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 TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR463/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 TR463 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 TR46 repeats. Importantly, in the laboratory, we recovered the TR463 mutation from a sexual cross between two TR46 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.

译文

这项研究调查了烟曲霉抗唑表型在两个具有相反唑暴露的堆肥堆中的动力学: 无唑和暴露的唑。热休克后,有性但非无性孢子对其具有高度抗性,无唑堆肥产生98% (49/50) 野生型和2% (1/50) 耐唑分离株,而含唑堆肥产生9% (4/45) 野生型和91% (41/45) 抗性分离株。从后者的堆肥中,80% (36/45) 分离株含有TR46/Y121F/T289A基因型,2% (1/45) 含有TR46/Y121F/M172I/T289A/G448S基因型,9% (4/45) 具有新的泛三唑耐药突变 (TR463/Y121F/M172I/T289A/G448S),具有三重46bp启动子重复。随后对一组代表性的临床烟曲霉分离株的筛选表明,新的TR463突变体已经存在于2012年收集的三个荷兰医疗中心的样品中。此外,在该组中发现了第二个新的抗性突变,其中包含四个TR46重复序列。重要的是,在实验室中,我们从来自同一含唑的堆肥的两个TR46分离株之间的有性杂交中恢复了TR463突变,可能是通过减数分裂期间双串联重复序列 (TRs) 之间的不相等杂交。含唑的堆肥中STR基因型的高度遗传多样性进一步暗示了有性生殖在突变出现中的可能作用。我们的研究证实,环境中继续出现唑类抗性突变,并表明含有唑类残基的堆肥可能是热点。需要更好地了解环境抗性选择的生物学,以保留用于食品生产和曲霉菌病治疗的唑类。含有唑残留的有机物堆肥的重要性对于烟曲霉的抗性发展和随后的抗性突变的传播可能是重要的。我们显示了在暴露于唑的堆肥中抗唑的烟曲霉的优势,并发现了具有临床相关性的新抗性突变。此外,我们的研究表明,随着新的抗性突变的不断出现,当前的杀菌剂应用是不可持续的,从而威胁到三唑在医学中的使用。我们提供的证据表明,真菌生命周期的有性部分可能在抗性突变的出现中起作用,因为在实验室条件下,我们通过来自同一堆肥堆的两种抗唑性烟曲霉分离株的有性杂交重建了抗性突变。需要了解环境中抗性选择的机制,以设计针对抗性突变积累的策略,以便保留azole类别以保护作物和治疗曲霉菌病。

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