0.05) showed that Ra of the silicone-based liner was lower than that of the PMMA-based liners (p < 0.05). The overall results showed high C. glabrata adherence (p < 0.001), while the lowest levels of remaining Candida cells were found for the treatment with 0.5% NaOCl (p = 0.0019). No difference among denture cleansers and control was found (p = 0.19). There was no correlation between Ra and C. albicans or C. glabrata adherence in all materials tested. The only treatment able to reduce both Candida species adherence on all materials tested was 0.5% NaOCl solution.     

Prevalence and antifungal resistance profile of Candida spp. oral isolates from patients with type 1 and 2 diabetes mellitus

Objective

The goal of the study was to measure the prevalence of Candida spp. in the oral cavity of patients with diabetes types 1 and 2 when compared to healthy individuals and to study antifungal resistance profile of the isolates.

Design

There were 162 subjects in the study: diabetes type 1 (n = 39); control group 1 (n = 50): healthy individuals matched in gender, age, and oral conditions to diabetes type 1 patients; diabetes type 2 (n = 37); control group 2 (n = 36) who were matched to each patient of the diabetes type 2 group. Stimulated saliva was collected and isolates were identified with phenotypic tests. The presence of C. dubliniensis was determined by multiplex PCR.

Results

There were no statistically significant differences in Candida spp. frequency between the diabetes 1 group and its control (p = 0.443) nor between the diabetes 2 group and its control (p = 0.429). C. albicans was the most frequently isolated yeast in all groups. In the diabetes groups, C. stellatoidea, C. parapsilosis, C. tropicalis, C. lipolytica, C. glabrata, and C. krusei were also identified. Additionally, in control groups, C. kefyr was also detected. None of the isolates were resistant to amphotericin B and flucytosine. A low percentage of the isolates were resistant to ketoconazole.

Conclusions

No differences were detected in colonization of Candida spp. oral isolates from type 1 and type 2 diabetes when compared to matched controls. The antifungal resistance of Candida spp. isolates for ketoconazole from type 1 diabetes patients was significantly higher than that of its matched control.     

Antiadherent activity of Schinus terebinthifolius and Croton urucurana extracts on in vitro biofilm formation of Candida albicans and Streptococcus mutans

Objective

To evaluate the antiadherent property of crude, methanol and acetate methanol extract fractions from Schinus terebinthifolius and Croton urucurana in hydroalcoholic (HA) and dimethylsulfoxide (DMSO) solvents on in vitro biofilms formed by Streptococcus mutans and Candida albicans strains.

Design

The minimal concentration of adherence (MICA) was determined to evaluate the antiadherent potential of extracts on the in vitro biofilm formation. The extracts of plants were subjected to thin layer chromatography (TLC) in order to detect what class of compounds was responsible for the antiadherent activity. Data were estimated by analysis of variance (ANOVA) complemented by Tukey test level of significance set at 5%.

Results

Both plants demonstrated inhibition of S. mutans and C. albicans on in vitro biofilm formation. The biofilms of C. albicans were more efficiently inhibited by the S. terebinthifolius fraction of acetate–methanol and methanol in hydroalcoholic solvents (p < 0.05). The S. mutans biofilms adherence was best inhibited by the S. terebinthifolius crude extract and its methanolic fraction, both in hydroalcoholic solvent (p < 0.05). TLC of crude extracts and fractions of S. terebinthifolius detected the presence of several active compounds, including phenolic compounds, anthraquinones, terpenoids, and alkaloids. C. urucurana extracts confirmed activity for both microorganisms (p < 0.05). However, higher concentrations were needed to achieve antiadherent activity, mainly to inhibit in vitro biofilm formation of C. albicans.

Conclusion

The antiadherent potential of both plants on in vitro biofilms formed by C. albicans and S. mutans were confirmed, suggesting the importance of studies about these extracts for therapeutic prevention of oral diseases associated with oral biofilms.     

