| 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. |
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