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.

Fungal biofilms are an escalating clinical problem associated with significant rates of mortality. Candida albicans is the most notorious of all fungal biofilm formers. However, non-Candida species, yeasts such as Cryptococcus neoformans, and filamentous moulds such as Aspergillus fumigatus, have been shown to be implicated in biofilm-associated infections. Fungal biofilms have distinct developmental phases, including adhesion, colonisation, maturation and dispersal, which are governed by complex molecular events. Recalcitrance to antifungal therapy remains the greatest threat to patients with fungal biofilms. This review discusses our current understanding of the basic biology and clinical implications associated with fungal biofilms.

These results significantly advance our understanding of the within-host population biology of P. aeruginosa during infection of patients with cystic fibrosis, and provide in vivo evidence for a link between pyocyanin production and patient morbidity.

Aspergillus fumigatus is an increasingly prevalent opportunistic fungal pathogen of various immunocompromised individuals. It has the ability to form filaments within the lungs, producing dense intertwined mycelial balls, which are difficult to treat. The aim of this study was to develop a suitable model of A. fumigatus to examine the effects of antifungal challenge on these intertwined filamentous communities. A. fumigatus NCPF 7367 growth conditions were optimized on both Thermanox coverslips and on flat-bottomed microtitre plates to establish optimal conidial seeding densities. Isolates were treated with itraconazole, voriconazole, amphotericin B and caspofungin and their overall killing efficiency was measured using an XTT formazan metabolic dye assay. This was compared with the CLSI (formerly NCCLS) methodology of broth microdilution of moulds (standard M38-A). It was shown that 1¾10 5 conidia ml "1 in RPMI 1640 was the optimum concentration of spores for biofilm formation. Filamentous growth characteristics were not observed until 10 h incubation, followed by an exponential increase in the biofilm biomass (hyphae and extracellular material) and cellular activity (metabolism). When susceptibility testing of biofilms was compared with that of planktonic cells by CLSI broth microdilution testing, all antifungal drugs were at least 1000 times less effective at reducing the overall metabolic activity of 90 % of the cells. Overall, this study showed that A. fumigatus has the ability to form coherent multicellular biofilm structures that are resistant to the effects of antifungal drugs.

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 since 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 (XTT) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately two days to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step towards the standardization of antifungal susceptibility testing of biofilms.