title: Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole.
abstract: BACKGROUND
  The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered.
  
  
  RESULTS
  MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation.
  
  
  CONCLUSIONS
  PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.
authors.forename: Isabel
authors.initials: I
authors.lastname: Faria-Ramos
authors.forename: Sofia
authors.initials: S
authors.lastname: Farinha
authors.forename: João
authors.initials: J
authors.lastname: Neves-Maia
authors.forename: Pedro Ribeiro
authors.initials: PR
authors.lastname: Tavares
authors.forename: Isabel M
authors.initials: IM
authors.lastname: Miranda
authors.forename: Letícia M
authors.initials: LM
authors.lastname: Estevinho
authors.forename: Cidália
authors.initials: C
authors.lastname: Pina-Vaz
authors.affiliation: Microbiology Department, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal. agr@med.up.pt.
authors.forename: Acácio G
authors.initials: AG
authors.lastname: Rodrigues
mesh_terms: D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification; D001393:Azoles / Q000494:pharmacology*; D003470:Culture Media / Q000737:chemistry; D025141:Drug Resistance, Fungal; D004783:Environmental Microbiology; D005659:Fungicides, Industrial / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D012692:Serial Passage; D013696:Temperature
chemical_list: D001393:Azoles; D003470:Culture Media; D005659:Fungicides, Industrial; D007093:Imidazoles; C045362:prochloraz