Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus
Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus
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DOI：
10.1016/j.ijpddr.2012.02.003
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发表时间：
2012-12-01
影响因子：
4
通讯作者：
Coleman, Glen T.
中科院分区：
文献类型：
--
作者：
Kotze, Andrew C.;Cowling, Katie;Coleman, Glen T.
虽然β-微管蛋白基因中的F200 Y SNP最常与线虫中的苯并咪唑抗性相关，但其他SNP以及药物外排途径与抗性有关。所有这些机制的相对贡献还不足以从分子数据中推断出预期的药物疗效。作为开发更好的手段来解释分子耐药性测试的一个组成部分，本研究利用了具有两个主要的苯并咪唑耐药SNP（E198 A和F200 Y）的药物抗性捻转血矛线虫分离株，以评估两个SNP赋予的相对耐药性程度。我们在体外发育试验中将幼虫暴露于一系列噻菌灵浓度，并收集每个药物浓度下存活的L3幼虫，以建立耐药性水平增加的亚群。然后，我们对混合幼虫样品中的同种型1 β-微管蛋白基因进行测序，并测量两个SNP位置的等位基因频率。随着噻菌灵浓度的增加，198位抗性等位基因的频率增加，而200位抗性等位基因的频率降低。个体幼虫的基因分型表明，最高药物浓度与除198位的纯合抗性以及200位的纯合易感性之外的所有基因型的去除相关。这表明，至少对于幼虫生命阶段，E198 A SNP能够赋予比F200 Y SNP更高水平的苯并咪唑药物抗性，并且在高度抗性个体中198处的纯合性与200位置处的杂合性或抗性纯合性是互斥的。这项研究表明，需要了解不同的耐药机制的相对贡献，以最大限度地提高分子检测能够告知耐药表型的程度。(C)2012年澳大利亚寄生虫学会出版的爱思唯尔有限公司保留所有权利。
While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype. (C) 2012 Australian Society for Parasitology Published by Elsevier Ltd. All rights reserved.