Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on growth phase.

Anders Folkesson,Rasmus Bojsen,Birgitte Regenberg

doi:10.1186/s12866-014-0305-4

Anders Folkesson, Rasmus Bojsen + Show 1 more

Open Access

https://doi.org/10.1186/s12866-014-0305-4

Copy DOI

Abstract

BackgroundBiofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. In spite of decades of research, the connection between biofilm and drug tolerance is not fully understood.ResultsWe used Saccharomyces cerevisiae as a model for drug susceptibility of yeast biofilms. Confocal laser scanning microscopy showed that S. cerevisiae and C. glabrata form similarly structured biofilms and that the viable cell numbers were significantly reduced by treatment of mature biofilms with amphotericin B but not voriconazole, flucytosine, or caspofungin. We showed that metabolic activity in yeast biofilm cells decreased with time, as visualized by FUN-1 staining, and mature, 48-hour biofilms contained cells with slow metabolism and limited growth. Time-kill studies showed that in exponentially growing planktonic cells, voriconazole had limited antifungal activity, flucytosine was fungistatic, caspofungin and amphotericin B were fungicidal. In growth-arrested cells, only amphotericin B had antifungal activity. Confocal microscopy and colony count viability assays revealed that the response of growing biofilms to antifungal drugs was similar to the response of exponentially growing planktonic cells. The response in mature biofilm was similar to that of non-growing planktonic cells. These results confirmed the importance of growth phase on drug efficacy.ConclusionsWe showed that in vitro susceptibility to antifungal drugs was independent of biofilm or planktonic growth mode. Instead, drug tolerance was a consequence of growth arrest achievable by both planktonic and biofilm populations. Our results suggest that efficient strategies for treatment of yeast biofilm might be developed by targeting of non-dividing cells.

Highlights

Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms
Mature green fluorescent protein (GFP)-tagged biofilm cells were challenged with an antifungal agent for 24 hours and stained with propidium iodide (PI) to identify dead cells
Biofilms treated with VOR, 5FC, or CAS had the same architecture and mixture of living and dead cells as untreated control cells (Figure 1), showing that the drugs were inactive against yeast biofilms

Summary

Introduction

Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. The azoles target cytochrome P450 and inhibit cell membrane ergosterol biosynthesis, resulting in accumulation of toxic ergosterol intermediates [5]. The number of nosocomial blood isolates of these non-susceptible Candida species has increased in the past decades, possibly because of the selection that frequent azole use impose [7]. The echinocandins inhibit 1,3-β-glucan synthases, resulting in a reduction in cell wall 1,3-β-glucan [8], and the polyenes target ergosterol and cause pore formation in the fungal cell membrane [9]. Flucytosine is deaminated upon uptake in susceptible cells and converted to 5-fluorouridine triphosphate, which is incorporated into RNA, inhibiting protein synthesis [10]. Despite the pronounced diversity in antifungal mechanism of action and chemical structure, most antifungal agents are inactive against fungal biofilms [11]

Methods

Results

Discussion

Conclusion