In vitro effects of micafungin against Candida biofilms on polystyrene and central venous catheter sections

https://doi.org/10.1016/j.ijantimicag.2006.07.024Get rights and content

Abstract

Long-term inserted and surgically implanted catheters can be colonised by Candida spp. Candida biofilms in vitro are often resistant to antifungal agents. The aim of this study was to investigate the in vitro activity of micafungin (MFG) against six Candida spp. biofilms on polystyrene (PS) and central venous catheter (CVC) sections. Safranin staining and differential interference contrast microscopy were used to demonstrate biofilm production. MFG activity was determined by the reduction in metabolic activity (%RMA) by tetrazolium reduction assay on both substrates. In vitro, Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida dubliniensis and Candida kefyr produced mature biofilms on PS and CVC sections. MFG was active against C. kefyr (0.5 μg/mL) and C. glabrata (<0.5 μg/mL) on PS. However, MFG displayed resistance (>16 μg/mL) against C. albicans, C. dubliniensis, C. tropicalis and C. parapsilosis. On CVC disks, MFG was active against C. glabrata (1 μg/mL) as well as C. parapsilosis and C. albicans (<0.5 μg/mL). MFG was resistant (>16 μg/mL) against C. dubliniensis, C. tropicalis and C. kefyr. MFG was active in vitro against all six Candida spp. on both substrates. However, MFG could not reduce the metabolic activity completely even at the highest concentration.

Introduction

Surgically implanted medical devices, such as Port or Hickman catheters, are easily colonised by the normal skin flora. Candida biofilms pose a reservoir from which Candida spp. can enter the bloodstream. In this state, immunocompromised and immunocompetent patients have an increased risk for candidaemia or invasive Candida infections [1].

Candida is able to attach to polymeric surfaces and generate a biofilm structure, protecting the yeast from host defences and antifungal drugs [2], [3]. Production of yeast biofilms is influenced by factors such as prostaglandins, growth rates, pH, dimorphism and liquid flow [4], [5], [6], [7]. Fungal biofilms represent a structure consisting of sessile fungi, a hyphal layer and a polysaccharide network of mannans and glucans known as the extracellular matrix (ECM) [8]. Inside biofilms, temperature, pH or oxygen concentrations can vary. Even flow of water or media is affected, therefore organisms live in a different environment that can support their growth and persistence [9]. Previous studies have shown that biofilms are less susceptible to antifungal drugs and that biofilm-protected Candida spp. are resistant to azoles [10], [11], [12], [13]. Once infected, the current clinical procedure is removal of the catheter [14]. Frequently, however, the device is essential for the patient and cannot be removed. A study by Bachman et al. [15] demonstrated the in vitro activity of the echinocandin caspofungin (CSP) against Candida albicans in biofilm. Cocuaud et al. [16] reported that CSP at the minimal inhibitory concentration (MIC) does not influence the metabolic activity of C. albicans in early, intermediate and mature biofilms. Kojic and Darouiche [17] reported that adherence does not differ between C. albicans and Candida parapsilosis on polystyrene and silicone catheters. However, C. parapsilosis more often colonises medical devices in vivo [18]. The new echinocandin micafungin (MFG) has so far only been tested in one small study by Kuhn et al. [8] with two clinical isolates of C. albicans and C. parapsilosis using tetrazolium (XTT)-based colorimetric assay in mature biofilms on silicone elastomer disks.

The aim of this study was to investigate the morphological characteristics of self-produced mature biofilms by six different Candida spp. on polystyrene (PS) and central venous catheter (CVC) sections using safranin staining and differential interference contrast microscopy (DIC-M). In addition, the in vitro antifungal activity of MFG was tested by XTT-based colorimetric assay on both substrates.

Section snippets

Strains

Clinical isolates of Candida spp. were originally obtained from human immunodeficiency virus (HIV)-infected children with azole-resistant oropharyngeal candidiasis (OPC) (isolates identified with superscript a), HIV-infected adults with OPC (isolates with superscript b), cystic fibrosis patients (isolates with superscript c) and pre-term infants with candidiasis (isolates with superscript d): C. albicans (B6a, B7a, CF 752419c), C. parapsilosis (B28a, B29a, CF 870714c), Candida glabrata (CF

Biofilm formation

Biofilm production was demonstrated for all six Candida spp. on PS. Safranin staining showed polysaccharides in the cell walls of blastospores, hyphae and the ECM of all tested Candida spp., as demonstrated for C. kefyr in Fig. 1. Additionally, the existence of ECM in the mature phase (24 h for C. albicans and 48 h for non-albicans species) was demonstrated by darkened spots on DIC-M. The structure of the water-containing ECM was amorphous. Polarised light was deflected by the polysaccharide

Discussion

Several studies have reported biofilm formation of C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. dubliniensis and C. kefyr on various surfaces from plastics to silastics, including silicone elastomer and CVCs. Hawser and Douglas [13], [22] formed biofilms of C. albicans on small disks of catheter material. They observed resistance to five clinically important antifungal agents as determined by tetrazolium reduction assay. A model system for studying C. parapsilosis, Candida

Acknowledgments

This study was supported by an unrestricted research grant by Fujisawa (now Astellas Pharma Inc., Tokyo, Japan). We are grateful to Prof. J. Morschhäuser, Würzburg, Germany, for providing azole-resistant Candida strains (B6, B7, B28 and B29). We thank Gil Holbrook for critical reading of the manuscript.

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