Kuhn D M, Chandra J, Mukherjee P K, Ghannoum M A
Case Western Reserve University. Division of Infectious DiseasesDepartment of Medicine, Department of Dermatology, University Hospitals of Cleveland, Cleveland, Ohio 44106.
Infect Immun. 2002 Feb;70(2):878-88. doi: 10.1128/IAI.70.2.878-888.2002.
Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.
关于真菌生物膜的了解甚少,真菌生物膜可能会导致感染和抗生素耐药性。在本研究中,通过使用医疗设备上念珠菌生物膜的临床相关模型,评估了不同念珠菌物种,特别是白色念珠菌和近平滑念珠菌的生物膜形成情况。念珠菌生物膜在硅橡胶弹性体上形成,并通过四氮唑(XTT)和干重(DW)测定进行定量。使用荧光显微镜和共聚焦扫描激光显微镜,分别用荧光增白剂(Sigma化学公司,密苏里州圣路易斯)、伴刀豆球蛋白A- Alexafluor 488(分子探针公司,俄勒冈州尤金)和FUN-1(分子探针公司)染料对形成的生物膜进行可视化观察。尽管在侵袭性白色念珠菌分离株中观察到生物膜产生的差异极小,但侵袭性和非侵袭性分离株之间存在显著差异(P < 0.001)。通过DW测定(所有比较的P均<0.001)和显微镜观察确定,白色念珠菌分离株比近平滑念珠菌、光滑念珠菌和热带念珠菌分离株产生更多的生物膜。有趣的是,非侵袭性分离株显示出比侵袭性分离株更高水平的XTT活性。在显微镜下,白色念珠菌生物膜的形态与其他物种不同,由一个基部芽生孢子层和一个由胞外多糖和菌丝组成的致密覆盖基质组成。相比之下,近平滑念珠菌生物膜的体积比白色念珠菌生物膜小,并且仅由聚集的芽生孢子组成。与浮游生长的细胞不同,念珠菌生物膜迅速(6小时内)产生氟康唑耐药性(MIC,>128μg/ml)。重要的是,XTT和FUN-1活性表明生物膜细胞具有代谢活性。总之,我们的数据表明,白色念珠菌比其他物种,特别是近平滑念珠菌,产生的生物膜在数量上更多且在质量上更复杂。