Krishnan Suganthini, Manavathu Elias K, Chandrasekar Pranatharthi H
Division of Infectious Diseases, Department of Internal Medicine, 427 Lande Building, Wayne State University, 550 E. Canfield, Detroit, MI 48201, USA.
J Antimicrob Chemother. 2005 Jun;55(6):914-20. doi: 10.1093/jac/dki100. Epub 2005 Apr 11.
To study the in vitro fungicidal activity of voriconazole against hyphae of Aspergillus fumigatus and compare the results with those obtained for the known fungicidal drug amphotericin B.
A. fumigatus mycelia were grown on Sabouraud dextrose agar and in peptone yeast extract glucose broth until the cultures reached a mid-logarithmic growth phase. The fungicidal activities of voriconazole and amphotericin B against actively growing hyphae of A. fumigatus were examined by a kill-curve experiment and a fungal cell viability test. For the kill-curve study, the drug-treated hyphae were washed, homogenized and resuspended in 1 mL of sterile water, diluted 10-1000 fold and aliquots of 0.1 mL were spread on Sabouraud dextrose agar and allowed to grow for 48 h at 35 degrees C. The cfu were determined and plotted against drug concentrations for each time of exposure to obtain the kill curve. The viability of drug-treated A. fumigatus hyphae was determined by their ability to reduce tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.
Exposure of A. fumigatus hyphae to several concentrations (1-16 mg/L) of voriconazole or amphotericin B for various time intervals killed the hyphae in a time- and drug concentration-dependent manner. Voriconazole at 1 mg/L killed >95% of the hyphae grown on Sabouraud dextrose agar after 48 h of exposure, whereas amphotericin B at the same concentration killed approximately 70% of the hyphae after exposure for the same duration. Approximately 99% killing of hyphae grown in peptone yeast extract glucose broth was obtained for voriconazole at 1 mg/L after 48 h of exposure, whereas amphotericin B at 1 mg/L yielded approximately 82% killing after 48 h. The fungal cell viability test by tetrazolium reduction assay showed that mycelia exposed to > or =1 mg/L (Sabouraud dextrose agar blocks) and > or =2 mg/L (broth cultures) of voriconazole for 48 h completely failed to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. At low concentrations (1-2 mg/L) amphotericin B had no detectable effect on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction by drug-treated mycelia, whereas mycelia treated with 16 mg/L for 48 h showed approximately 50% inhibition of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction compared with the control.
Voriconazole possesses excellent fungicidal activity against actively growing hyphae of A. fumigatus. A comparison of results with those obtained for the known fungicidal drug amphotericin B shows that, in peptone yeast extract glucose broth, voriconazole has superior fungicidal activity against A. fumigatus hyphae compared with that of amphotericin B.
研究伏立康唑对烟曲霉菌丝的体外杀真菌活性，并将结果与已知的杀真菌药物两性霉素B的结果进行比较。
烟曲霉菌丝在沙氏葡萄糖琼脂和蛋白胨酵母提取物葡萄糖肉汤中培养，直至培养物达到对数生长期中期。通过杀灭曲线实验和真菌细胞活力测试，检测伏立康唑和两性霉素B对烟曲霉活跃生长菌丝的杀真菌活性。对于杀灭曲线研究，将经药物处理的菌丝洗涤、匀浆并重悬于1 mL无菌水中，稀释10 - 1000倍，取0.1 mL等分试样涂布于沙氏葡萄糖琼脂上，在35℃下培养48 h。测定菌落形成单位（cfu），并针对每次暴露时间的药物浓度进行绘制，以获得杀灭曲线。通过检测经药物处理的烟曲霉菌丝还原四氮唑化合物3 -（4,5 - 二甲基 - 2 - 噻唑基）- 2,5 - 二苯基 - 2H - 四氮唑溴化物的能力，来确定其活力。
将烟曲霉菌丝暴露于不同浓度（1 - 16 mg/L）的伏立康唑或两性霉素B中不同时间间隔，菌丝的死亡呈时间和药物浓度依赖性。暴露48 h后,浓度为1 mg/L的伏立康唑可杀死在沙氏葡萄糖琼脂上生长的>95%的菌丝，而相同浓度的两性霉素B在相同暴露时间后可杀死约70%的菌丝。暴露48 h后，浓度为1 mg/L的伏立康唑可杀死在蛋白胨酵母提取物葡萄糖肉汤中生长的约99%的菌丝，而浓度为1 mg/L的两性霉素B在48 h后可杀死约82%的菌丝。通过四氮唑还原试验进行的真菌细胞活力测试表明，暴露于≥1 mg/L（沙氏葡萄糖琼脂块）和≥2 mg/L（肉汤培养物）伏立康唑中的菌丝48 h完全不能还原3 -（4,5 - 二甲基 - 2 - 噻唑基）- 2,5 - 二苯基 - 2H - 四氮唑溴化物。在低浓度（1 - 2 mg/L）时，两性霉素B对经药物处理的菌丝还原3 -（4,5 - 二甲基 - 2 - 噻唑基）- 2,5 - 二苯基 - 2H - 四氮唑溴化物没有可检测到的影响，而用16 mg/L处理48 h的菌丝与对照相比，3 -（4,5 - 二甲基 - 2 - 噻唑基）- 2,5 - 二苯基 - 2H - 四氮唑溴化物还原受到约50%的抑制。
伏立康唑对烟曲霉活跃生长的菌丝具有优异的杀真菌活性。将结果与已知的杀真菌药物两性霉素B的结果进行比较表明，在蛋白胨酵母提取物葡萄糖肉汤中，伏立康唑对烟曲霉菌丝的杀真菌活性优于两性霉素B。
