Laboratoire de Parasitologie et Mycologie Médicales, Centre Hospitalier Universitaire La Milétrie, BP 577, 86021 Poitiers Cedex, France
Received 12 September 2003; returned 22 October 2003; revised 19 November 2003; accepted 6 December 2003
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Abstract |
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Methods: Eleven strains of C. albicans were studied: six strains were susceptible to fluconazole in vitro and five strains were resistant to this antifungal agent.
Results: Caspofungin induced a decrease in the adherence of all the tested strains that were susceptible to fluconazole but induced a decrease in the adherence of only 60% of the fluconazole-resistant strains.
Conclusions: This study demonstrated the anti-adherent activity of caspofungin but indicated a reduced effect in the case of in vitro fluconazole resistance. These results indicated a possible relationship between the efficiency of caspofungin to inhibit the first step of the development of C. albicans biofilm and the resistance of C. albicans to fluconazole in vitro.
Keywords: yeast, adhesion, antifungal agents, candidiasis
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Introduction |
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A small number of antifungal drugs can be used to treat candidiasis associated with implanted medical devices, and infected devices generally need to be removed.4 This type of infection can result in serious medical complications, expensive care and is noted as the most frequent factor limiting the prolonged use of central venous catheters.5
Echinocandins represent a new class of antifungal drug and act by inhibiting the synthesis of ß-D-glucan in fungal cell walls.6 The cell wall is highly implicated in the adherence process of C. albicans, and so, in the first step of biofilm formation.3 Caspofungin is the first representative of echinocandins and shows in vitro antifungal activity against C. albicans.6 The aim of this study was to determine whether caspofungin, used in a concentration below its corresponding sub-inhibitory concentration, could prevent adherence of C. albicans to plastic coated with extracellular matrix proteins. This action could be effective before the emergence of infection by preventing the formation of C. albicans biofilm on implanted medical devices.
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Materials and methods |
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Eleven isolates of C. albicans were studied: five strains (92, 109, 163, 182 and 240) were isolated in our laboratory, from patients with candidaemia. The identification of these clinical isolates was carried out by using conventional physiological and morphological studies such as the germ-tube test in serum, agglutination (Bichro-Latex, Fumouze, Levallois Perret, France) and metabolic properties (API 20C, bioMérieux, Marcy-LEtoile, France). The 1066 strain of C. albicans, originally isolated from a patient with septicaemia, was kindly provided by Professor R. Robert (Laboratory of Immunology, Parasitology and Mycology, Angers, France). These six strains were susceptible to fluconazole (MIC < 8 mg/L, Etest method).
Five other strains were obtained from IHEM (Biomedical Fungi and Yeasts Collection, Brussels, Belgium) and were originally isolated from human mouth (IHEM-9581, IHEM-9582, IHEM-9584, IHEM-9586) or human blood (IHEM-10266); these strains were used in this study because they showed high MIC values of fluconazole by Etest method. These MICs have also been determined by the microdilution method (Table 1).
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Before use in the adherence experiments, blastospores were harvested, washed twice in 0.1 M phosphate-buffered saline (PBS, pH 7.2; BioMérieux) and adjusted to 1.5 x 107 blastospores/mL.
MICs of caspofungin and fluconazole
Standard antifungal powder of caspofungin (caspofungin acetate, Merck, NJ, USA) and fluconazole (Pfizer, Orsay, France) was kindly provided by the manufacturers. Caspofungin was prepared as a stock solution of 10 mg/mL in DMSO and aliquots stored at 80°C. Fluconazole was prepared as a stock solution of 10 mg/mL in water, and aliquots stored at 80°C.
The MICs of antifungals were determined using YNB-Glu medium, in experimental conditions related to adherence assays and were denoted MICYNB-glu. C. albicans inocula were prepared by suspension of the yeasts in YNB-Glu, adjusted to a final concentration of 104 cfu/mL and the MICs were determined after incubation for 24 and 48 h at 37°C, without shaking.8
The MIC was defined as the lowest drug concentration showing no visible fungal growth. Caspofungin was then used in sub-MIC conditions, and arbitrarily set as MICYNB-glu/2.
The MICs of caspofungin and fluconazole were also determined with the broth microdilution method carried out using RPMI-1640 medium with L-glutamine but without bicarbonate, buffered with 0.165 M MOPS at pH 7 (MICRPMI). All tests were carried out in duplicate.
Immobilized extracellular matrix proteins
Extracellular matrix gel (ECM gel, Sigma) was coated onto wells of 96-well tissue culture plates (polystyrene, Evergreen Scientific, USA) according to the manufacturers instructions. This gel was composed primarily of laminin, collagen type IV, heparan sulphate proteoglycan and entactin. Briefly, the wells of microtitre plates were coated with 300 µL of ECM gel (10 µg/mL); after overnight incubation at 4°C, plates were washed twice with PBS.8,9
Adherence of Candida albicans to polystyrene coated with extracellular matrix proteins
Adherence experiments were carried out in untreated 96-well tissue culture plates as previously described.8 Tetrazolium salt XTT was used to assess the adherence of C. albicans blastospores to wells of tissue culture plates: the principle was based upon the reduction of XTT tetrazolium to tetrazolium formazan by mitochondrially active C. albicans blastospores in the presence of an electron-coupling agent, menadione. Briefly, C. albicans blastospores pre-incubated or not for 18 h with sub-MIC of caspofungin were added to 96-well tissue culture plates at an inoculum of 1.5 x 107 cells/mL in 150 µL of PBS and were allowed to adhere to the polystyrene coated with extracellular matrix proteins for 2 h at 37°C; half of the wells were then washed twice with PBS to remove the non-adherent yeasts. Thereafter, 300 mg/L XTT (Sigma) and 0.13 mM menadione (Sigma) were added to all wells. Plates were incubated for 3 h at 37°C without shaking, then gently agitated and XTT formazan measured at A492nm (micro-plate reader LP400, Sanofi Diagnostics Pasteur) in washed and unwashed wells. The percentage adherence capacity of each isolate was calculated as a mean of absorbance units in washed wells/absorbance units in unwashed wells.
