Silver nanoparticles : influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

Aim : The purpose of this work was to evaluate the size-dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. Methods and Results:  The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4–3·3 μg ml−1). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml−1. In general, all SN suspensions promoted significant log10 reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml−1. Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. Conclusions:  This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. Significance and Impact of the Study:  SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.This study was supported by the Coordenacao de Aperfeicoamento de Pessoal de Nivel superior (CAPES), Brazil, grant BEX 1221/10-8. The authors are indebted to LIEC-CMDMC and INCTMN/FAPESP-CNPq for preparing and characterizing the colloidal suspensions of SN. The authors also thank George Duchow for the English review

Effect of Macroscopic Grooves on Bone Formation and Osteoblastic Differentiation.

Objectives: The aim of this study is to investigate the effect of macroscopic grooves on bone formation in vivo and differentiation of human mesenchymal stem cells (hMSCs) in vitro. Materials and Methods: The effects of macroscopic grooves on titanium alloy implants and disks were tested in rabbit tibiae and cultured hMSCs. The bone-to-implant contact (BIC) and bone area were evaluated in rabbit tibiae at 6 and 24 weeks after implant insertion. Osteoblastic differentiation was assessed by alkaline phosphatase (ALP) activity and real-time reverse-transcription polymerase chain reaction (RT-PCR) on days 7, 14, and 21. All values were statically analyzed. Results: BIC and bone area inside the grooves were significantly higher than those of control implants (P < 0.05). ALP activity was significantly higher for titanium disks with macroscopic grooves than without grooves on day 14 (P < 0.05). Real-time RT-PCR showed that the expression of osteogenic genes was significantly higher for disks with grooves on day 7 (P < 0.01). Conclusions: Macroscopic grooves accelerate osteoblastic differentiation in vitro and stimulate direct bone growth and deposition within the grooves in vivo

Silver colloidal nanoparticle stability: influence on Candida biofilms formed on denture acrylic

Our aim in this study was to evaluate how the chemical stability of silver nanoparticles (SNs) influences their efficacy against Candida albicans and C. glabrata biofilms. Several parameters of SN stability were tested, namely, temperature (50ºC, 70ºC, and 100ºC), pH (5.0 and 9.0), and time of contact (5 h and 24 h) with biofilms. The control was defined as SNs without temperature treatment, pH 7, and 24 h of contact. These colloidal suspensions at 54 mg/L were used to treat mature Candida biofilms (48 h) formed on acrylic. Their efficacy was determined by total biomass and colony-forming unit quantification. Data were analyzed using analysis of variance and the Bonferroni post hoc test (=0.05). The temperature and pH variations of SNs did not affect their efficacy against the viable cells of Candida biofilms (P > 0.05). Moreover, the treatment periods were not decisive in terms of the susceptibility of Candida biofilms to SNs. These findings provide an important advantage of SNs that may be useful in the treatment of Candida-associated denture stomatitis.We thank Dr David Williams, Cardiff University, Cardiff, UK, for providing the strain 324LA/94. The authors also thank Sao Paulo Research Foundation (FAPESP, process 2009/15146-5), Brazil, for supporting the work of D. R. M. The authors are indebted to Laboratorio Interdisciplinar de Eletroquimica e Ceramica, Federal University of Sao Carlos, Brazil, in the name of Andressa Kubo, for preparing and characterizing the colloidal suspensions of silver nanoparticles

Candida albicans forms biofilms on the vaginal mucosa

Current understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1)

Screening of Pharmacologically Active Small Molecule Compounds Identifies Antifungal Agents Against Candida Biofilms

