Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus

Abstract

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.

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

“…However, Ͼ95% of the reduction in metabolic activity was obtained with Af293 at the highest concentrations (32 g/ml for AMB and 16 g/ml for LAMB), and there was still 10% activity 24 h after challenging CG261 with 32 g/ml of both AMB and LAMB ( Fig. 1 and 2), in agreement with previous studies (6,13,18). Conversely, AlgL did not affect the time-kill curve of A. fumigatus Af293 and CG261 biofilms challenged with 1 U/ml of the compound, while a minimal reduction in metabolic activity over the 24-h period was shown following exposure to 10 U/ml AlgL, with a maximal 26% metabolism reduction displayed after 24 h ( Fig.…”

“…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.…”

“…After biofilm formation, the medium was aspirated and the plates were washed in sterile phosphate-buffered saline (PBS) to remove planktonic and/or nonadherent cells. Biofilm biomass was assessed as described elsewhere (6). Briefly, biofilms were stained with 0.5% (wt/vol) crystal violet solution for 5 min, rinsed with distilled water, and destained with 95% ethanol.…”

“…However, high concentrations of AMB (at least 8 g/ml) are needed to inhibit hyphal clumps (6,18) or mature mycelia in vitro (13), although AMB was seen to be significantly more effective against each phase of A. fumigatus biofilm development and to display more rapid effects than other antifungals, such as voriconazole or caspofungin (13). In contrast to AMB deoxycholate, unique efficacy against Candida (C. albicans and C. parapsilosis) biofilms grown on a bioprosthetic model was displayed by lipid-formulated AMB (19), probably due to the higher doses of drug present in these formulations.…”

“…S imilar to biofilm-forming bacteria or yeasts (1), Aspergillus fumigatus, the most prevalent airborne fungal pathogen (2), is now largely acknowledged to be an organism able to grow and develop as a multicellular community (3), in which the hyphae are cohesively bonded together by a hydrophobic extracellular matrix (ECM) (4), under the aerial and static conditions found by the fungus either in vitro or in vivo (5)(6)(7)(8)(9). This growth phenotype, which complies with the definition of a biofilm (10), may help A. fumigatus to colonize the host substratum and to resist phagocytic and antimicrobial attacks, mimicking the typical Candida albicans or bacterial biofilm (8,11).…”