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.

We discuss in this work the role of Aspergillus biofilms in the clinical setting by reviewing the most recent findings on this topic. Aspergillus fumigatus can produce in vitro an extracellular hydrophobic matrix with typical biofilm characteristics under all static conditions tested, i.e., agar media, polystyrene and bronchial epithelial cells. Under static conditions the mycelial growth is greater than in shaken, submerged conditions. The extracellular matrix (ECM) is composed of galactomannan, α-1,3-glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. Typical biofilm structures were observed in the aspergillomas from two patients and in a murine model of invasive pulmonary aspergillosis. The results indicate that α-1,3-glucans plays a predominant role in the agglutination of the hyphae together in aerial conditions, and that nutrient starvation was responsible for mycelial death in aspergilloma. Melanin was produced during the infection, suggesting that this pigment is necessary for lung tissue invasion. All antifungal drugs are significantly less effective when A. fumigatus is grown under biofilm vs. planktonic conditions. Chronic persistence of a unique genotype of A. fumigatus in the respiratory tract of CF-patients and the presence of an ECM in vivo may have some therapeutical application for aspergillosis. The most appropriate antifungal drug should not be selected only on the basis of its efficiency to kill in vitro grown fungal cells, but also on its ability to penetrate the ECM.

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.

Aspergillus fumigatus is a chronic colonizer of the respiratory tract of patients with cystic fibrosis (CF). A total of 204 A. fumigatus isolates from 36 CF patients from three different medical centers, collected over a period of four months till 9.5 years, were genotyped using the short tandem repeat panel for A. fumigatus (STRAf assay). Four different colonization patterns were observed. Colonization patterns with only unique genotypes were found in 36% of the patients. In contrast 17% of the patients were chronically colonized with a single genotype. The remaining patients showed a predominant genotype or genotypes that succeed each other. In this collection no relation was found between colonization patterns and allergic bronchopulmonary aspergillosis.