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

:这项研究调查了两个堆肥堆中烟熏曲霉的耐唑性表型的动态,这两种堆肥与对照暴露的吡咯不同:无吡咯和暴露于吡咯。在热休克后,对性但非无性孢子具有很高的抵抗力,无唑堆肥产生98%(49/50)野生型和2%(1/50)对唑的抗药性,而含吡咯的堆肥产生9%(4/45)野生型和91%(41/45)抗性分离株。在后一种堆肥中,80%(36/45)的分离株含有TR46 / Y121F / T289A基因型,2%(1/45)具有TR46 / Y121F / M172I / T289A / G448S基因型,9%(4 / 45)具有新的泛三唑抗性突变(TR463 / Y121F / M172I / T289A / G448S),具有三重46 bp启动子重复序列。随后对代表性的临床烟曲霉菌株进行了筛选,结果显示自2012年以来从三个荷兰医学中心收集的样品中已经存在新的TR463突变体。此外,在该菌株中发现了第二个新的耐药突变,其中包含四个TR46重复序列。重要的是,在实验室中,我们可能是通过减数分裂过程中双串联重复序列(TR)之间的不相等交换,从同一个含唑的堆肥中的两个TR46分离株之间的有性杂交中恢复了TR463突变。在含唑的堆肥中,STR基因型的高水平遗传多样性进一步暗示了性繁殖在突变出现中的这种可能作用。我们的研究证实,环境中仍会继续出现唑类抗性突变,并表明含有唑类残留物的堆肥可能是热点。需要更好地了解环境抗性选择的生物学以保持唑类用于食品生产和治疗曲霉病。重要事项含吡咯残留物的有机物的堆肥对于烟曲霉的抗性发展和随后的抗性突变的传播可能很重要。在本文中,我们显示了对吡唑有抵抗力的烟曲霉在暴露于吡咯的堆肥中的优势地位,并发现了与临床相关的新的抗药性突变。此外,我们的研究表明,随着新的耐药性突变的不断出现,当前杀菌剂的应用是不可持续的,从而威胁到三唑类药物的使用。我们提供的证据表明,真菌生命周期的性部分可能在耐药性突变的出现中起作用,因为在实验室条件下,我们通过对两个具有唑类抗性的A进行有性杂交来重建耐药性突变。来自同一堆肥堆的烟熏分离株。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,以保留用于农作物保护和曲霉病治疗的唑类。

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