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Moon, Shin, Kim, Kee, Kim, Shin, Suh, and Ryang: Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis
Original Article

Korean J Leg Med 2009;29(2):127-134.

Published online: 14 February 2009

DOI: https://doi.org/10.3343/kjlm.2009.29.2.127

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

Chang Jin Moon, M.D., Jong Hee Shin, M.D., Da Woon Kim, M.D., Seung Jung Kee, M.D., Soo Hyun Kim, M.D., Myung Geun Shin, M.D., Soon Pal Suh, M.D., Dong Wook Ryang, M.D.

Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea

Corresponding author: Jong Hee Shin, M.D. Department of Laboratory Medicine, Chonnam National University Medical School, 671 Jaebong-ro, Dong-gu, Gwangju 501-757, Korea Tel: +82-62-220-5342, Fax: +82-62-224-2518 E-mail: shinjh@chonnam.ac.kr

Received 12 November 200820 January 2009       Accepted 20 January 2009

Copyright © 2009 by the Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Candida, Flow cytometry, Rhodamine 6G, Azole resistance, Mechanism

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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
Fig. 2.
Comparison of mean of rhodamine 6G GMF among clinical isolates of different Candida species.
kjlm-29-127f2.tif

∗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
ATCC strain Azole MIC (μg/mL) at 48 hr R6G accumulation (GMF)
Fluconazole Itraconazole Voriconazole
C. albicans ATCC 90028 0.5 0.125 0.03 160.4
C. albicans ATCC 64550 16 0.5 0.25 92.2
C. glabrata ATCC 90030 16 1 0.5 58.0
C. parapsilosis ATCC 22019 4 0.25 0.06 60.0
C. parapsilosis ATCC 90018 0.5 0.06 0.03 87.7
C. krusei ATCC 6258 32 0.5 0.5 194.2
C. krusei ATCC 14243 8 0.03 0.06 200.5
C. haemulonii ATCC 22991 0.25 0.03 0.06 30.0

Abbreviations: MIC, minimal inhibitory concentration; GMF, geometric mean of the fluorescence intensity.

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§

∗ , Susceptible after 24-hr incubation (MIC ≤8 μg/mL) and resistant after 48-hr incubation (MIC ≥64 μg/mL);

† , susceptible dose dependent on fluconazole (MIC 16-32 μg/mL);

‡ , resistant to more than two of the three azole antifungal agents (fluconazole, itraconazole and voriconazole) tested;

§ , values represent reduced R6G accumulation for corresponding Candida species.

Abbreviations: R6G, rhodamine 6G; SDD, dose dependent susceptible; See Table 1

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

∗ Values represent increases in the levels of gene expression relative to that of C. glabrata ATCC 90030.

Abbreviations: R6G, rhodamine 6G; See Table 1.

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