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Open Access Research article

Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole

Isabel Faria-Ramos1, Sofia Farinha1, João Neves-Maia1, Pedro Ribeiro Tavares1, Isabel M Miranda123, Letícia M Estevinho4, Cidália Pina-Vaz1235 and Acácio G Rodrigues1236*

Author Affiliations

1 Microbiology Department, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

2 Cardiovascular Research & Development Unit, Faculty of Medicine, University of Porto, Porto, Portugal

3 CINTESIS - Center for Health Technology and Services Research, Faculty of Medicine of the University of Porto, Porto, Portugal

4 Department of Biology and Biotechnology, CIMO-Mountain Research Center, Agricultural College of Bragança, Polytechnic Institute of Bragança, Porto, Portugal

5 Microbiology Department, Centro Hospitalar de São João, Porto, Portugal

6 Burn Unit, Centro Hospitalar de São João, Porto, Portugal

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BMC Microbiology 2014, 14:155  doi:10.1186/1471-2180-14-155

Published: 11 June 2014

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.

Keywords:
Aspergillus fumigatus; Cross-resistance; Clinical and agricultural azoles