【作者】 曾荣；

【导师】 刘维达；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学， 2014， 博士

【摘要】 第一章烟曲霉生物膜体外模型构建及其药物敏感性研究第一节烟曲霉生物膜体外模型构建及结构观察目的通过构建体外烟曲霉生物膜模型来研究生物膜的形成和结构特点。方法在预先放置细胞爬片的24孔组织培养板中进行烟曲霉生物膜构建(使用烟曲霉标准菌株AF293)。在不同的培养时间(O、2、4、6、8、10、12、16、24、48、72h)取出细胞爬片,进行FUN-1荧光染色。然后通过共聚焦激光扫描显微镜(CLSM)对生物膜的形成过程进行观察,并使用甲基四氮盐(XTT)减低法评价生物膜生长过程中活力的变化。结果烟曲霉生物膜形成过程中4h开始出现孢子的黏附；8h开始出现孢子的萌发；随后的10-16h菌丝迅速生长,相互交错,出现生物膜的立体三维结构；16-24h生物膜厚度继续快速生长；24h-72h其厚度不再增加。XTT检测结果发现在0-24h之间,生物膜活力随着生物膜成长时间而增加,直至24h后不再显著变化。结论烟曲霉能够在体外形成典型生物膜结构,该结构的形成过程及特征与其他微生物生物膜相似。第二节烟曲霉生物膜体外药物敏感性研究目的探讨烟曲霉生物膜对临床常用抗真菌药物的药物敏感性。方法建立96孔细胞培养板的烟曲霉生物膜模型。参照CLSI M-38A2推荐的丝状真菌微量液基稀释法对15株烟曲霉的悬浮状态及其生物膜状态进行药物敏感性测定。抗真菌药物包括两性霉素B、伏立康唑和米卡芬净。生物膜药物敏感性采用甲基四氮盐(XTT)减低法进行评价。结果烟曲霉悬浮状态对3种抗真菌药物的敏感性结果：15株菌株对三种抗真菌药物两性霉素B、伏立康唑、米卡芬净均为敏感；烟曲霉生物膜状态对3种抗真菌药物的敏感性结果：只有2株菌对伏立康唑敏感；15株菌对两性霉素B、米卡芬净都不敏感。结论烟曲霉生物膜的药物敏感性较游离状态明显降低,抑制生物膜的形成将有助于提高药物敏感性。第二章低热联合抗真菌药物对烟曲霉生物膜形成影响的研究第一节低热对烟曲霉生物膜形成影响的动态研究目的探索不同低热效应对烟曲霉生物膜形成的影响。方法将烟曲霉AF293的菌悬液(1×105孢子/m1)接种于预先放置细胞爬片的24孔组织培养板中,分别在三种不同低热效应下(37℃、39℃和41℃)进行培养,构建烟曲霉生物膜。分别于培养4、8、10、16、24、48、72h后使用FUN-1荧光染料染色,并运用激光共聚焦扫描显微镜(CLSM)观察各组生物膜生长情况及其厚度变化。实验数据采用t检验法进行统计分析。结果不同温度低热作用下对孢子的黏附率、黏附浓度无差异。但高温热效应能够加速孢子萌芽,加快菌丝的生长速度。但是同时高温也会导致菌丝的极性生长减弱,最终导致生物膜形成厚度的减小。结论低热效应能够减弱烟曲霉的极性生长状态,减弱其垂直方向生长的能力,进而将减少生物膜厚度形成。第二节低热联合抗真菌药物对体外烟曲霉生物膜药物敏感性影响的研究目的探讨不同低热作用联合不同抗真菌药物对烟曲霉生物膜活力的影响。方法建立96孔组织培养板烟曲霉生物膜模型。参照CLSI M-38A推荐的微量液基稀释法,同时联合不同的低热(37℃、39℃和41℃)对生物膜形成的早期阶段(4h)及晚期阶段(24h)进行药物敏感性测定。三种抗真菌药物浓度范围均为0.06～2.0μg/mL。使用甲基四氮盐(XTT)减低法和结晶紫分光光度法分别对生物膜活力和生物膜量进行检测。结果生物膜形成的早期阶段米卡芬净、伏立康唑各个药物浓度组在39℃或41℃作用下与37℃组无显著差异。然而两性霉素B组在37℃作用下,相对于39℃或41℃组能够更好的抑制生物膜的活力及其形成量。生物膜形成的晚期阶段,米卡芬净、伏立康唑和两性霉素B在39℃或41℃组,相对于37℃各个药物浓度组都能够更加有效的抑制生物膜活力及形成量。结论在生物膜形成的晚期阶段使用低热效应联合抗真菌药物将有可能成为有效的烟曲霉生物膜治疗手段。第三章卡泊芬净、米卡芬净对念珠菌药物敏感性实验的动态研究第一节卡泊芬净、米卡芬净对念珠菌体外药物敏感性的动态研究目的动态研究卡泊芬净、米卡芬净体外对念珠菌的药物敏感性。方法参照CLSI公布M-27A2方案微量液基稀释法分别测定卡泊芬净、米卡芬净、氟康唑对85株念珠菌的体外药物敏感性,并连续7d观测结果。结果48h卡泊芬净对白念珠菌、光滑念珠菌及其他念珠菌MIC50.MIC90中位数分别为0.030μg/mL.0.030μg/mL、0.060μg/mL、0.125μg/mL、0.125μg/mL、0.500μg/mL。48h米卡芬净对白念珠菌、光滑念珠菌及其他念珠菌MIC50、MIC90中位数分别为0.030μg/mL、0.060μg/mL、0.060μ/mL、0.250μg/mL、0.500μg/mL。48h氟康唑对白念珠菌、光滑念珠菌及其他念珠菌MIC80、MIC10o中位数分别128μg/mL、64μg/mL、128μg/mL、2μg/mL、32μg/mL。85株念珠菌中未见对3种药物同时耐药的菌株。卡泊芬净组白念珠菌MIC50、MIC9024h后不再升高；光滑念珠菌MIC5072h后不再升高,MIC90120h后不再升高；其他念珠菌组MIC50168h、MIC9096h后不再升高。米卡芬净组白念珠菌、光滑念珠菌MIC50、MIC9024h后不再升高；其他念珠菌MIC50、MIC9。在72h后不再升高。结论卡泊芬净、米卡芬净对念珠菌属有较好的抗菌作用,其中对白念珠菌、光滑念珠菌作用更强,且MICs随着作用时间延长而升高并存在药物特异性和念珠菌种属特异性。第二节卡泊芬净、米卡芬净对念珠菌体外药敏实验产生“矛盾现象”的动态研究目的：观察念珠菌在卡泊芬净、米卡芬净低浓度时被抑制、高浓度时出现菌落生长的“矛盾现象”的动态变化。方法在体外药敏实验中,采用CLSI公布的M-27A2方案微量液基稀释法分别测定85株念珠菌对卡泊芬净、米卡芬净产生“矛盾现象”的发生率。连续观察7d。结果48h,白念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌、都柏林念珠菌及其他念珠菌种产生“矛盾现象”的发生率在卡泊芬净组分别为90.0%、20.0%、41.7%、37.5%、33.3%、28.6%,各菌出现该现象的起始,终点药物浓度分别为4/16、8/32、8/32、2/8、2/8、8/32μg/ml;48h后,仅近平滑念珠菌、光滑念珠菌和其他念珠菌种该现象发生率仍升高。48h,在米卡芬净组仅白念珠菌、热带念珠菌、都柏林念珠菌产生“矛盾现象”,发生率分别为5.O%、25.0%、33.3%,各菌出现该现象的起始/终点药物浓度分别为4/16、4/32、1/8μg/m1；72h,光滑念珠菌出现该现象。结论“矛盾现象”的产生及出现的时间存在念珠菌种间差异性和棘白菌素类药物特异性。卡泊芬净出现“矛盾现象”的发生率高于米卡芬净。各菌株对卡泊芬净、米卡芬净产生“矛盾现象”的发生率与MIC的高低无明显相关性。更多还原

