Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
PLoS One. 2012;7(3):e31801. doi: 10.1371/journal.pone.0031801. Epub 2012 Mar 1.
Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14α-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR(34)/L98H). We investigated if TR(34)/L98H could have developed through exposure to DMIs.
Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in The Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR(34)/L98H isolate in 1998. Through microsatellite genotyping of TR(34)/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR(34)/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes.
Our findings support a fungicide-driven route of TR(34)/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs.
唑类药物在曲霉菌病的治疗中起着重要作用。唑类药物耐药性是烟曲霉菌中一个新兴的全球性问题，可能通过患者治疗而发展。此外，环境耐药发展途径已通过暴露于 14α-脱甲基酶抑制剂（DMIs）提出。与这种假定的杀真菌剂驱动途径相关的主要耐药机制是 Cyp51A 基因（TR(34)/L98H）的改变组合。我们调查了 TR(34)/L98H 是否可以通过暴露于 DMIs 而发展。
研究了 1970 年至 2005 年期间在荷兰获准用作杀真菌剂、除草剂、除草剂安全剂和植物生长调节剂的 31 种化合物对医学三唑类药物的交叉耐药性。此外，进行了 CYP51 蛋白同源建模和分子比对研究，以鉴定分子结构和对接模式的相似性。五种三唑类 DMI（丙环唑、溴菌唑、戊唑醇、环氧康唑和三氟甲戊唑醇）的分子结构与医学三唑类药物非常相似，并且在对接蛋白时采用了相似的构象。这些 DMI 还表现出最大的交叉耐药性，重要的是，它们在 1990 年至 1996 年之间获得批准使用，直接在前 1998 年恢复的第一个临床 TR(34)/L98H 分离株之前。通过 TR(34)/L98H 分离株的微卫星基因分型，我们能够计算出第一个分离株将出现在 1997 年，证实了上述实验的结果。最后，我们进行了诱导实验，以研究在实验室条件下是否可以诱导 TR(34)/L98H 的出现。一个分离株从两个串联重复序列进化到三个，表明杀真菌剂压力确实可以导致这些基因组变化。
我们的发现支持烟曲霉菌中 TR(34)/L98H 的杀真菌剂驱动途径的发展。五种三唑类 DMI 和三种医学三唑类药物的相似分子结构特征似乎是交叉耐药性发展的潜在机制。我们的发现对评估与使用三唑类 DMI 相关的健康风险具有重大意义。
