Flow Cytometry-Based Method To Detect Persisters in Candida albicans

Candida albicans biofilms contain a subpopulation whose members are defined as persisters, displaying great tolerance of fungi-cides. To directly observe such persisters, an effective method using green fluorescent protein (GFP) strain labeling by mutation of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (TDH3), combined with propidium iodide (PI) staining, was established. Amphotericin B-tolerant persisters harbor the characteristics of both GFP positivity [GFP ()] and propidium io-dide (PI) negativity [PI ()], which are easily visualized using a fluorescence microscope andmeasured by flow cytometry. Candida albicans, a common pathogenic fungus in the hospital,has caused high mortality and morbidity and has had a great economic impact (1–3). C. albicans-caused infection is most fre-quently associated with biofilm formation in immunocompro-mised patients (3). The formed biofilm is highly resistant to the antifungal treatments (4). One of the most important factors con-tributing to the resistance is the existence of a subpopulation whose members are defined as persisters and which highly tolerate agents with fungicidal activity such as amphotericin B (5). The presence of persisters often causes infection relapse when the an-tifungal treatment is terminated and often leads to recalcitran