Aspergillus fumigatus is a leading cause of death in immunocompromised patients and a frequent colonizer of the respiratory tracts of asthma and cystic fibrosis (CF) patients. Biofilms enable bacteria and yeasts to persist in infections and can contribute to antimicrobial resistance. We investigated the ability of A. fumigatus to form biofilms on polystyrene (PS) and human bronchial epithelial (HBE) and CF bronchial epithelial (CFBE) cells. We developed a novel in vitro coculture model of A. fumigatus biofilm formation on HBE and CFBE cells. Biofilm formation was documented by dry weight, scanning electron microscopy (SEM), and confocal scanning laser microscopy (CSLM). The in vitro antifungal activities of seven antifungal drugs were tested by comparing planktonic and sessile A. fumigatus strains. A. fumigatus formed an extracellular matrix on PS and HBE and CFBE cells as evidenced by increased dry weight, SEM, and CSLM. These biofilms exhibited decreased antifungal drug susceptibility and were adherent to the epithelial cells, with fungi remaining viable throughout 3 days. These observations might have implications for treatment of A. fumigatus colonization in chronic lung diseases and for its potential impact on airway inflammation, damage, and infection.

The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and in part immunocompetent patients. A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic- and biofilm-grown A. fumigatus mycelium after 24 and 48 h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24 h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes, which code for hydrophobins, and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by real-time PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may also play a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

Effects of voriconazole combined with micafungin against 101 isolates of Candida spp. and 100 isolates of filamentous fungi have been evaluated by in vitro checkerboard analysis. The combination was indifferent for 97% of the Candida isolates and synergistic for 64% of the filamentous fungi (79% for Aspergillus fumigatus).The combination of two antifungal agents, voriconazole (VRC) and micafungin (MFG), was assessed for synergism against clinically relevant yeasts and molds. So far, only one little in vitro study including 10 isolates of Aspergillus fumigatus has been performed on the specific combination of VRC and MFG (6,9). In the present study, we have investigated the in vitro interaction between VRC and MFG by checkerboard assays against 196 clinical isolates from patients, including fluconazole-resistant strains from human immunodeficiency virus-infected patients (15) The assay preparation for Candida was performed in accordance to NCCLS document M27-A2 (17). Since the NCCLS M38-A (16) reference method for testing filamentous fungi does not give recommendations for in vitro testing of echinocandins, minor modifications were necessary. Several preliminary tests demonstrated an incubation temperature of 37°C and an incubation time of 24 h (11, 12) as optimal conditions. Conidia were counted on a hemocytometer. Measuring the metabolic activity by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTT) colorimetric method was used to determine the MIC endpoints (10). Standard antifungal powder of VRC was provided by Pfizer (New York, N.Y.), while MFG was provided by Fujisawa (now Astellas Pharma Inc., Tokyo, Japan). The final concentration of the antifungal agents ranged from 0.0156 to 4 g/ml for VRC and from 0.002 to 128 g/ml for MFG. The interactions were investigated by a checkerboard titration broth microdilution-based method. 96-well round-bottom plates (Corning BV, SchipholRijk, The Netherlands) were prepared with twofold concentrated RPMI 1640 medium (with L-glutamine, without bicarbonate)

This article was originally published in Proteomics 2010, 10, 3097–3107, DOI 10.1002/pmic.201000129

Cross-resistance among Candida albicans isolates from the oropharynges of human immunodeficiency virus-infected patients (n = 16) and environmental yeast strains of various species (n = 54) to medical and agricultural azole drugs was observed. Precautions against the unnecessary widespread use of azoles in the environment and human medicine are strongly recommended to prevent patients from acquiring azole-resistant yeasts.