ORIGINAL ARTICLE /j x

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1 ORIGINAL ARTICLE /j x The effect of aminocandin (HMR 3270) on the in-vitro adherence of Candida albicans to polystyrene surfaces coated with extracellular matrix proteins or fibronectin E. Cateau 1, P. Levasseur 2, M. Borgonovi 2 and C. Imbert 1 1 Laboratoire de Microbiologie Fondamentale et Appliquée, UMR 6008, Service de Parasitologie, Centre Hospitalier Universitaire La Milétrie, Poitiers and 2 Anti-infective Pharmacology, Novexel SA, Parc Biocitech, Romainville, France ABSTRACT Aminocandin is a new representative of the echinocandins that could potentially affect the cellular morphology and metabolic status of Candida albicans cells within biofilms. This study investigated the influence of a sub-inhibitory concentration (MIC 2) of aminocandin on in-vitro growth of C. albicans and subsequent fungal adherence to plastic surfaces coated with fibronectin or extracellular matrix (ECM) proteins. Eleven strains of C. albicans were studied, of which six were susceptible and five were resistant to fluconazole. All 11 strains were susceptible to aminocandin in vitro, regardless of the culture medium used for the microdilution method. Aminocandin induced a significant (p <0.005) decrease in adherence when polystyrene was coated with ECM gel (ten strains) or fibronectin (seven strains). Growth in medium containing aminocandin (MIC 2) decreased the adherence of five (ECM gel) or three (fibronectin) of the six strains susceptible to fluconazole, and inhibition was observed for all five (ECM gel) or four (fibronectin) of the five fluconazole-resistant strains. Overall, the study demonstrated the anti-adherent properties of aminocandin with fluconazole-susceptible strains, and suggested that this activity was at least equivalent with fluconazole-resistant strains. Thus, the ability of aminocandin to inhibit the first step in the development of C. albicans biofilms appeared to be independent of the in-vitro resistance of C. albicans to fluconazole. Keywords Adhesion, aminocandin, biofilm, Candida albicans, echinocandins, inhibition Original Submission: 20 June 2006; Revised Submission: 21 September 2006; Accepted: 22 September 2006 Clin Microbiol Infect 2007; 13: INTRODUCTION Candida infections have been associated with various invasive indwelling medical devices, including central venous catheters, joint prostheses, and dialysis access and cardiovascular devices, as well as with superficial devices such as dentures and dental implants [1,2]. Catheterrelated infections are a major cause of morbidity and mortality among hospitalised patients, and microbial biofilms formed on catheter surfaces are associated with 90% of such infections [3]. Corresponding author and reprint requests: C. Imbert, Laboratoire de Microbiologie Fondamentale et Appliquée, UMR 6008, Service de Parasitologie, Centre Hospitalier Universitaire La Milétrie, BP 577, Poitiers Cedex, France Christine.Imbert@univ-poitiers.fr Adherence of Candida albicans to implanted medical devices is a prerequisite for colonisation, and is therefore considered to be a prerequisite for the process leading to infection. Medical devices introduced into the body are rapidly coated by adsorbed proteins [4], and this is followed by initial attachment of C. albicans blastospores and the rapid formation of a biofilm layer on the surface of the device. Individual cells in the biofilm are embedded within a matrix of frequently slimy, extracellular polymers, and typically display a phenotype that is very different from that of planktonic cells. In particular, cells in biofilms are less susceptible to antifungal agents, with the degree of resistance increasing in association with the maturation of biofilm [1,5 7]. Higher MICs and minimal fungicidal concentrations of amphotericin B, miconazole, ketoconazole Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 312 Clinical Microbiology and Infection, Volume 13 Number 3, March 2007 and itraconazole have been reported for C. albicans cells that have adhered to silicone for 2 h, compared with planktonic cells, attesting to the implications of the adherence step [8]. Only a limited number of antifungal drugs can be used to treat candidiasis associated with implanted medical devices, and infected devices generally need to be removed [9]. This type of infection can result in serious medical complications and additional treatment costs, and is a major factor limiting the prolonged use of central venous catheters. Echinocandins represent a new class of antifungal drug, with a mode of action that leads to cell-wall damage [10,11]. The cell wall is highly involved in the adherence process of C. albicans, and thus is important in the first step of biofilm formation [5]. Previous results have shown that low concentrations of caspofungin, which was the first member of the echinocandins licensed for clinical use, can modulate the in-vitro adherence of C. albicans to polystyrene coated with extracellular matrix (ECM) [12]. The present study concerns aminocandin, which is a new antifungal agent that also belongs to the echinocandin class [13]. The aim of the study was to determine whether aminocandin, used at a concentration below the MIC, could prevent adherence of C. albicans to plastic coated with ECM proteins or fibronectin. Theoretically, such activity could prevent, or at least slow down, the formation and the maturation of C. albicans biofilms on implanted medical devices. MATERIALS