Background formazan values were determined with plates which contained PBS only or PBS, XTT and menadione; these values did not exceed 0.005 absorbance units and therefore were not significant. All experiments were carried out twice with six replicates.
Statistical analyses
An analysis of variance (ANOVA, P < 0.05) and a Scheffés test were conducted to determine differences among the test groups.
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Results and discussion |
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It is important to understand the mechanisms involved in biofilm formation on implanted medical devices, which could improve the prevention and the treatment of systemic candidiasis. Previous studies have demonstrated the intrinsic resistance of C. albicans biofilms to the most commonly used antifungal agents, fluconazole and amphotericin B.10 These authors recently suggested that caspofungin could affect the cellular morphology and the metabolic status of C. albicans cells within the biofilm. Results obtained in a previous study demonstrated that the activity of some antifungal agents on adherence and on metabolic activity could be different and even opposite, depending on the tested molecule.8 Caspofungin is the first representative of a new antifungal class, and its influence on fungal colonization is not yet well characterized. This paper deals with the effect of caspofungin on the adherence capacity of C. albicans blastospores to plastic coated with ECM proteins, which is considered as the first step in the development of C. albicans biofilm.3
Our results showed that the growth of C. albicans blastospores in medium containing a sub-inhibitory concentration (MIC/2) of caspofungin significantly inhibited (Scheffés test, P 0.001) the adherence capacity of yeasts to plastic coated with ECM proteins (Figure 1): nine of the 11 strains used (81.8%) were less adherent (P
0.001) than without contact with caspofungin during growth.
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In the experimental conditions of this work, the anti-adherent activity of caspofungin was less obvious when C. albicans strains were resistant to fluconazole in vitro: only three of the five fluconazole-resistant strains tested (60%) were significantly less adherent (P 0.001) in this case. So, this result suggested that the anti-adherent activity of caspofungin could be reduced in the case of in vitro fluconazole resistance and suggested a possible relationship between in vitro fluconazole resistance and the activity of caspofungin. Our results also indicated that under these experimental conditions, caspofungin (MIC/2) did not modify the metabolic activity of the yeasts independently of their resistance to fluconazole (data not shown).
In conclusion, this study demonstrated the efficiency of caspofungin in preventing adherence of C. albicans to plastic coated with proteins. This indicates that this antifungal drug could be a good candidate in the prevention of the early stage of C. albicans biofilm development and so in the prevention of candidiasis related to medical devices. Our results indicated a reduced anti-adherent activity of caspofungin when the C. albicans strains were resistant to fluconazole in vitro; further studies will be conducted to confirm this relationship.
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Acknowledgements |
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Footnotes |
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References |
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2 . Gristina, A. (1987). Biomaterial-centered infection: microbial adhesion versus tissue integration. Science 237, 158895.[ISI][Medline]
3
.
Chaffin, W. L., Lopez-Ribot, J. L., Casanova, M. et al. (1998). Cell wall and secreted proteins of Candida albicans: identification, function, and, expression. Microbiology and Molecular Biology Reviews 62, 13080.
4 . Mermel, L. A., Farr, B. M., Sherertz, R. J. et al. (2001). Guidelines for the management of intra-vascular catheter-related infections. Clinical Infectious Diseases 32, 124972.[CrossRef][Medline]
5 . Pittet, D., Tarara, D. & Wenzel, R. P. (1994). Nosocomial bloodstream infection in critically ill patients: excess length of stay, extra costs, and attributable mortality. Journal of the American Medical Association 271, 1598601.[Abstract]
6
.
Letscher-Bru, V. & Herbrecht, R. (2003). Caspofungin: the first representative of a new antifungal class. Journal of Antimicrobial Chemotherapy 51, 51321.
7 . Imbert, C., Imbert, S., Rodier, M. H. et al. (2001). Influence of sub-inhibitory concentrations of systemic antifungal agents on adherence, filamentation and mitochondrial metabolism of Candida albicans. Journal of Medical Mycology 11, 148.
8 . Imbert, C., Rodier, M. H., Daniault, G. et al. (2002). Influence of sub-inhibitory concentrations of conventional antifungals on metabolism of Candida albicans and on its adherence to polystyrene and extracellular matrix proteins. Medical Mycology 40, 1239.[ISI][Medline]
9
.
Yan, S., Rodrigues, R. G., Cahn-Hidalgo, D. et al. (1998). Hemoglobin induces binding of several extracellular matrix proteins to Candida albicans. Identification of a common receptor for fibronectin, fibrinogen, and laminin. Journal of Biological Chemistry 273, 563844.
10
.
Bachmann, S. P., VandeWalle, K., Ramage, G. et al. (2002). In vitro activity of Caspofungin against Candida albicans biofilms. Antimicrobial Agents and Chemotherapy 46, 35916.