Publicação
Candida dubliniensis versus Candida albicans : adhesion and biofilm formation
| Resumo: | Candida species are opportunistic yeasts that colonize the human body. Among the more well known and more virulent is Candida albicans. Although for several years, until 1995, another species, Candida dubliniensis, was mismatched with that one. Both Candida species possess similar phenotypic and genotypic characteristics. As this species has not been fully studied, it became important to understand its behaviour and its properties. Candida dubliniensis is mainly found in the mouth of some HIV patients. So, its behaviour in the oral cavity was chosen as the objective of the present work. The main goal of this work was the comparison of those two species in terms of the ability to adhere to inert surfaces and epithelial cells, to form biofilms and susceptibility to antifungal agents. The characteristics and behaviours under concern were assayed in two strains of each species. Artificial saliva solution was used in order to simulate the oral conditions. Yeast cells and inert surfaces were characterized in terms of physico-chemical surface properties (surface tension parameters, degree of hydrophobicity and zeta potential) as well as elemental surface composition determined by X-ray Photoelectron Spectroscopy. Adhesion to inert surfaces was assayed on acrylic, that is commonly used in prosthetic devices and hydroxyapatite (HAP), which mimics tooth enamel. The adhesion was performed during one hour, both in ultrapure water and artificial saliva. The number of adhered cells was determined by direct enumeration using fluorescence microscopy. Adhesion of yeast cells was also performed to epithelial cells (HeLa cell line) and was quantified by direct enumeration, after Gram staining. Concerning the biofilm formation, the profiles of biofilm evolution were determined for biofilms formed on acrylic and grown in Sabouraud dextrose broth or artificial saliva growth medium. The biofilm formation was evaluated by quantifying total biomass (crystal violet staining) and activity (XTT/formazan salts formation). Two different antifungal agents were used, one fungistatic – fluconazole – and one fungicidal – amphotericin B. The yeast cells response to subinhibitory concentrations of both antifungal agents was analysed in terms of adhesion and biofilm formation (either in SDB or artificial saliva growth medium), using cells grown in media containing those agents. The influence of subinhibitory concentrations of the antifungal agents was also studied in biofilm formation along 78 h in the presence of the antifungal agents. The assessment was performed in terms of biofilm biomass and activity. Considering cell surface properties (surface tension parameters, degree of hydrophobicity), there were no significative differences among the four strains assayed, neither when these properties were measured with cells conditioned by different solutions (water, artificial saliva solution, saline solution or after growth in subMIC concentrations of fluconazole and amphotericin B). The results obtained lead to the conclusion that although adhesion to inert surfaces is not strain dependent, adhesion to epithelium and biofilm formation are species and strain dependent. The effect of sub inhibitory concentrations of the antifungal agents on the growth of yeast cells and consequent extent of adhesion and biofilm formation was not notorious. |
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| Autores principais: | Henriques, Mariana |
| Ano: | 2005 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Candida species are opportunistic yeasts that colonize the human body. Among the more well known and more virulent is Candida albicans. Although for several years, until 1995, another species, Candida dubliniensis, was mismatched with that one. Both Candida species possess similar phenotypic and genotypic characteristics. As this species has not been fully studied, it became important to understand its behaviour and its properties. Candida dubliniensis is mainly found in the mouth of some HIV patients. So, its behaviour in the oral cavity was chosen as the objective of the present work. The main goal of this work was the comparison of those two species in terms of the ability to adhere to inert surfaces and epithelial cells, to form biofilms and susceptibility to antifungal agents. The characteristics and behaviours under concern were assayed in two strains of each species. Artificial saliva solution was used in order to simulate the oral conditions. Yeast cells and inert surfaces were characterized in terms of physico-chemical surface properties (surface tension parameters, degree of hydrophobicity and zeta potential) as well as elemental surface composition determined by X-ray Photoelectron Spectroscopy. Adhesion to inert surfaces was assayed on acrylic, that is commonly used in prosthetic devices and hydroxyapatite (HAP), which mimics tooth enamel. The adhesion was performed during one hour, both in ultrapure water and artificial saliva. The number of adhered cells was determined by direct enumeration using fluorescence microscopy. Adhesion of yeast cells was also performed to epithelial cells (HeLa cell line) and was quantified by direct enumeration, after Gram staining. Concerning the biofilm formation, the profiles of biofilm evolution were determined for biofilms formed on acrylic and grown in Sabouraud dextrose broth or artificial saliva growth medium. The biofilm formation was evaluated by quantifying total biomass (crystal violet staining) and activity (XTT/formazan salts formation). Two different antifungal agents were used, one fungistatic – fluconazole – and one fungicidal – amphotericin B. The yeast cells response to subinhibitory concentrations of both antifungal agents was analysed in terms of adhesion and biofilm formation (either in SDB or artificial saliva growth medium), using cells grown in media containing those agents. The influence of subinhibitory concentrations of the antifungal agents was also studied in biofilm formation along 78 h in the presence of the antifungal agents. The assessment was performed in terms of biofilm biomass and activity. Considering cell surface properties (surface tension parameters, degree of hydrophobicity), there were no significative differences among the four strains assayed, neither when these properties were measured with cells conditioned by different solutions (water, artificial saliva solution, saline solution or after growth in subMIC concentrations of fluconazole and amphotericin B). The results obtained lead to the conclusion that although adhesion to inert surfaces is not strain dependent, adhesion to epithelium and biofilm formation are species and strain dependent. The effect of sub inhibitory concentrations of the antifungal agents on the growth of yeast cells and consequent extent of adhesion and biofilm formation was not notorious. |
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