AND METHODS Organisms and growth conditions Eleven strains of C. albicans were studied, of which five (92, 109, 163, 182 and 240) were clinical isolates from patients with candidaemia. These strains were identified by conventional physiological and morphological methods, e.g., the germ tube test in serum, agglutination (Bichro-Latex; Fumouze, Levallois Perret, France) and metabolic properties (API 20C; bio- Mérieux, Marcy-L Etoile, France). C. albicans strain 1066, isolated originally from a patient with septicaemia, was kindly provided by R. Robert. These six strains were susceptible to fluconazole (MIC <8 mg L by Etest). Five other strains were obtained from (Biomedical Fungi and Yeasts Collection, Brussels, Belgium) and were isolated originally from the human mouth (-9581, -9582, -9584, ) or human blood (-10266); these strains had fluconazole MICs >256 mg L by Etest. C. albicans strains were first grown for 48 h at 28 C on Sabouraud agar slants (Sanofi Diagnostics Pasteur, Marnes-La- Coquette, France) to obtain a culture of synchronous stationary-phase C. albicans yeast cells [14]. A loopful of this culture was transferred to 25 ml of Yeast Nitrogen Base medium (YNB; Difco, Detroit, MI, USA), supplemented with 50 mm glucose (YNB-glu; Sigma, St Louis, MO, USA), with or without sub-mics of aminocandin, and was incubated for 18 h at 37 C without shaking. Before use in the adherence experiments, blastospores were harvested, washed twice in 0.1 M phosphate-buffered saline, ph 7.2 (PBS; biomérieux) and adjusted to a concentration of blastospores ml. MICs of aminocandin Standard aminocandin powder was kindly provided by Indevus Pharmaceuticals (Lexington, MA, USA) and was prepared as a 10 mg ml stock solution in dimethylsulphoxide, and then aliquoted and stored at )80 C. MICs of aminocandin were determined using YNB-Glu medium in experimental conditions related to adherence assays (MIC YNBglu). C. albicans inocula were prepared by suspension of cells in YNB-Glu to a final concentration of 10 4 CFU ml. MICs were determined after incubation for 48 h at 37 C without shaking [15]. The MIC was defined as the lowest drug concentration at which there was no visible fungal growth. Aminocandin was then used at a sub-mic, arbitrarily set at MIC YNB-glu 2. MICs of aminocandin were also determined using the broth microdilution method in RPMI-1640 medium with L-glutamine, but without bicarbonate, buffered with M MOPS at ph 7 (MIC RPMI ). All tests were performed in duplicate. Immobilised ECM proteins ECM gel (Sigma) or fibronectin (Sigma) were coated on the wells of polystyrene 96-well tissue culture plates (Evergreen Scientific, Los Angeles, CA, USA). ECM gel was coated according to the manufacturer s instructions. This gel was composed primarily of laminin, collagen type IV, heparin sulphate, proteoglycan and entactin. In brief, the wells of the microtitre plates were coated with 300 ll of ECM gel (10 mg L) or 300 ll of fibronectin (10 mg L), incubated overnight at 4 C, and washed twice with PBS [15,16]. Adherence of C. albicans to polystyrene coated with ECM proteins Adherence experiments were performed in untreated 96-well tissue culture plates as described previously [15]. Tetrazolium salt XTT was used to assess the adherence of C. albicans blastospores to the wells of tissue culture plates, based on the reduction of XTT tetrazolium to tetrazolium formazan by mitochondrially active C. albicans blastospores in the presence of an electron-coupling agent, menadione [12]. In brief, C. albicans blastospores, with or without pre-incubation for 18 h with a sub-mic of aminocandin, were added to 96-well tissue culture plates ( CFU ml) in 150 ll of PBS, and were allowed to adhere to the polystyrene, coated with either ECM proteins or fibronectin, for 2 h at 37 C. Half of the wells were then washed twice with PBS to remove the non-adherent yeasts. Then, XTT (Sigma) 300 mg L and 0.13 mm menadione (Sigma) were added to all wells. The plates were incubated for 3 h at 37 C without shaking, and then gently agitated before measurement of the XTT formazan concentration at A 492 nm (Micro-plate Reader LP400; Sanofi Diagnostics Pasteur) in washed and unwashed wells. The adherence capacity of each

3 Cateau et al. Effect of aminocandin on C. albicans adherence 313 isolate was calculated as a mean of absorbance units in washed wells divided by absorbance units in unwashed wells. Background formazan values were determined using plates that contained only PBS or PBS, XTT and menadione; these values did not exceed absorbance units and were therefore not significant. All experiments were performed twice with six replicates. Statistical analyses An analysis of variance (ANOVA, p <0.05) and a Scheffé test were conducted to determine differences among the test groups. RESULTS AND DISCUSSION All the isolates of C. albicans investigated were susceptible to aminocandin. Regardless of the culture medium used for microdilution, the MICs did not exceed 0.25 mg L; however, MIC YNB-glu values were higher than MIC RPMI values, with a difference of between one and four dilutions (Table 1). This is consistent with previous studies showing that the composition and the ph of the culture medium can influence MIC values [17]. The MIC RPMI values of aminocandin were in accordance with the MICs determined by Andes et al. [13] using similar experimental conditions. Previous studies have demonstrated the intrinsic resistance of C. albicans in biofilms to the most commonly used