dTo understand the process of Candida biofilm development and the effects of antifungal agents on biofilms, we analyzed realtime data comprising time-lapse images taken at times separated by brief intervals. The growth rate was calculated by measuring the change of biofilm thickness every hour. For the antifungal study, 5-h-old biofilms of Candida albicans were treated with either micafungin (MCFG) or fluconazole (FLCZ). MCFG began to suppress biofilm growth a few minutes after the initiation of the treatment, and this effect was maintained over the course of the observation period. In contrast, the suppressive effects of FLCZ on biofilm growth took longer to manifest: biofilms grew in the first 5 h after treatment, and then their growth was suppressed over the next 10 h, finally producing results similar to those observed with MCFG. MCFG was also involved in the disruption of cells in the biofilms, releasing string-like structures (undefined extracellular component) from the burst hyphae. Thus, MCFG inhibited the detachment of yeast cell clusters from the tips of hyphae. In contrast, FLCZ did not disrupt biofilm cells. MCFG also showed fast antifungal activity against Candida parapsilosis biofilms. In conclusion, our results show that inhibition of glucan synthesis due to MCFG contributed not only to fungicidal activity but also to the immediate suppression of biofilm growth, while FLCZ suppressed growth by inhibiting ergosterol synthesis. Therefore, those characteristic differences should be considered when treating clinical biofilm infections. P athogenic fungi in the genus Candida can cause both superficial and serious systemic diseases and are now widely recognized as important agents of hospital-acquired infection (1). Candida albicans is known as a biofilm former. Bloodstream infections are frequently associated with the use of a catheter, and the catheter can be a scaffold for biofilms (2). Once biofilms are formed, the biofilms continuously supply detaching cells as a source of infection; thus, biofilm-related infection is associated with a poor prognosis (2, 3). Recently, research into molecular mechanisms related to biofilm formation has revealed transcriptional regulation (4); however, the majority of these related studies lack realtime observation of the development process, and therefore, the fate of the biofilm and the effects of antifungals against biofilms are not clearly understood. Knowing the mechanism of antifungal action against biofilms would provide us with key information when considering therapeutic strategy against clinical infections related to biofilms. In this study, we analyzed images obtained by time-lapse photography to investigate the developmental process and detachment of biofilms, as well as the antibiofilm effects of the echinocandin micafungin (MCFG), which inhibits cell wall glucan synthesis, and the azole fluconazole (FLCZ), which inhibits cell membrane ergosterol synthesis. MATERIALS AND METHODSChemicals. All general chemicals used in this study were purchased from Wako ...

The thallus diameter is commonly used as a quantitative parameter to evaluate hyphal growth. However, a different parameter is required to evaluate hyphal growth more precisely. The hyphal growth of Trichophyton rubrum in the presence of antimycotic agents was evaluated using the number of hyphal crossings as a quantitative parameter. Continuous video images of hyphal growth were taken for 48 h. Culture medium contained 0.4 μg ml(-1) of terbinafine (TBF) and itraconazole (ITCZ). Image analyses were performed every 6 h using a 50 μm square grid. The mean density of the hyphal crossings in each sampling frame was used as a parameter of hyphal growth. The mean ratio of hyphal crossings on distressed hyphae to total hyphal crossings was used as a parameter representing the antimycotic effects of TBF and ITCZ. The mean density of total hyphal crossings in the TBF group was significantly lower than in the control and ITCZ groups. The ratio of distressed hyphae significantly increased during the 48-h time course in the TBF group, but not in the ITCZ group. Counting the number of hyphal crossings provides a new method for assessing hyphal growth and antimycotic activity quantitatively.