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Keywords:

  • Candida spp.;
  • antifungal susceptibility testing;
  • cytometry;
  • FUN-1;
  • propidium iodide;
  • JC-1

Objective  To achieve a fast and reliable determination of the susceptibility of Candida strains to amphotericin B (Am B), fluconazole (Flu) and 5-fluorocytosine (5-FC), using cytometric methods as an alternative to the classical dilution method.

Methods  Twenty-three clinical isolates of Candida with different susceptibility 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 32 mg/L), followed by staining with three different fluorochromes, under conditions previously defined through an optimisation study. These were 1 mg/L propidium iodide (PI)/106 cells for 30 min at 30 °C (a marker that only penetrates cells with severe lesions of the membrane); 0.5 µm FUN-1/106 cells for 30 min at 30 °C (a fluorescent probe which after entering the yeast cell is converted, by metabolically active yeasts, from a diffuse cytosolic pool with a yellow-green fluorescence into red cylindrical intravacuolar structures) and 0.25 µm of JC-1/106 cells for 15 min at 37 °C (a monomer that changes reversibly from green to red the J-aggregates, with the increased membrane potential). About 50 000 yeast cells were analysed by flow cytometry (FCM), at FL3 (red, 620 nm) 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 microscopy (EPM). Viability studies were performed in parallel to count the number of colony forming units.

Results  Susceptible (S) strains exposed to Am B and stained with JC-1 showed a dose-dependent decrease in the mitochondrial potential, i.e. a decreased ratio between red/green fluorescence by FCM and a decrease in J-aggregates by EPM. Neither FUN-1 nor PI was useful in the study of Am B activity. Susceptibility to Flu and 5-FC could be detected with FUN-1 staining: metabolic changes were detected by an increase in yellow–green intensity of fluorescence by FCM or a decrease of cylindrical intravacuolar structure formation by EPM, although no decrease in total viability was registered. Staining with JC-1 could predict resistance to both drugs, but did not allow distinction between sensitive dose-dependent strains (S-DD) or intermediate (I) resistance to Flu or 5-FC, respectively, from S strains. PI did not stain Candida cells treated with Flu or 5-FC under our experimental conditions.

Conclusion  Susceptibility patterns of Candida strains to Am B can be determined 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 conditions described here.