title: Prior in vitro exposure to voriconazole confers resistance to amphotericin B in Aspergillus fumigatus biofilms.
abstract: Triazoles are the mainstay of treatment for aspergillosis, although resistance to these antifungal agents may be associated with treatment failure. Refractory infections often necessitate a switch to other antifungal agents, including amphotericin B (AmB), although these infections may not resolve. The aim of this study was to investigate the effect of prior azole exposure on AmB sensitivity in Aspergillus fumigatus biofilms. It was hypothesised that sequential antifungal therapy has the potential to impact adaptive resistance mechanisms. Antifungal sensitivity was determined for each isolate against AmB±voriconazole (VRZ) exposure by a broth microdilution method and an XTT metabolic assay. To analyse the role of extracellular DNA (eDNA) and Hsp90 activation, sensitivity to AmB±DNA-digesting enzyme (DNase) or Hsp90 inhibitor [geldanamycin (GDA)] was also tested. Finally, scanning electron microscopy was performed to assess phenotypic changes. The in vitro data revealed that A. fumigatus sensitivity to AmB was decreased when it was tested in combination with VRZ. In addition, a two- to four-fold decreased sensitivity to AmB was recorded against VRZ-exposed germlings compared with controls. It was also shown that depletion of eDNA by DNase treatment enhanced AmB activity against VRZ-exposed cells by eight-fold, which visually could be explained by destabilisation of the biofilm when examined microscopically. Pharmacological inhibition of Hsp90 by GDA significantly improved biofilm susceptibility to AmB by four- to eight-fold. In conclusion, A. fumigatus pre-exposure to VRZ concomitantly induces eDNA release and activates the stress response, which collectively confers AmB resistance in vitro.
authors.affiliation: School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, Scotland, UK.
authors.forename: Ranjith
authors.initials: R
authors.lastname: Rajendran
authors.affiliation: School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, Scotland, UK.
authors.forename: Eilidh
authors.initials: E
authors.lastname: Mowat
authors.affiliation: Microbiology Department, Glasgow Royal Infirmary, Glasgow, Scotland, UK.
authors.forename: Brian
authors.initials: B
authors.lastname: Jones
authors.affiliation: Institute of Healthcare Associated Infection, School of Health, Nursing and Midwifery, University of the West of Scotland, Paisley, Scotland, UK.
authors.forename: Craig
authors.initials: C
authors.lastname: Williams
authors.affiliation: School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, Scotland, UK. Electronic address: gordon.ramage@glasgow.ac.uk.
authors.forename: Gordon
authors.initials: G
authors.lastname: Ramage
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000502:physiology*; D018441:Biofilms / Q000187:drug effects*; D004271:DNA, Fungal / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000187:drug effects*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests; D008855:Microscopy, Electron, Scanning; D013778:Tetrazolium Salts / Q000378:metabolism; D065819:Voriconazole / Q000494:pharmacology*
chemical_list: D004271:DNA, Fungal; D018841:HSP90 Heat-Shock Proteins; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D000666:Amphotericin B; D065819:Voriconazole
keywords: Aspergillus fumigatus; Azole; Biofilm; Hsp90; Polyene; eDNA