A Novel Environmental Azole Resistance Mutation in
Aspergillus fumigatus
and a Possible Role of Sexual Reproduction in Its Emergence
Authors: Zhang Jianhua , Snelders Eveline , Zwaan Bas J. , Schoustra Sijmen E. , Meis Jacques F. , van Dijk Karin , Hagen Ferry , van der Beek Martha T. , Kampinga Greetje A. , Zoll Jan , Melchers Willem J. G. , Verweij Paul E. , Debets Alfons J. M. 
Published: June 2017

This study investigated the dynamics of
Aspergillus fumigatus
azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR
46
/Y121F/T289A genotype, 2% (1/45) harbored the TR
46
/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR
46
3
/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical
A. fumigatus
isolates showed that the novel TR
46
3
mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR
46
repeats. Importantly, in the laboratory, we recovered the TR
46
3
mutation from a sexual cross between two TR
46
isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of
Aspergillus
diseases.

IMPORTANCE
Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in
Aspergillus fumigatus
. In this article, we show the dominance of azole-resistant
A. fumigatus
in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant
A. fumigatus
isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of
Aspergillus
diseases.