Real-Time Microscopic Observation of Candida Biofilm Development and Effects Due to Micafungin and Fluconazole

Abstract

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 ...

Candida Biofilms: Development, Architecture, and Resistance

“…These investigators reported that all Candida isolates (except C. tropicalis ) displayed a paradoxical effect more frequently when grown as biofilms compared to planktonic cells. A paradoxical effect of echinocandin can also be discerned in the study by Kaneko et al ( 130 ), who compared the antibiofilm activity of micafungin against early-phase biofilms after continued exposure for up to 24 h (described above). These investigators reported that after 15 h of incubation, micafungin-exposed biofilms exhibited some regrowth, compared to exposure for 5 min, when almost all the biofilm was inhibited.…”

“…Aminocandin, a newer echinocandin, has also been shown to exhibit anti-biofilm properties ( 129 ). Recently, Kaneko et al ( 130 ) performed time-lapse microscopic observation of the effect of micafungin (an echinocandin) and fluconazole on Candida biofilms formed for up to 24 h on silicon disks in RPMI medium under flow (20 ml/h). These investigators showed that Candida biofilms grew at a uniform rate in the absence of drugs (17.2 ± 1.3 μm/h) and observed detachment of clusters of fungal cells from the hyphal tips in mature biofilms.…”

Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms

“…One of the final stages of BF production is its maturation through excretion of matrix material . Use of VRC for the treatment of C. albicans cells in BF results in the formation of cells with altered structure and function of plasma membranes .…”

“…One of the final stages of BF production is its maturation through excretion of matrix material. [18][19][20] Use of VRC for the treatment of C. albicans cells in BF results in the formation of cells with altered structure and function of plasma membranes. 21 In our study, treatment of intermediate BFs with VRC resulted in down-regulation of SUN41 and BGL2 genes, whereas the opposite effect was observed when mature biofilms were treated with VRC.…”

Detection The Prevalence of Adhesins and Extracellular hydrolytic enzymes genes in Candida albicans Biofilm Formation

“…albicans biofilm formation characterized as a series of sequential steps: (1) biofilm formation begins with adherence step which cell-wall protein-mediated adherence of yeast cells to a surface (2) Initiation step, the yeast cells proliferate across the surface and produce elongated projections that grow into filamentous forms, including hyphae or/ and pseudohyphae (3) Maturation step extracellular matrix accumulates as the biofilm matures, and high-level drug resistance is also acquired Finally, (4) non-adherent yeast cells are released from the biofilm into the surrounding medium (dispersal step). While these steps may occur concurrently rather than sequentially during natural biofilm formation [12,13].…”