유세포 분석에 의한 각종 칸디다 균종의 Rhodamine 6G 축적 분석
Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis
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배경 : 형광물질인 rhodamine 6G (R6G)는 칸디다의 다약제 내성 펌프를 통해 균 밖으로 배출되는데, 다약제 내성 유출 유전 자가 과발현된 azole 내성 칸디다 균주에서 R6G의 세포내 축적 이 감소됨이 밝혀졌다. 칸디다의 azole 약제에 대한 감수성 정도 는 균종에 따라 서로 다름이 관찰되고 있는데, 서로 다른 칸디다 균종 간의 세포내 R6G 축적을 비교한 연구는 아직 시행된 바가 없다. 본 연구에서는 6종의 칸디다 균종을 대상으로 세포내 R6G 축적양을 측정하여 비교하여 보았다. 방법 : 칸디다 임상균주 및 표준균주 48주(Candida albicans 14주, Candida tropicalis 9주, Candida glabrata 8주, Candida krusei 8주, Candida parapsilosis 7주 및 Candida haemulonii 2주)를 대상으로 하였다. 3가지 azole 항진균제(fluconazole, itraconazole, voriconazole)에 대한 감수성 검사는 CLSI M27-A2의 broth microdilution법을 이용하여 minimal inhibitory concentration (MIC)를 측정하였고, 칸디다 균 주의 세포내 R6G 축적 정도는 FACscan flow cytometer (BD biosciences, USA)를 이용하여 geometric mean of fluorescence intensity (GMF)를 측정하였다. 결과 : 세포내 R6G 축적을 측정하여 얻은 GMF의 평균은 균 종별로 C. tropicalis, C. albicans, C. krusei 및 C. parapsilosis에서 각각 167.3±18.5, 126.9±6.6, 88.5±18.5 및 50.8± 7.0순이었고, 가장 낮은 GMF를 보인 균종은 C. glabrata로서 (평균 38.1±3.9) 다른 균종에 비해 의의 있게 낮았다(P<0.05). Trailing성장을 보인 5주와 fluconazole에서만 약제의존성 내 성(MIC 16-32 μg/mL)을 보이는 2주에서는 GMF의 감소가 관 찰되지 않은 반면, 세 가지 azole 항진균제 모두에 대한 MIC가 증가된 4주 중 3주(C. glabrata 2주 및 C. parapsilosis 1주)에 서 낮은 GMF가 관찰되어 다약제 유출에 의한 내성기전이 시사 되었다. 결론 : 6종의 칸디다를 대상으로 세포내 R6G 축적량을 측정 한 결과, GMF의 평균값은 다른 칸디다 균종에 비해 C. glabrata에서 낮게 측정되어 칸디다 균종에 따라 R6G 축적량에 차이 가 있음을 알 수 있었다. 또한, R6G의 축적 검사는 azole 계열 132 문창진∙신종희∙김다운 외 5인 항진균제에 내성을 보이는 칸디다 균주에서 다약제 약물 유출에 의한 내성기전을 조사하는데도 이용될 수 있으리라 생각된다.
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.
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