曲霉生物膜对常用抗真菌药物的敏感性测定及其耐药的分子机制初探

摘要/Abstract

摘要：

目的探讨曲霉生物膜对常用抗真菌药物的敏感性及其耐药的分子机制。方法利用微量液基稀释法测定22株曲霉游离孢子对两性霉素B、卡泊芬净和伏立康唑的敏感性；同时建立22株曲霉生物膜的体外模型，用抗真菌药物敏感试验联合甲基四氮盐（XTT）减低法来评价曲霉生物膜对两性霉素B、卡泊芬净和伏立康唑的敏感性。通过实时RT-PCR测定烟曲霉生物膜形成过程中药物外排泵基因及唑类药物靶酶基因的相对表达量。结果对于曲霉的游离孢子，两性霉素B的最低抑菌浓度（MIC）范围是0.5 ～ 2 mg/L，卡泊芬净的最低有效浓度（MEC）范围是0.125 ～ 0.25 mg/L，伏立康唑的MIC范围是0.5 ～ 1 mg/L；对于曲霉生物膜，两性霉素B的SMIC50（抑制50%固着性曲霉生物膜生长的最小药物浓度）和SMIC80（抑制80%固着性曲霉生物膜生长的最小药物浓度）范围分别是2 ～ > 32 mg/L和8 ～ > 32 mg/L，卡泊芬净的SMIC50和SMIC80范围分别是32 ～ > 256 mg/L和256 ～ > 256 mg/L，伏立康唑的SMIC50和SMIC80范围分别是4 ～ > 256 mg/L和32 ～ > 256 mg/L。烟曲霉生物膜形成过程中药物外排泵基因及唑类药物靶酶基因相对表达量测定显示，与0 h相比，7个待测基因在4 h时表达量没有变化；8 h时AfuMDR1、CYP51A、CYP51B表达量升高，CYP51A表达量升高最明显；12、16、24 h时AfuMDR1、AfuMDR2、AfuMDR4、CYP51A、CYP51B表达量升高，其中CYP51A表达量升高的最明显；AfuMDR3和atrF在生物膜形成过程中表达量没有明显变化。结论与游离孢子相比，曲霉生物膜对两性霉素B、卡泊芬净和伏立康唑的敏感性明显下降；烟曲霉形成生物膜以后，药物外排泵基因和唑类靶酶基因的表达量升高。

关键词: 药物敏感试验

Abstract:

Objective To explore the susceptibility of Aspergillus biofilms to common antifungal drugs and molecular mechanisms of antifungal drug resistance of Aspergillus biofilms. Methods The susceptibility of planktonic spores of 22 clinical isolates of Aspergillus spp. to amphotericin B, caspofungin and voriconazole was evaluated by a standard broth microdilution method. Meanwhile, in vitro model of Aspergillus spp. biofilm was established for the 22 isolates, then the susceptibility of Aspergillus spp. biofilm to amphotericin B, caspofungin and voriconazole was evaluated by a method for antifungal susceptibility testing combined with colorimetric XTT-reduction assay. In addition, real-time reverse transcription （RT）-PCR was performed to determine the relative expressions of drug efflux pump genes and azole target enzyme genes during the formation of Aspergillus spp. biofilms. Results In terms of planktonic spores of Aspergillus spp., the minimal inhibitory concentration （MIC） range was 0.5 to 2 mg/L for amphotericin B, 0.5 to 1 mg/L for voriconazole, and the minimal effective concentration （MEC） range was 0.125 to 0.25 mg/L for caspofungin. As far as Aspergillus spp. biofilms was concerned, the sessile minimum concentration required to inhibit the growth of 50% organisms （SMIC50） and 80% organisms （SMIC80） ranged from 2 to﹥32 mg/L and from 8 to﹥32 mg/L, respectively, for amphotericin B, from 32 to﹥256 mg/L and from 256 to >256 mg/L respectively for caspofungin, from 4 to﹥256 mg/L and from 32 to﹥256 mg/L, respectively for voriconazole. During the formation of Aspergillus spp. biofilms, no change was observed for the expression of any of the 7 tested drug efflux pump genes or azole target enzyme genes at 4 hours, while a significant increase was noted in the expression of AfuMDR1, CYP51B and CYP51A genes at 8 hours, as well as in the expression of AfuMDR1, AfuMDR2, AfuMDR4, CYP51A, CYP51B at 12, 16 and 24 hours. Of these genes, CYP51A showed the strongest increase in expression at the above 4 time points. The expression of AfuMDR3 and atrF experienced no significant change during the formation of Aspergillus spp. biofilms. Conclusions Compared to planktonic spores, Aspergillus spp. biofilms exhibit a decreased susceptibility to amphotericin B, caspofungin and voriconazole. After the formation of biofilms, the expression of drug efflux pump genes and azole target enzyme genes is elevated in Aspergillus spp.

Key words: antifungal susceptibility test

李丽娟陈伟许辉万喆李若瑜刘伟. 曲霉生物膜对常用抗真菌药物的敏感性测定及其耐药的分子机制初探[J]. 中华皮肤科杂志, 2011, 44(6): 382-386.

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