【Abstract】 Chapter I Construction of aspergillus fumigates biofilm in vitro and its susceptibility to antifungal agentsSection I The construction and architecture study of aspergillus fumigates biofilm in vitroObjective To study the development and architecture characteristics of aspergillus fumigates biofilm in vitro. Method Aspergillus fumigates AF293was used to construct the aspergillus fumigates biofilm in vitro on a plastic cell culture coverslip in24-well microculture plates. The biofilms were checked at the time points0、2、4、6、8、10、12、16、24、48、72h. After stained by FUN-1, they were recorded by scanning laser confocal microscopy (CLSM). XTT assay was used to evaluate the metabolic activity of biofilm at various time points during biofilm formation. Result A.fumigatus conidial began to adhere to the support at4h and began to germinate at8h; Hyphae began to intertwine forming a monolayer after the quick hyphae growth (10-16h); The complex three-dimensional architecture was set up at24h without depth increase from24h to72h. The metabolic activity of biofilm was increasing as time went until24h. Conclusion A.fumigates biofilm was successfully established. The process of biofilm formation and the architecture characteristics were much the same as biofilm of other microbes.Section II Study on susceptibility of antifungal drugs against biofilm of aspergillus fumigatesObjective To study the susceptibility of aspergillus fumigates biofilm to amphotericin B, voriconazole and micafungin. Method15strains suspension and biofilm of aspergillus fumigates were tested by a modified M38-A2method of CLSI biofilm in96-well microculture plates against amphotericin B, voriconazole and micafungin in vitro. XTT reduction assay was used to evaluate the effect of antifungal effect on biofilm. Result The susceptibility of the suspension of aspergillus fumigates to three antifungal agents was listed as follows:15strains were all susceptible to amphotericin B, voriconazole and micafungin; The susceptibility of the biofilm of aspergillus fumigates to three antifungal agents were listed as follows:only2strains were susceptible to voriconazole;15strains were all resistant to amphotericin B and micafungin. Conclusion Compared with suspension, mild heat stress can greatly reduce the susceptibility to antifungal agents.Chapter Ⅱ In vitro analyses of mild heat stress in combination with antifungal agents against Aspergillus fumigatus biofilmSection Ⅰ Dynamic study on the effect of mild heat stress against Aspergillus fumigatus biofilm