Candida species have emerged as important and common opportunistic human pathogens, particularly in immunocompromised individuals. The current antifungal therapies either have toxic side effects or are insufficiently effect. The aim of this study is develop new small-molecule antifungal compounds by library screening methods using Candida albicans, and to evaluate their antifungal effects on Candida biofilms and cytotoxic effects on human cells. Wild-type C. albicans strain SC5314 was used in library screening. To identify antifungal compounds, we screened a small-molecule library of 1,280 pharmacologically active compounds (LOPAC1280TM) using an antifungal susceptibility test (AST). To investigate the antifungal effects of the hit compounds, ASTs were conducted using Candida strains in various growth modes, including biofilms. We tested the cytotoxicity of the hit compounds using human gingival fibroblast (hGF) cells to evaluate their clinical safety. Only 35 compounds were identified by screening, which inhibited the metabolic activity of C. albicans by >50%. Of these, 26 compounds had fungistatic effects and nine compounds had fungicidal effects on C. albicans. Five compounds, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate, ellipticine and CV-3988, had strong fungicidal effects and could inhibit the metabolic activity of Candida biofilms. However, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine were cytotoxic to hGF cells at low concentrations. CV-3988 showed no cytotoxicity at a fungicidal concentration. Four of the compounds identified, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine, had toxic effects on Candida strains and hGF cells. In contrast, CV-3988 had fungicidal effects on Candida strains, but low cytotoxic effects on hGF cells. Therefore, this screening reveals agent, CV-3988 that was previously unknown to be antifungal agent, which could be a novel therapies for superficial mucosal candidiasis

Antifungal resistance gene expressions in various lifestyles of Candida albicans

Oral Presentation: abstract no. 74Objectives: Lifestyle change of Candida albicans from planktonic to biofilm is one way of acquiring higher antifungal resistance. Therefore, the purpose of present study was to investigate the expressions of antifungal resistance genes in three growth modes, i.e. planktonic, adhesion and biofilm. Furthermore, we investigated the effect of ...link_to_OA_fulltextThe International Association for Dental Research (IADR) 88th General Session and Exhibition 2010, Barcelona, Spain, 14-17 July 2010

Shotgun Proteomics elucidates the regulatory pathway of Candida biofilms

Objectives: Biofilm formation and filamentation are major virulent attributes of the fungal pathogen Candida albicans. Mitogen Activated Proteins Kinase (MAPK) and cAMP/Protein Kinase (PKA) pathways are the two major biological pathways that regulate these attributes. Therefore, aim of the present study was to elucidate the role of MAPK, PKA pathways using Candida mutants lacking CPH1 and EFG1; major regulators of MAPK and PKA pathways, respectively. Methods: Initially, biofilm formation, anti-oxidative capacities and antifungal susceptibility of Candida mutants were studied using standard methodology. Thereafter, protein lysates of respective biofilms were quantified, denatured, and cysteines blocked. Each sample was then digested with trypsin and labeled with the iTRAQ tags. Differentially labeled digests were subjected to LC-MALDI-TOF/TOF analysis. The MS/MS peptides and phosphorylation patterns were identified by bioinformatics tools and further validated using Candida Genome Database (CGD). Results: The mutant lacking EFG1 was devoid of filaments and unable to form mature biofilms, and more susceptible to oxidative stress and antifungals, compared to the wild type strain. Totally, 861 proteins over 95% C.I. which are related to MAPK and PKA pathways were identified. In particular out of 452 known and characterized proteins, 133 related to PKA pathway were down-regulated more than two folds compared to 20 proteins down-regulated in MAPK pathway. Of these down-regulated proteins a considerable number was related to the stress response and drug resistance. Of the characterized proteins, 317 were phosphorylated and 24 were fungal specific. Further, 409 yet uncharacterized proteins were also identified. . Conclusion: cAMP/PKA pathway, but not MAPK pathway governs the filamentation and biofilm formation of Candida albicans. PKA related stress response proteins may potentially mediate the higher antifungal resistance seen in Candida biofilms. Fungal specific, PKA pathway biomarkers, and their phosphorylation profiles are likely to be of use in the search for the novel antifungals. (RGC-Grant HKU-7624/06M

Susceptibility of Candida albicans filamentation-defective mutants to clinical biocides

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