Candida glabrata and Candida albicans co‐infection of an in vitro oral epithelium

J Oral Pathol Med (2010) 40 : 421–427 Background: Candida albicans is regarded as the leading of candidosis. However, Candida glabrata has emerged as an important pathogen of oral mucosa, occurring both singly or in mixed species infections, often with C. albicans. Compared with C. albicans, little is known about the role of C. glabrata in oral infection. The aim of this study was to examine single and mixed species infection of oral epithelium involving C. glabrata and establish its ability to invade and damage tissue. Methods: A reconstituted human oral epithelium (RHOE) was infected only with C. glabrata, or simultaneously with C. glabrata and C. albicans. The ability of both species to invade the tissue was examined using species specific peptide nucleic acid (PNA) probe hybridization and confocal laser scanning microscopy. Epithelial damage was assessed by measuring lactate dehydrogenase (LDH) activity. Results: Candida glabrata strains were able to colonize the RHOE, in a strain dependent manner. Candida glabrata single infection after 12 h, generally revealed no invasion of the RHOE, which contrasted with extensive tissue invasion demonstrated by C. albicans. Mixed infection showed that C. albicans enhanced the invasiveness of C. glabrata, and led to increased LDH release by the RHOE, which paralleled the observed histological damage. Conclusions: The results obtained demonstrating enhanced invasion and increased tissue damage caused by mixed C. glabrata and C. albicans infections have important clinical significance and highlight the need to identify Candida species involved in oral candidosis.     

Biofilm-forming ability and adherence to poly-(methyl-methacrylate) acrylic resin materials of oral Candida albicans strains isolated from HIV positive subjects

PURPOSE

This study evaluated the adhesion to acrylic resin specimens and biofilm formation capability of Candida albicans strains isolated from HIV positive subjects'' oral rinse solutions.

MATERIALS AND METHODS

The material tested was a heat-cured acrylic resin (Acron Duo). Using the adhesion and crystal violet assays, 14 oral Candida albicans isolated from HIV-positive subjects and 2 references Candida strains (C. albicans ATCC 90028 and C. albicans ATCC 90128) were compared for their biofilm production and adhesion properties to acrylic surfaces in vitro.

RESULTS

There were no significant differences in adhesion (P=.52) and biofilm formation assays (P=.42) by statistical analysis with Mann-Whitney test.

CONCLUSION

Denture stomatitis and increased prevalence of candidal carriage in HIV infected patients is unlikely to be related to the biofilm formation and adhesion abilities of C. albicans to acrylic resin materials.     

Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites

Objectives

The aim of this study was to evaluate the acid production, acid tolerance and composition of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites.

Methods

S. mutans biofilms were formed on saliva-coated discs prepared from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing materials (Filtek Z350, GRADIA DIRECT and hydroxyapatite). To assess the level of acid production and acid tolerance, glycolytic pH drop and proton permeability assays were performed using 94 h old S. mutans biofilms. To evaluate the biofilm composition, the biomass (total dry-weight), colony forming unit (CFU), water-insoluble extracellular polysaccharides (EPS), water-soluble EPS and intracellular iodophilic polysaccharides (IPS) of 94 h old S. mutans biofilms were analysed. The amount of fluoride of old culture medium released from the materials during the experimental period was also determined. Each assay was performed in duplicate in at least four different experiments (n = 8).

Results

All biofilms showed similar initial rates of acid production (0.083–0.089 pH drop/min) and proton permeability (0.025–0.036 pH increase/min), irrespective of fluoride release from the materials. On the other hand, the amount of biomass, water-insoluble EPS and IPS of the biofilms on Unifil Flow, which releases a larger amount of fluoride in the early stages of biofilm formation, were significantly lower than those on the other materials (up to 27%, 38% and 36% reduction in biomass, water-insoluble and IPS, respectively).

Conclusions

Our finding suggests that fluoride releasing resin composites might contribute to the decrease in cariogenic composition of S. mutans biofilms if an appropriate amount of fluoride is released in the early stages of biofilm formation.     

Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine

Objectives

Candida albicans cells form biofilms on polymeric surfaces of dentures and other prostheses introduced into the oral cavity. Many biofilm microorganisms exhibit resistance to antimicrobial agents; C. albicans cells may also develop resistance to naturally occurring antifungal peptides in human saliva including histatins (Hsts) and defensins (hBDs). Therefore, we evaluated Hst 5 activity on C. albicans biofilm cells compared to planktonic cells and measured whether surface treatment of denture acrylic with Hst 5, hBD-3, or chlorhexidine gluconate could inhibit in vitro biofilm development.

Methods

Acrylic disks were preconditioned with 500 μl saliva for 30 min, and inoculated with C. albicans cells (10⁶ cells/ml) for 1 h, at 37 °C. Non-adherent cells were removed by washing and disks and were incubated in YPD growth medium for 24, 48, and 72 h at 37 °C. Candidacidal assays were performed on 48-h-biofilms and on planktonically grown cells using Hst 5 (15.5, 31.25, and 62 μM). Cell adhesion was compared on disks pre-coated with 0.12% chlorhexidine gluconate, 50 μM Hst 5, or 0.6 μM hBD-3 after 24, 48, and 72 h growth.

Results

No significant difference was observed in sensitivity to Hst 5 of biofilm cells compared to planktonic cells (p > 0.05). Pre-coating disks with hBD-3 did not inhibit biofilm development; however, Hst 5 significantly inhibited biofilm development at 72 h, while 0.12% chlorhexidine significantly inhibited biofilm development at all time intervals (p < 0.05).

Conclusions

C. albicans biofilm cells grown on denture acrylic are sensitive to killing by Hst 5. Surface coating acrylic with chlorhexidine or Hst 5 effectively inhibits biofilm growth and has potential therapeutic application.     

Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms

Objective

This study aimed to evaluate the influence of NaF (2, 10, 50 and 125 ppm F⁻) on the virulence factors and composition of Streptococcus mutans biofilms.

Methods

S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite discs. To assess the influence of NaF on the virulence factors of S. mutans biofilm cells, glycolytic pH drop, proton-permeability and F-ATPase activity assay were performed using 74 h old S. mutans biofilms. Glucosyltransferase (GTF) activity assay in suspension was also performed. To examine the influence of NaF on S. mutans biofilm composition, the biofilms were treated twice daily (5 min exposure/treatment) a total of five times during biofilm formation. After a total of 5 treatments, the biomass, colony forming unit (CFU) and polysaccharide composition of the treated 74 h old S. mutans biofilms were analysed by microbiological and biochemical methods, and scanning electron microscopy.

Results

NaF showed inhibitory effects on the acid production and acid tolerance of S. mutans biofilm cells at 10, 50 and 125 ppm F⁻, compared to the vehicle control (P < 0.05) and the treatments at these concentrations also affected the biomass, water-insoluble extracellular polysaccharides and intracellular iodophilic polysaccharides of the biofilms, compared to the vehicle control (P < 0.05).

Conclusions

These results indicate that NaF (10, 50 and 125 ppm F⁻) has inhibitory effects on the virulence factors and composition of S. mutans biofilms, suggesting the potential use of these concentrations as an effective measure for controlling dental biofilms.     

