Bouchara Jean-Philippe, Zouhair Rachid, LE Boudouil Sandrine, Renier Gilles, Filmon Robert, Chabasse Dominique, Hallet Jean-Noel, Defontaine Alain
Groupe d'Etude des Interactions Hôte-Parasite, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, 4 rue Larrey, 49033 Angers Cedex 01, *Laboratoire de Biotechnologie, UPRES 2161 Biocatalyse, 2 rue de la Houssinière, 44322 Nantes Cedex 03, †Laboratoire d'Immunologie, Centre Hospitalier Universitaire, 4 rue Larrey, 49033 Angers Cedex 01 and ‡Service Commun de Microscopie Electronique, Faculté de Médecine, rue Haute de Reculée, 49045 Angers, France.
J Med Microbiol. 2000 Nov;49(11):977-984. doi: 10.1099/0022-1317-49-11-977.
Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.
对一名接受氟康唑治疗的骨髓移植受者的同一粪便样本中分离出的两株光滑念珠菌进行了分析，其中大菌落的菌株命名为1084-L（在酵母提取物-蛋白胨-葡萄糖琼脂上生长），小菌落的菌株命名为1084-S。采用市售的纸片扩散法进行体外药敏试验，结果显示，菌株1084-L具有光滑念珠菌野生型菌株典型的药敏模式，对多烯类敏感，除噻康唑外，所有唑类化合物的抑菌圈内均有随机分布的耐药菌落。相比之下，通过脉冲场凝胶电泳和随机扩增多态性DNA发现与菌株1084-L基因密切相关的菌株1084-S，对除噻康唑外的唑类化合物表现出交叉耐药性。用E-test法测定最低抑菌浓度（MIC）证实了这些结果，表明菌株1084-S对两性霉素B更敏感，对酮康唑和氟康唑完全耐药。在以葡萄糖或甘油作为唯一碳源的琼脂平板上生长表明，耐药菌株存在呼吸缺陷，用罗丹明123染色的芽生孢子进行流式细胞术分析进一步证实了这一点。线粒体DNA（mtDNA）的限制性内切酶分析确定了呼吸缺陷的线粒体起源。然而，用引物ML1和ML6对mtDNA进行PCR扩增以及透射电子显微镜观察表明，mtDNA存在部分缺失，类似于酿酒酵母rho-小菌落突变体所描述的情况。这些结果共同提供了证据，表明在给予氟康唑后，体内可能会选择光滑念珠菌的唑类耐药小菌落突变体，这可能因此解释了抗真菌治疗的临床失败。
