A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

Abstract

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.

In Vitro Interaction between Alginate Lyase and Amphotericin B against Aspergillus fumigatus Biofilm Determined by Different Methods

“…All the isolates were retrieved from their frozen glycerol stocks and were streaked on fresh Sabouraud dextrose agar (Kima, Padua, Italy) plates, until good sporulation was achieved following incubation at 37°C. For all experiments, conidial suspensions in RPMI 1640 medium (Sigma-Aldrich, Milan, Italy) were obtained as described elsewhere (12,28) and used to prepare the inocula (see below).…”

“…This method, originally developed for investigating yeast adhesion and susceptibility but subsequently adapted to A. fumigatus (6,31), is based on the reduction of 2,3-bis{2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium-hydroxide} (XTT) by metabolically active sessile cells (28). Briefly, biofilm-containing wells were filled with RPMI 1640 medium which contained doubling concentrations of the antifungal drugs, whereas untreated biofilm wells (positive controls) were filled with the same medium without drugs for 24 h at 37°C.…”

Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

“…In vitro biofilm assay -The protocol for biofilm growth followed that of Pierce et al (2008) with slight modifications. Briefly, Candida biofilms were formed on commercially available, presterilised, polystyrene, flatbottomed, 96-well microtitre plates (Corning Inc, Corning, NY, USA).…”

“…The level [optical density (OD)] of the formazan present in the destaining solution was measured at 540 nm using a microplate reader (Asys) (Eugendorf, Salzburg, Austria). Candida albicans SC5314 was used as a biofilm control strain (Ramage et al 2002, Pierce et al 2008.…”

“…The emergence of C. parapsilosis strains that have decreased susceptibility to azoles and that may be transmitted throughout the hospital environment may be related to the extensive use of FLC in combination with suboptimal hand hygiene and central venous catheter care in a population of seriously ill patients. Moreover, it is known that biofilms have increased levels of resistance to the most commonly used antifungal agents (Ramage et al 2001, Pierce et al 2008.…”