Oral epithelium–Candida glabrata interactions in vitro

Background: Oropharyngeal candidiasis is a common opportunistic infection and Candida glabrata is the second or third most frequently isolated species from oropharyngeal candidiasis lesions, after Candida albicans. The aim of this study was to study the cytokine‐inducing and cell‐damaging potential of C. glabrata in oral epithelial cells and compare this to C. albicans. Methods: Oral epithelial cell lines and primary gingival epithelial cells were cocultured with C. glabrata strains GDH2269 and 94‐11 or C. albicans strains SC5314 and ATCC28366. Supernatants were analysed for the presence of interleukin‐1α (IL‐1α), IL‐8 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) by enzyme‐linked immunosorbent assay. The cytotoxity of different strains was determined using the CytoTox‐96 assay. Results: Compared to C. albicans, C. glabrata induced different proinflammatory cytokine responses in oral epithelial cells; a high level of GM‐CSF induction was only detected in C. glabrata‐infected cells and not in C. albicans‐infected cells, regardless of the origin of these cells (cell lines or primary cells) or the strain used. Like C. albicans, C. glabrata induced an IL‐1α response by oral epithelial cells, but this response was both strain‐dependent and epithelial cell origin‐dependent. Unlike C. albicans, C. glabrata failed to induce a strong IL‐8 response in any of the cell systems studied. Finally, in these studies C. glabrata showed lower cytotoxicity than C. albicans. Conclusions: C. glabrata is less cytotoxic than C. albicans and induces different proinflammatory cytokine responses in oral epithelial cells.     

Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans

Objective

To assess antimicrobial activities of nanoemulsion (NE) to control the adhesion and biofilm formation by Streptococcus mutans by in vitro.

Design

In vitro antimicrobial susceptibility of nanoemulsion was determined as per National Committee for Clinical Laboratory Standards guidelines and agar diffusion, serial dilution technique for the determination of minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). Efficacy was tested by kinetics of killing, biofilm assay and scanning electron microscopy.

Results

: NE concentrations ranging from 1:100 to 1:10,000 dilutions were effective against S. mutans as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. 4-day-old S. mutans biofilms were treated with NE; subsequent reductions of bacterial cell counts were noticed with decreasing dilutions. Staining of NE-treated biofilms with LIVE/DEAD BacLight resulted in dead cell areas of up to 48% in 1 min, 84% at 1 h and significant (<0.05) increases in dead cell counts at all time points. Damage to cell membranes and cell walls of S. mutans by NE was demonstrated using scanning electron microscopy (SEM).

Conclusion

These results suggest that nanoemulsion has effective antibacterial activity against S. mutans and may be a useful medication in the prevention of dental caries.     

Role of efflux pump inhibitors on the antibiofilm efficacy of calcium hydroxide, chitosan nanoparticles, and light-activated disinfection

Introduction

The purpose of this study was to evaluate the role of efflux pumps in altering the susceptibility of Enterococcus faecalis biofilms to calcium hydroxide (Ca(OH)₂), chitosan nanoparticles, and light-activated disinfection (LAD).

Methods

E. faecalis as 4-day-old biofilms and biofilm-derived cells were tested with aqueous Ca(OH)₂ in concentrations of 25%, 50%, and 100%; chitosan nanoparticles in concentrations of 10 and 20 mg/mL (3, 12, and 24 hours); and methylene blue (MB) mediated LAD at an energy dose range of 2–40 J/cm². An efflux pump inhibitor (EPI) was incorporated into all 3 modalities of treatment. The antimicrobial activity was assessed by determining the colony-forming units.

Results

E. faecalis biofilms, in contrast to the biofilm-derived cells, were found to persist even after 24-hour treatment with different concentrations of Ca(OH)₂ and chitosan nanoparticles. LAD at an energy dose of 40 J/cm² completely inactivated 4-day-old E. faecalis biofilms. The addition of EPI improved the antibiofilm efficacy of Ca(OH)₂ at lower concentrations (P < .001) and LAD (P < .001). EPI did not influence the antibiofilm effect of chitosan nanoparticles and Ca(OH)₂ at higher concentrations.

Conclusions

E. faecalis biofilms were more susceptible to killing by LAD, when compared with the tested concentrations of Ca(OH)₂ and chitosan nanoparticles. The effect of EPI was more significant with LAD, when compared with Ca(OH)₂ and chitosan nanoparticles. This study highlighted the role of biofilm matrix in providing resistance to antimicrobials.     

