Korean J Lab Med.  2009 Apr;29(2):127-134. 10.3343/kjlm.2009.29.2.127.

Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis

• Moon CJ
• Shin JH
• Kim DW
• Kee SJ
• Kim SH
• Shin MG
• Suh SP
• Ryang DW

• KMID: 854970
• DOI: http://doi.org/10.3343/kjlm.2009.29.2.127

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. CONCLUSIONS: This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.

Keyword

Candida; Flow cytometry; Rhodamine 6G; Azole resistance; Mechanism

MeSH Terms

Antifungal Agents/pharmacology
Azoles/pharmacology
Candida/chemistry/isolation & purification/*metabolism
Candidiasis/drug therapy
Drug Resistance, Fungal
Flow Cytometry/*methods
Fluconazole/pharmacology
Fluorescent Dyes/*analysis
Humans
Microbial Sensitivity Tests
Rhodamines/*analysis
Species Specificity

Figure

• 

  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).

• 

  Fig. 2. Comparison of mean of rhodamine 6G GMF among clinical isolates of different Candida species.

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