Development of a 96-well catheter-based microdilution method to test antifungal susceptibility of Candida biofilms.
Development of a 96-well catheter-based microdilution method to test antifungal susceptibility of Candida biofilms.
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开发基于 96 孔导管的微量稀释方法来测试念珠菌生物膜的抗真菌敏感性。
DOI：
10.1093/jac/dkr429
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Mukherjee,PranabK
中科院分区：
文献类型：
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作者：
Nweze,EmekaI;Ghannoum,Adam;Chandra,Jyotsna;Ghannoum,MahmoudA;Mukherjee,PranabK
背景：念珠菌膜通常与器械相关感染（包括导管相关血流感染）有关，对常用的抗真菌药物具有耐药性。目前基于微滴（96孔）板的方法来确定这些生物膜的抗真菌敏感性，不涉及临床相关的底物（例如导管），也不适合评估生物膜的表面形貌和三维结构。我们描述了一种简单，可重复的基于导管的微滴板方法来形成生物膜并评估其抗真菌敏感性。方法念珠菌在放置于微滴板上的5 mm导管圆盘上形成生物膜，并利用甲醛染料（XTT）的代谢转化进行定量。利用荧光显微镜、激光共聚焦扫描显微镜和扫描电镜对生物膜的形貌、表面形貌和三维结构进行了表征。采用优化后的XTT法评价形成的念珠菌膜对氟康唑、伏立康唑、伊曲康唑和安哌唑芬的抗真菌敏感性。结果XTT活性在90 min内达到最大值。所有测试的念珠菌在24和48 h时均在导管盘上形成坚固的生物膜（P= 0.66）。生物膜的大体形态、表面形貌和结构具有典型特征，生物膜厚度差异无统计学意义（P= 0.37）。3种唑类药物对生物膜均无活性（MIC≥64 mg/L），而阿尼达拉芬素对生物膜具有较强的活性（MIC = 0.063 ~ 0.125 mg/L）。结论该方法简便、快速、重复性好，所需药物量较小。它可以用于对生物膜的形貌和结构进行高分辨率显微分析，并评估其抗真菌敏感性。
BackgroundCandidabiofilms, which are often associated with device-related infections, including catheter-related bloodstream infections, are resistant to commonly used antifungal agents. Current microtitre (96-well) plate-based methods to determine the antifungal susceptibility of these biofilms do not involve clinically relevant substrates (e.g. catheters), and are not well suited for evaluating the surface topography and three-dimensional architecture of biofilms. We describe a simple, reproducible catheter-based microtitre plate method to form biofilms and evaluate their antifungal susceptibility.MethodsBiofilms were formed byCandidaspecies on 5 mm catheter discs placed in microtitre plates and quantified using metabolic conversion of a formazan dye (XTT). The morphology, surface topography and three-dimensional architecture of these biofilms were evaluated by fluorescence, confocal scanning laser and scanning electron microscopy, respectively. The optimized XTT method was used to evaluate the antifungal susceptibility of formedCandidabiofilms to fluconazole, voriconazole, itraconazole and anidulafungin.ResultsMaximum XTT activity was achieved within 90 min. All testedCandidastrains formed robust biofilms on catheter discs at both 24 and 48 h (P= 0.66). Biofilms exhibited typical gross morphology, surface topography and architecture, and no difference in biofilm thickness (P= 0.37). The three tested azoles were not active against the biofilms (MIC ≥64 mg/L), but anidulafungin possessed potent activity against them (MIC = 0.063–0.125 mg/L).ConclusionsThe developed method is simple, rapid and reproducible, and requires relatively small amounts of drug. It can be used to perform both high-resolution microscopic analysis of the topography and architecture of biofilms, and evaluation of their antifungal susceptibility.