The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces

Although Candida containing biofilms contribute to the development of oral candidosis, the characteristics of multi-species Candida biofilms and how oral bacteria modulate these biofilms is poorly understood. The aim of this study was to investigate interactions between Candida albicans and either Candida glabrata or Streptococcus mutans in biofilms grown on various surfaces, with or without saliva. Hydroxyapatite (HA), polymethylmetacrylate (PMMA) and soft denture liner (SL) discs were used as substratum. Counts of viable micro-organisms in the accumulating biofilm layer were determined and converted to colony forming units per unit surface area. Confocal laser scanning microscopy was used to characterize biofilms and to quantitate the number of hyphae in each condition tested. Viable counts of C. albicans and C. glabrata per mm(2) decreased in the order HA>PMMA>SL (p<0.05). Biofilms grown on saliva-coated specimens harboured fewer C. glabrata than uncoated specimens (p<0.05). Glucose and the presence of S. mutans suppressed C. albicans hyphal formation. Dual Candida species biofilms did not show competitive interaction between the two species. We conclude that Candida biofilms are significantly affected by saliva, substratum type and by the presence of other micro-organisms.     

Parylene coating hinders Candida albicans adhesion to silicone elastomers and denture bases resin

Objective

To investigate whether parylene coatings over denture bases and silicone elastomers can effectively reduce Candida albicans adhesion and thus to decrease the incidence of denture stomatitis.

Design

Specimens of silicone elastomers A-2186 or lucitone 199 resin were prepared, and the measurements of contact angle, assay of XTT reduction and cell count of C. albicans adhesion were taken before and after parylene treatment. Furthermore, morphology of C. albicans adhesion for 48 h was observed by scanning electron microscope (SEM), and C. albicans adhesion for 4 h was illustrated by confocal scanning laser microscopy (CLSM) in combination with fluorescent dyes FUN-1 and Concanavalin A.

Result

There was a statistical difference between mean contact angles of silicone elastomer A-2186 before and after parylene coating (P < 0.05). The amount of C. albicans adhesion to the surface of silicone elastomer A-2186 and lucitone 199 resin after parylene treatment was significantly less than before parylene treatment by cells count and XTT reduction assay (P < 0.05). In SEM and CLSM analysis, C. albicans biofilm was more apt to generate on the surface of silicone elastomer A-2186 than other three groups, and more C. albicans aggregation formed on the surface of silicone elastomer A-2186 and lucitone 199 resin before parylene treatment than after parylene treatment.

Conclusion

Parylene coating reduced C. albicans adhesion and aggregation on the surface of silicone elastomer A-2186 and lucitone 199 resin, and improved the wettability of silicone elastomer A-2186.     

The effect of different titanium nitride coatings on the adhesion of Candida albicans to titanium

Objectives

The aim of the present study was an in vitro evaluation of the effects of different titanium nitride (TiN_x) coatings on Candida albicans (C. albicans) adhesion to titanium and to correlate these findings to differences in specific surface characteristics (surface topography, roughness, chemical component, and surface free energy).

Methods

TiN_x coatings were prepared by physical vapour deposition (PVD), a plasma nitriding process or a dual nitriding process. Surface properties were analysed by the optical stereoscopic microscopy, scanning electron microscopy, roughmeter, and drop shape methods. Quantity comparisons of C. albicans on the four surfaces were assessed by cell count and XTT reduction assays. Types of adhesive C. albicans were explored by SEM and confocal laser scanning microscope.

Results

The nitrided modifications were found to influence the surface properties and fungal susceptivity of flat titanium. Compared to flat titanium, fewer adhered C. albicans in yeast form were observed on the TiN-coated surface, whereas the plasma nitrided surface did not show any reduced potential to adhere C. albicans in hyphal or yeast form. The dual nitrided coating showed anti-fungal characteristics, although a small quantity of hyphae were identified. Our findings indicate that the Ti₂N phase is prone to C. albicans hyphae, while the TiN phase inhibits their adhesion.

Conclusions

Different TiN_x phases could influence the characteristics of C. albicans adhesion. TiN coating by PVD could be a potential modification to inhibit C. albicans.