antifungal agents, namely fluconazole and amphotericin B [1,18,19], and it has been suggested that echinocandins could affect the cellular morphology and the metabolic status of C. albicans cells within biofilms. Echinocandins block the production of 1,3-b-D-glucan, a fundamental component of the fungal cell wall, and Table 1. MICs of aminocandin for the Candida albicans strains investigated C. albicans strains Aminocandin (mg L) a MICYNB-Glu MICRPMI b b b b b a MICs were determined with a broth microdilution method performed in YNB-glu medium (MIC YNB-Glu ) or in RPMI medium (MIC RPMI ). Results are representative of two separate experiments. b Resistant to fluconazole (MIC >256 mg L by Etest). cell-wall polysaccharides are important components of the Candida biofilm ECM [20]. The influence of echinocandins on fungal colonisation has not yet been well-characterised, although previous results have demonstrated the ability of caspofungin to prevent adherence of C. albicans to polystyrene surfaces coated with proteins. The present study investigated the effect of aminocandin on the adherence of C. albicans blastospores to plastic surfaces coated with ECM proteins or fibronectin. The aminocandin concentrations tested were below the MIC, as it has been shown previously that the effects of sub-inhibitory concentrations of caspofungin on planktonic cells destined to form biofilms have important implications [12,21]. The results of the present study showed that the growth of C. albicans blastospores in medium containing a sub-inhibitory concentration (MIC 2) of aminocandin significantly inhibited (Scheffé s test, p 0.005) the adherence of yeasts to plastic coated with ECM proteins or fibronectin (Figs 1 and 2). Thus, ten (plastic coated with ECM) and seven (plastic coated with fibronectin) of the 11 strains investigated were less adherent (p 0.005) than controls that had not been in contact with aminocandin during growth. Aminocandin significantly inhibited (p 0.005) the adherence capacity of all five strains that were resistant to fluconazole when polystyrene was coated with ECM proteins, and four of these strains when polystyrene was coated with fibronectin. Similarly, the adherence capacity was inhibited significantly for five and three, respectively, of the six fluconazole-susceptible strains of C. albicans. Thus the anti-adherent activity of aminocandin did not appear to be influenced by the in-vitro resistance to fluconazole. The anti-adherent activity of aminocandin can be explained by the mechanism of action of echinocandins on the yeast cell wall, which contains adhesins implicated in the contact of C. albicans with fibronectin and or the different components of the ECM [5]. Thus, disruption of the parietal surface could easily perturb the cellular adherence process. In a previous study, performed using the same experimental conditions, the anti-adherent activity of caspofungin (MIC 2) was evaluated with the same strains of C. albicans, with the results indicating a reduced anti-adherent activity of caspofungin when the strains were resistant to

4 314 Clinical Microbiology and Infection, Volume 13 Number 3, March 2007 Control Aminocandin - MIC/2 Adherence capacity (%) * Candida albicans strains 9582* 9584* 9586* 10266* Fig. 1. Effect of sub-mic aminocandin (MIC 2) on the capacity of Candida albicans strains to adhere to plastic coated with extracellular matrix proteins. The data represents average and standard deviations for two independent experiments carried out with six replicates. Aminocandin significantly inhibited (p <0.005) the adherence capacity of all strains except strain 163. Control Aminocandin - MIC/2 90 Adherence capacity (%) * 9582* Candida albicans strains 9584* 9586* 10266* Fig. 2. Effect of sub-mic aminocandin (MIC 2) on the capacity of Candida albicans strains to adhere to plastic coated with fibronectin. The data depict averages and standard deviations for two independent experiments carried out with six replicates. Aminocandin significantly inhibited (p <0.005) the adherence capacity of strains 92, 109, 240, 9581, 9582, 9584 and fluconazole in vitro [12]. This suggested a possible relationship between in-vitro fluconazole resistance and caspofungin activity. In contrast, aminocandin demonstrated anti-adherent activity against both fluconazole-susceptible and -resistant strains. These observations suggest that aminocandin could be a good candidate for preventing the early stages of C. albicans biofilm development. Further studies are required to determine the significance of preventing biofilm formation in the clinical setting. ACKNOWLEDGEMENTS The authors thank (Biomedical Fungi and Yeasts Collection, Brussels, Belgium) for providing C. albicans strains. This study was supported by Novexel S.A. REFERENCES 1. Douglas LJ. Candida biofilms and their role in infection. Trends Microbiol 2003; 11: Kojic EM, Darouiche RO. Candida infections of medical devices. Clin Microbiol Rev 2004; 17: Mukherjee PK, Zhou G, Munyon R, Ghannoum MA. Candida biofilm: a well-designed protected environment. Med Mycol 2005; 43: Gristina A. Biomaterial centered infection: microbial adhesion versus tissue integration. Science 1987; 237: Chaffin WL, Lopez-Ribot JL, Casanova M et al. Cell wall and secreted proteins of Candida albicans: identification, function, and, expression. Microbiol Mol Biol Rev 1998; 62: Mah TFC, O Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001; 9:

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