In vitro evolution of itraconazole resistance in Aspergillus fumigatus involves multiple mechanisms of resistance
In vitro evolution of itraconazole resistance in Aspergillus fumigatus involves multiple mechanisms of resistance
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DOI：
10.1128/aac-.48.11.4405-4413.2004
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发表时间：
2004-11-01
影响因子：
4.9
通讯作者：
Goldman, GH
中科院分区：
文献类型：
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作者：
Ferreira, MED;Capellaro, JL;Goldman, GH
我们研究了抗真菌药物伊曲康唑在烟曲霉重复种群中的耐药性的演变，烟曲霉重复种群是从对单一药物敏感的基因型菌株中建立的，然后在统一条件下繁殖。对于每个群体，将分生孢子连续转移到含有或不含伊曲康唑的琼脂培养基中10次。在添加伊曲康唑的培养基中转移10次后，从两个群体中分离出10株伊曲康唑抗性突变株。这些突变株具有不同的生长速率和不同的伊曲康唑抗性水平。对这些突变体的麦角固醇含量的分析表明，当它们在伊曲康唑存在下生长时，它们积累麦角固醇。只有当CYP 51 A基因的N22 D和M220 I突变被用作选择标记基因时，野生型菌株的CYP 51 A基因的替换才改变了该菌株对伊曲康唑的敏感性模式。实时定量逆转录-PCR用于评估这些突变菌株中Afumdr 1、Afumdr 2、Afumdr 3、Afumdr 4、AtrF转运蛋白、CYP 51 A和CYP 51 B基因的表达水平。大多数突变体在暴露于伊曲康唑后显示出组成型高水平表达或诱导Afumdr 3、Afumdr 4和AtrF。我们的研究结果表明，药物外排泵的过度表达和/或药物靶点突变的选择是至少部分负责伊曲康唑耐药，并可能被认为是出现这种药物的临床耐药的机制。
We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.