School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, Scotland, UK.
Microbiology Department, Glasgow Royal Infirmary, Glasgow, Scotland, UK.
Int J Antimicrob Agents. 2015 Sep;46(3):342-5. doi: 10.1016/j.ijantimicag.2015.03.006. Epub 2015 Apr 24.
Triazoles are the mainstay of treatment for aspergillosis, although resistance to these antifungal agents may be associated with treatment failure. Refractory infections often necessitate a switch to other antifungal agents, including amphotericin B (AmB), although these infections may not resolve. The aim of this study was to investigate the effect of prior azole exposure on AmB sensitivity in Aspergillus fumigatus biofilms. It was hypothesised that sequential antifungal therapy has the potential to impact adaptive resistance mechanisms. Antifungal sensitivity was determined for each isolate against AmB±voriconazole (VRZ) exposure by a broth microdilution method and an XTT metabolic assay. To analyse the role of extracellular DNA (eDNA) and Hsp90 activation, sensitivity to AmB±DNA-digesting enzyme (DNase) or Hsp90 inhibitor [geldanamycin (GDA)] was also tested. Finally, scanning electron microscopy was performed to assess phenotypic changes. The in vitro data revealed that A. fumigatus sensitivity to AmB was decreased when it was tested in combination with VRZ. In addition, a two- to four-fold decreased sensitivity to AmB was recorded against VRZ-exposed germlings compared with controls. It was also shown that depletion of eDNA by DNase treatment enhanced AmB activity against VRZ-exposed cells by eight-fold, which visually could be explained by destabilisation of the biofilm when examined microscopically. Pharmacological inhibition of Hsp90 by GDA significantly improved biofilm susceptibility to AmB by four- to eight-fold. In conclusion, A. fumigatus pre-exposure to VRZ concomitantly induces eDNA release and activates the stress response, which collectively confers AmB resistance in vitro.
唑类药物是治疗曲霉菌病的主要药物，尽管这些抗真菌药物的耐药性可能与治疗失败有关。难治性感染通常需要改用其他抗真菌药物，包括两性霉素 B（AmB），尽管这些感染可能无法解决。本研究旨在研究唑类药物暴露前对烟曲霉生物膜中 AmB 敏感性的影响。假设序贯抗真菌治疗有可能影响适应性耐药机制。通过肉汤微量稀释法和 XTT 代谢测定法，针对每个分离株对抗 AmB±伏立康唑（VRZ）暴露的敏感性进行了测定。为了分析细胞外 DNA（eDNA）和 Hsp90 激活的作用，还测试了对抗 AmB±DNA 消化酶（DNase）或 Hsp90 抑制剂[格尔德霉素（GDA）]的敏感性。最后，进行扫描电子显微镜检查以评估表型变化。体外数据显示，当与 VRZ 联合测试时，烟曲霉对 AmB 的敏感性降低。此外，与对照相比，暴露于 VRZ 的芽管对 AmB 的敏感性降低了两到四倍。还表明，用 DNase 处理耗尽 eDNA 可使 AmB 对暴露于 VRZ 的细胞的活性增强八倍，当用显微镜检查时，这可以通过生物膜的不稳定来直观地解释。通过 GDA 对 Hsp90 的药理抑制可使 AmB 对生物膜的敏感性显著提高四到八倍。总之，烟曲霉预先暴露于 VRZ 会同时诱导 eDNA 释放并激活应激反应，这共同导致体外 AmB 耐药性。
