Martinez Luis R, Casadevall Arturo
Department of Medicine and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, USA.
Antimicrob Agents Chemother. 2006 Mar;50(3):1021-33. doi: 10.1128/AAC.50.3.1021-1033.2006.
Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.
微生物生物被膜通过分别保护微生物细胞免受宿主防御和抗菌药物的影响，从而促进毒力和抗生素耐药性。已证明新生隐球菌可在聚苯乙烯微量滴定板中形成生物被膜。使用分散生物被膜的菌落形成单位数量、2,3-双（2-甲氧基-4-硝基-5-磺基苯基）-5-[(苯胺基)羰基]-2H-氢氧化四唑还原法以及光学显微镜和共聚焦显微镜，分别测量真菌量、代谢活性和新生隐球菌生物被膜的外观。新生隐球菌生物被膜的形成遵循标准的事件序列：真菌表面附着、微菌落形成和基质产生。研究了生物被膜和浮游表型的新生隐球菌细胞对四种抗真菌药物的敏感性。新生隐球菌细胞或预先形成的隐球菌生物被膜暴露于氟康唑或伏立康唑，分别不会导致酵母生长抑制，也不会影响生物被膜的代谢活性。相比之下，新生隐球菌细胞和预先形成的生物被膜对两性霉素B和卡泊芬净均敏感。然而，新生隐球菌生物被膜对两性霉素B和卡泊芬净的耐药性明显高于浮游细胞，如果隐球菌细胞含有黑色素，它们对这些药物的敏感性会进一步降低。使用斑点酶联免疫吸附测定法以及光学显微镜和共聚焦显微镜来研究抗真菌药物如何影响新生隐球菌生物被膜的形成。两性霉素B和卡泊芬净干扰新生隐球菌生物被膜形成的机制涉及荚膜多糖释放和黏附。我们的结果表明，生物被膜形成可能会降低隐球菌感染期间某些抗真菌药物的疗效。
