Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy

The susceptibility of clinical Candida isolates to fluconazole was assayed by flow cytometry (FCM) and epifluorescence microscopy (EFM), with FUN-1 st aining. In all, 25 clinical isolates of Candida spp. (12 sensitive, 3 dose- dependently sensitive and 10 resistant to fluconazole according to the NCCL S M27-A protocol) were treated with increasing concentrations of fluconazol e during 1 or 2 h staining with FUN-1 for 30 min and analysed, respectively , by FCM at 575 nm (FL2) and by EFM. Fluconazole-susceptible strains showed an increased accumulation of FUN-1 in comparison with controls as determin ed by FCM and a reduced metabolic processing of the probe, confirmed by EFM . Conversely, resistant strains showed decreased FUN-1 staining and were ab le to process the probe. The fluconazole minimal inhibitory concentrations (MICs) determined by FCM or EFM after FUN-1 staining compared very well wit h the corresponding values determined by the M27-A protocol, indicating tha t FUN-1 staining can be used as an alternative to the conventional method. MIC values of resistant strains, with the exception of C. krusei, were lowe r when treatment with fluconazole followed pre-incubation with 0.1 mM sodiu m azide, a concentration known to inhibit the activity of efflux pumps. The se results show that FUN-1 staining can be used as an alternative and rapid method for the assessment of susceptibility of Candida clinical isolates t o fluconazole. Furthermore, the results suggest that resistance of Candida cells to fluconazole, with the exception of C. krusei strains, is likely to be due to the activity of efflux pumps.