Assessment of the Effect of Amphotericin B on the Vitality of Candida albicans

Abstract

The processes involved in cell death are complex, and individual techniques measure specific fractions of the total population. The interaction of Candida albicans with amphotericin B was measured with fluorescent probes with different cellular affinities. These were used to provide qualitative and quantitative information of physiological parameters which contribute to fungal cell viability. SYBR Green I and 5,(6)-carboxyfluorescein were used to assess membrane integrity, and bis-(1,3-dibutylbarbituric acid)trimethine oxonol and 3,3-dihexyloxacarbocyanine iodide were used to evaluate alterations in membrane potential. The fluorescent indicators were compared with replication competency, the conventional indicator of viability. By using these tools, the evaluation of the response of C. albicans to amphotericin B time-kill curves delineated four categories which may represent a continuum between alive and dead. The data showed that replication competency (CFU per milliliter) as determined by conventional antifungal susceptibility techniques provided only an estimate of inhibition. Interpretation of fluorescent staining characteristics indicated that C. albicans cells which were replication incompetent after exposure to greater than 0.5 μg of amphotericin B per ml still maintained degrees of physiological function.

Antifungal activity againstScedosporiumspecies and novel assays to assess antifungal pharmacodynamics against filamentous fungi

“…The carboxyfluorescein is retained by cells with intact membranes and can be observed by fluorescent microscopy. In contrast, the morbidity dye DiBAC ((1,3-dibutylbarbnituric acid) trimethine oxonol), an anionic lipophilic dye sensitive to membrane potential, is excluded by viable cells with a net negative internal charge [64,65]. Upon damage caused by antifungal exposure the fungal cells depolarize allowing the dye to penetrate and bind to lipid-rich components and fluoresce.…”

“…Fluorescent dyes that have been used against filamentous fungi include CFDA DiBAC, and FUN-1. The lipophilic, non-polar viability dye CFDA (5,(6)-carboxyfluorescein diacetate) relies on the esterase activity of viable cells to cleave the acetate moiety from the molecule producing the free anionic carboxyfluorescein [12,64,65]. The carboxyfluorescein is retained by cells with intact membranes and can be observed by fluorescent microscopy.…”

Antifungal activity and mode of action of silver nano-particles on Candida albicans

“…C. albicans cells were cultured in the presence of nano-Ag or CCCP used as a positive control, and the amounts of accumulated DiBAC 4 (3) in the cells were measured via flow cytometry by staining with DiBAC 4 (3). DiBAC 4 (3) has a high voltage sensitivity and it enters depolarized cells, where it binds to lipidrich intracellular components (Liao et al 1999). Therefore, the fluorescence of DiBAC 4 (3) increases upon membrane depolarization.…”

“…After incubation for 3 h, the cells were washed three times with a PBS. To detect any depolarization of the cell membrane, 1-ml of a PBS, containing 50 lg of bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol [DiBAC 4 (3)], was added and the samples were incubated for 1 h at 4°C in the dark (Liao et al 1999). Flow cytometric analysis was performed via a FACSCalibur flow cytometer.…”

Fluconazole susceptibility testing of Candida species by flow cytometry

“…Many fluorescence probes have been used for antifungal drug susceptibility testing of Candida spp., such as PI, 8,22,26,27 acridine orange (AO), 28,29 FDA, 28,30,31 3,3 0 -dipentyloxacarbocyanine iodide [DiOC 5 (3)], 32,33 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC 4 (3)]. 30 PI is one of the most popular fluorescence probes used for susceptibility testing. Ramani et al used PI to stain yeast cells and sodium deoxycholate to facilitate the diffusion of PI into the yeast cell membranes damaged by amphotericin B or fluconazole.…”