Abstract
Background
Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species.
Methods
The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C.haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates.
Results
The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3±18.5, 126.9±6.6, 88.5±18.5, 50.8±7.0, and 38.1±3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF.
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Fig. 1.
Representative histograms of rhodamine 6G accumulation obtained by flow cytometric analysis for (A) Candida albicans ATCC 90028, and (B) Candida haemulonii ATCC 22991. Histograms are given for cells incubated with rhodamine 6G for 4 hr (black area) and unlabeled controls (grey line).
![kjlm-29-127f1.tif](/upload/SynapseXML/0039kjlm/thumb/kjlm-29-127f1.gif)
Fig. 2.
Comparison of mean of rhodamine 6G GMF among clinical isolates of different Candida species.
![kjlm-29-127f2.tif](/upload/SynapseXML/0039kjlm/thumb/kjlm-29-127f2.gif)
∗P<0.05, significant difference between a Candida species and all other Candida species by Student's t-test.
Abbreviations: GMF, geometric mean of the fluorescence intensity; Flu MIC, MIC of fluconazole.
Table 1.
Intracellular accumulation of Rhodamine 6G (R6G) in Candida ATCC strains
Table 2.
Antifungal susceptibility and R6G accumulation in some Candida isolates showing a trailing growth or decreased fluconazole susceptibility
Isolate No. | Species | Interpretation | Azole MIC (μg/mL) at the indicated time | R6G accumulation (GMF) | |||||
---|---|---|---|---|---|---|---|---|---|
Fluconazole | Itraconazole | Voriconazole | |||||||
24 hr | 48 hr | 24 hr | 48 hr | 24 hr | 48 hr | ||||
1 | C. albicans | Trailer∗ | 1 | 128 | 0.03 | 0.03 | 0.03 | 0.03 | 144.4 |
2 | C. albicans | Trailer∗ | 2 | 64 | 0.03 | 0.06 | 0.03 | 0.06 | 156.0 |
3 | C. albicans | Flu SDD† | 16 | 16 | 0.03 | 0.03 | 0.03 | 0.03 | 125.5 |
4 | C. tropicalis | Trailer∗ | 0.25 | 64 | 0.03 | 16 | 0.06 | 16 | 112.4§ |
5 | C. tropicalis | Trailer∗ | 0.25 | 128 | 0.03 | 0.125 | 0.03 | 32 | 173.6 |
6 | C. tropicalis | Trailer∗ | 0.5 | 64 | 0.03 | 16 | 0.06 | 16 | 222.6 |
7 | C. tropicalis | Flu SDD† | 8 | 16 | 0.03 | 0.03 | 0.125 | 0.125 | 205.4 |
8 | C. glabrata | Azole R‡ | 32 | 64 | 2 | 2 | 1 | 4 | 33.2§ |
9 | C. glabrata | Azole R‡ | 32 | 64 | 0.5 | 1 | 1 | 2 | 39.5 |
10 | C. glabrata | Azole R‡ | 64 | 128 | 2 | 32 | 2 | 4 | 22.7§ |
11 | C. parapsilosis | Flu SDD† | 8 | 32 | 0.25 | 0.25 | 0.03 | 0.25 | 32.6§ |
Table 3.
Expression profiles of resistance-related genes for the 7 Candida glabrata isolates
Isolate No. | Fluconazole MIC (μg/mL) | R6G accumulation (GMF) | Gene expression (fold increase)∗ | |
---|---|---|---|---|
CgCDR1 | CdCDR2 | |||
8 | 64 | 33.2 | 1.12 | 2.05 |
9 | 64 | 39.5 | 1.69 | 2.35 |
10 | 128 | 22.7 | 3.03 | 4.74 |
12 | 16 | 36.8 | 0.83 | 3.90 |
13 | 16 | 46.5 | 0.53 | 2.35 |
14 | 8 | 29.1 | 0.59 | 2.93 |
15 | 16 | 42.6 | 0.48 | 2.48 |