title: The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus.
abstract: OBJECTIVE
  Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that >50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations.
  
  
  METHODS
  Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT-PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates.
  
  
  RESULTS
  All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (>5-fold), 3 of which showed >30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a >500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters.
  
  
  CONCLUSIONS
  The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus.
authors.affiliation: National Aspergillosis Centre, University Hospital of South Manchester, University of Manchester, Institute of Inflammation and Repair, Manchester Academic Health Science Centre, Manchester M23 9LT, UK.
authors.forename: Marcin G
authors.initials: MG
authors.lastname: Fraczek
authors.forename: Michael
authors.initials: M
authors.lastname: Bromley
authors.forename: Ahmed
authors.initials: A
authors.lastname: Buied
authors.forename: Caroline B
authors.initials: CB
authors.lastname: Moore
authors.forename: Ranjith
authors.initials: R
authors.lastname: Rajendran
authors.forename: Riina
authors.initials: R
authors.lastname: Rautemaa
authors.forename: Gordon
authors.initials: G
authors.lastname: Ramage
authors.forename: David W
authors.initials: DW
authors.lastname: Denning
authors.forename: Paul
authors.initials: P
authors.lastname: Bowyer
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000935:Antifungal Agents / Q000494:pharmacology*; D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D002648:Child; D025141:Drug Resistance, Fungal; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D006801:Humans; D017964:Itraconazole / Q000494:pharmacology*; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D006113:United Kingdom
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D017964:Itraconazole
keywords: antifungal; aspergillosis; azole resistance; drug resistance; efflux pumps