Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus.
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 1x10(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.
Mowat
Eilidh
E
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK.
Butcher
John
J
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK.
Lang
Sue
S
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK.
Williams
Craig
C
Microbiology Department, Yorkhill Hospital, Glasgow, UK.
Ramage
Gordon
G
Section of Infection and Immunity, Glasgow University Dental School and Hospital, Glasgow, UK.
D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects; D005562:Formazans / Q000378:metabolism; D025301:Hyphae / Q000254:growth & development; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability
D000935:Antifungal Agents; D005562:Formazans; C059088:1,5-bis(2-methoxy-4-nitro-5-sulfophenyl)-3-((phenylamino)carbonyl)formazan