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S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PM Introduction Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are commonly co-isolated from the airways of cystic fibrosis patients, especially in mixed biofilms. A mixed Af-Sm biofilm model, developed by our lab, demonstrated that Sm exhibits an antibiosis effect on Af by (1) inhibiting fungal growth, (2) rendering the fungal mycelia highly branched, and (3) increasing the fungal cell wall thickness.1 The presence of three Af cell wall polysaccharides, galactomannan (GM), galactosaminogalactan (GAG), and α-1,3-glucan, have been described in the extracellular matrix (ECM) of Af biofilms.2 GM and GAG are known to be released out from the cell wall of Af.3 GAG contains N-acetylgalactosamines (GalNAc) which undergo partial deacetylation extracellularly, to be converted to GalN, and thus transform this polysaccharide into a polycation with strong surface binding properties.4 Aim of the study Analyze the structure of fungal cell wall polysaccharides in the formation of Af biofilm alone or mixed with Sm. Methods The Af-Sm co-culture was prepared on an 8-chambers Lab-Tek slide as described previously1. Anti-GAG and anti-GM monoclonal antibodies produced at the Aspergillus Unit at Pasteur Institute of Paris, were used to focus on the 3D structure of GAG and GM in the ECM of the biofilm. These polysaccharides were analyzed by fluorescence confocal microscopy at 24 h. Results and Conclusion GM was found, as expected, in the hyphal Af cell wall but was very little in the ECM. GAG was also found in the cell wall but mainly formed a beautiful fibrillary network between the hyphae, showing the importance of this polysaccharide in cell-cell interaction and in the structuration of Af biofilm. GAG could be the surface receptor for Sm, which would promote strong adhesion between Sm and Af in the biofilm. Sources 1. Melloul E et al. Front Microbiol. 2018; 2. Beauvais A et al. Microbiol Spectr. 2015; 3. Fontaine T et al. J Fungi. 2020; 4. Briard B et al. Mycologia 2016.

Aspergillosis is pervasive in bird populations, especially those under human care. Its management can be critically impacted by exposure to high levels of conidia and by resistance to azole drugs. The fungal contamination in the environment of a Humboldt penguin (Spheniscus humboldti) group, housed in a French zoological park next to numerous large crop fields, was assessed through three serial sessions of surface sampling in nests, in 2018–20: all isolates were counted and characterized by sequencing. When identified as Aspergillus fumigatus, they were systematically screened for resistance mutations in the cyp51A gene and tested for minimal inhibitory concentrations (MICs) determination. At the same time, the clinical incidence of aspergillosis was evaluated in the penguin population by the means of systematic necropsy and mycological investigations. A microsatellite-based analysis tracked the circulation of A. fumigatus strains. Environmental investigations highlighted the substantial increase of the fungal load during the summer season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati, ranging from 22.7 to 94.6% relative prevalence. Only one cryptic species was detected (A. nishimurae), and one isolate exhibited G138S resistance mutation with elevated MICs. The overall incidence of aspergillosis was measured at ∼3.4% case-years, and mostly in juveniles. The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus clinical isolates. In contrast, one environmental strain appeared largely overrepresented during the summer sampling session. In all, the rural location of the zoo did not influence the emergence of resistant strains. Lay summary