title: Azole resistance of Aspergillus fumigatus biofilms is partly associated with efflux pump activity.
abstract: This study investigated the phase-dependent expression and activity of efflux pumps in Aspergillus fumigatus treated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P<0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P<0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for the A. fumigatus germling cells by 2- to 8-fold. Quantitative expression analysis of AfuMDR4 mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P<0.001). Finally, an in vivo mouse biofilm model was used to evaluate efflux pump expression, and it was shown that AfuMDR4 was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P<0.01). Our results demonstrate that efflux pumps are expressed in complex A. fumigatus biofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis.
authors.affiliation: School of Medicine, College of Medicine, Veterinary and Life Sciences, University of Glasgow, and Microbiology Department, Royal Hospital for Sick Children, 378 Sauchiehall Street, Glasgow G2 3JZ, United Kingdom.
authors.forename: Ranjith
authors.initials: R
authors.lastname: Rajendran
authors.forename: Eilidh
authors.initials: E
authors.lastname: Mowat
authors.forename: Elaine
authors.initials: E
authors.lastname: McCulloch
authors.forename: David F
authors.initials: DF
authors.lastname: Lappin
authors.forename: Brian
authors.initials: B
authors.lastname: Jones
authors.forename: Sue
authors.initials: S
authors.lastname: Lang
authors.forename: Jayesh B
authors.initials: JB
authors.lastname: Majithiya
authors.forename: Peter
authors.initials: P
authors.lastname: Warn
authors.forename: Craig
authors.initials: C
authors.lastname: Williams
authors.forename: Gordon
authors.initials: G
authors.lastname: Ramage
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology / Q000627:therapeutic use; D001228:Aspergillosis / Q000188:drug therapy; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D001393:Azoles / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008826:Microbial Sensitivity Tests; D011743:Pyrimidines / Q000494:pharmacology / Q000627:therapeutic use; D014230:Triazoles / Q000494:pharmacology / Q000627:therapeutic use; D065819:Voriconazole
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D011743:Pyrimidines; D014230:Triazoles; D065819:Voriconazole