formationObjective To study the effect of different kinds of mild heat stress aganist biofilm formation. Method Aspergillus fumigatus biofilm was formed on the support of plastic cell culture coverslips in24-well tissue culture plates after adding cell suspension of AF293. Confocal laser scanning microscopy (CLSM) was used to image and quantify Aspergillus fumigatus biofilm formation under three different regimens of continuous mild heat stress: at37,39, and41℃respectively. Results of statistical analyses were tested using Student’s/test. Result CLSM analysis showed that higher temperatures induce earlier germination and greater hyphal elongation, but poorer polar growth and reduced biofilm thickness. Compared to37℃, the adhesion rate of conidia to coverslip and the conidial concentration on a coverslip at39℃or41℃is not different from a statistical point of view. Conclusion Mild heat stress has a negative regulation effect on polar growth of hyphae which will reduce the depth of biofilm.Section Ⅱ Study on mild heat stress in combination with antifungal drugs against Aspergillus fumigatus biofilm in vitroObjective To study the effect of mild heat stress in combination with antifungal agents against Aspergillus fumigatus biofilm. Method Fungal growth has been investigated under the above conditions in combination with antifungal drugs (amphotericin B (AMB), micafungin (MCF), voriconazole (VOC)) at early and late stages. A. fumigatus biofilms were evaluated in vitro using XTT formazan metabolic dye assay and crystal violet (CV) colorimetric assay. Result In the early stage (4h) of biofilm formation, we found that the combination of39or41℃treatment with antifungal drugs (MCF and VOC), produced no visible difference in biomass formation from similar treatments at37℃with the same antifungal drugs (MCF and VOC). Interestingly, AMB treatment at37℃inhibited early stage biofilm formation to a much greater extent than at39and41℃. On the other hand, at the late stage (24h) of biofilm formation, the mild heat treatments at39and41℃with AMB, MFC, and VOC inhibited biomass formation compared to37℃, suggesting a synergistic action of mild heat stress to enhance antifungal drug activity.Conclusion Antifungal drug improvement with mild heat treatment at late stage biofilm formation provides useful indications of possible effective strategies in clinical management of aspergillosis.Chapter Ⅲ Dynamic drug sensitivity test of caspofungin and micafungin to common pathogenic CandidaSection I Dynamic study on the susceptibilities of caspofungin and micafungin to Candida spp. in vitroObjective To assess the dynamic susceptibilities of Candida spp. to caspofungin (CAS) and micafungin (MCF). Method Susceptibilities of Candida isolates to CAS、 MCF and