Cytometric approach for a rapid evaluation of susceptibility of Candida strains to antifungals

Objective To achieve a fast and reliable determination of the susceptibilit y of Candida strains to amphotericin B (Am B), fluconazole (Flu) and 5-fluo rocyrosine (5-FC), using cytometric methods as an alternative to the classi cal dilution method. Methods Twenty-three clinical isolates of Candida with different susceptibi lity patterns were treated for 1 h with two concentrations each of Am B (2 and 8 mg/L), Flu (8 and 64 mg/L) and 5-FC (4 and 321 mg/L), followed by sta ining with three different fluorochromes, under conditions previously defin ed through an optimisation study. These were 1 mg/L propidium iodide (PI)/1 0(6) cells for 30 min at 30 degreesC (a marker that only penetrates cells w ith severe lesions of the membrane) 0.5 muM FUN-1/10(6) cells for 30 min at 30 degreesC (a fluorescent probe which after entering the yeast cell is co nverted, by metabolically active yeasts, from a diffuse cytosolic pool with a yellow-green fluorescence into red cylindrical intravacuolar structures) and 0.25 muM JC-1/10(6) cells for 15 min at 37 degreesC (a monomer that ch anges reversibly from green to red the J-aggregates, with the increased mem brane potential). About 50000 yeast cells were analysed by flow cytometry ( FCM), at FL3 (red, 620 mm) for PI and FL2 (yellow-green, 575 nm) for FUN-1 and the ratio of FL3 to FL1 was determined (red, 620 nm/green, 525 nm) for JC-1; 200 cells of each suspension were also analysed by epifluorescence mi croscopy (EPM). Viability studies were performed in parallel to count the n umber of colony forming units. Results Susceptible (S) strains exposed to Am B and stained with JC-1 showe d a dose-dependent decrease in the mitochondrial potential, i.e. a decrease d ratio between red/green fluorescence by FCM and a decrease in J-aggregate s by EPM. Neither FUN-1 nor PI was useful in the study of Am B activity. Su sceptibility to Flu and 5-FC could be detected with FUN-1 staining: metabol ic changes were detected by an increase in yellow-green intensity of fluore scence by FCM or a decrease of cylindrical intravacuolar structure formatio n by EPM, although no decrease in total viability was registered. Staining with JC-1 could predict resistance to both drugs, but did not allow distinc tion between sensitive dose-dependent strains (S-DD) or intermediate (1) re sistance to Flu or 5-FC, respectively, from S strains. PI did not stain Can dida cells treated with Flu or 5-FC under our experimental conditions. Conclusion Susceptibility patterns of Candida strains to Am B can be determ ined by using JC-1, and to Flu and 5-FC by using FUN-1. PI was not a useful probe with which to study the effect of such antifungals under the conditi ons described here.