fluconazole (FLZ) was determined by Microdilution Method based on CLSI M27-A2. The results were recorded for seven days. Result After48hours, the median of MIC50and MIC90of C. albicans, C. glabrata, other Candida spp to CAS were0.03and0.03μg/mL,0.06and0.125μg/mL,0.125and0.25μg/mL. The median of MIC50and MIC90of C. albicans, C. glabrata, other Candida spp to MCF were0.03and0.03μg/mL,0.06and0.06μg/mL,0.5and0.25μg/mL. The median of MIC50and MIC90of C. albicans, C. glabrata, other Candida spp. to FLZ were2and 128μg/mL,64and128μg/mL,2and32μg/mL. None of the85isolates of Candida spp. showed cross-resistances to CAS, MCF and FLZ. To CAS, there is no change on MIC50,MIC90of C. albicans after24hous.There was no increase on MIC50,MIC9o of C. glabrata after72hours and120hours respectively. There was no increase on MIC50, MIC90of other Candida spp. after168hours and96hours respectively. In the group of MCF, there was no change on MIC50, MIC90of C. albicans and C. glabrata. There was no increase on MIC50, MIC90of other Candida spp after72hours. Conclusion CAS and MCF may have a great antifungal activity to all of the Candida spp., especially to C. albicans, C. glabrata and the change of MICs which may be related to different kinds of echinocandin and different species of Candida would increase with the time of exposure to the drug.Section II Dynamic study on the "paradoxical effect" of echinocandins across candida species in vitroObjective To test the incidence of "Paradoxical effect" of candida species across caspofungin (CAS) and micafungin (MCF) in vitro susceptibilities dynamically.Method "Paradoxical effecf" of85isolates of different candida species to CAS and MCF was determined by M27-A Microdilution Method based on CLSI in vitro. The results were recorded for seven days. Result After48h, the incidence of "Paradoxical effecf" of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. dubliniensis and other kinds of Candida species was90%,20%,41.7%,37.5%,33.3%,28.6%to CAS, and5%,0%,0%,25%,33.3%,0%to MCF, respectively. The startpoint/endpoint of this effect were4/16,8/16,8/32,2/8,2/8,8/32μg/ml to CAS, and4/16, NA, NA,4/32,1/8, NA (μg/ml) to MCF. After48h, the incidence of this effect of C. parapsilosis, C. glabrata, other kinds of Candida species in the group of CAS were still increasing. After48h, the incidence of this effect of C. albicans, C. glabratas in the group of MCF were still increasing. Conclusions "Paradoxical effect" of Candida species to CAS, MCF in vitro and the time to completely occur was specific in different candida species and different echinocandins. The incidence of "Paradoxical effecf" of CAS was higher than MCF. The incidence of "Paradoxical effecf" of Candida species to CAS, MCF had no relation with MICs.更多还原