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Hyun Woo Choi, Jong Hee Shin, Sook In Jung, Kyung Hwa Park, Duck Cho, Seung Jung Kee, Myung Geun Shin, Soon Pal Suh and Dong Wook Ryang Antimicrob. Agents Chemother. 2007, 51(4):1520. DOI: 10.1128/AAC.01141-06. Published Ahead of Print 5 February 2007. Downloaded from http://aac.asm.org/ on March 1, 2012 by guest
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-D-glucan synthase. No. Published ahead of print on 5 February 2007. ﬂuconazole. The MICs for sessile cells (bioﬁlms) were determined using a microtiter-based assay (3.00 0 doi:10. albicans is the most commonly isolated Candida species.1128/AAC. micafungin. 1. 1520 . Vol. 2007. 12 Candida parapsilosis.1 Seung Jung Kee. The inhibitory effects of the antifungals were measured as the optical densities (ODs) of the antifungal-treated wells relative to those of the control (antifungal-free) wells (considered to be 100%) as determined TABLE 1. albicans C. other non-C. Distributions of ﬂuconazole MICs for different Candida species strains under planktonic or bioﬁlm (sessile-cell) growth conditions Species No. While C. glabrata but not those formed by C.2 Duck Cho.03 g/ml. We compared the in vitro activities of caspofungin. We examined 43 Candida species isolates. South Korea.1 and Dong Wook Ryang1 Departments of Laboratory Medicine1 and Internal Medicine. tropicalis C. there have been few comparisons of the activities of echinocandins against bioﬁlms formed by different Candida species.1 Myung Geun Shin. Phone: 82 (62) 2205342. parapsilosis C. Fax: 82 (62) 224-2518.024 to 2 g/ml. However. 51. 4 Species-Speciﬁc Differences in the Susceptibilities of Bioﬁlms Formed by Candida Bloodstream Isolates to Echinocandin Antifungals Hyun Woo Choi. * Corresponding author.06 No. Downloaded from http://aac. Gwangju 501-757.2 Kyung Hwa Park.01141-06 Copyright © 2007. In this system. The drugs were prepared in a series of twofold dilutions as follows: ﬂuconazole. albicans. South Korea Received 11 September 2006/Returned for modiﬁcation 26 October 2006/Accepted 28 January 2007 The echinocandin susceptibilities of bloodstream Candida isolates growing in a bioﬁlm was investigated. Within the therapeutic range of concentrations of each drug. and the two echinocandins.03 0. amphotericin B. of isolates tested Type of MICa 0. mature bioﬁlms were allowed to form in 96-well microtiter plates for 48 h and the cell densities of the bioﬁlms were estimated using the 2.12 0. between January 1999 and December 2003. 16). E-mail: shinjh@chonnam. albicans species have been increasingly recognized as catheter-related BSI pathogens (4.1* Sook In Jung. Gwangju. South Korea. American Society for Microbiology. Apr. 8 Hakdong Dongku.1 Jong Hee Shin. including 12 C.3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrozolium5-carboxanilide (XTT) absorbance assay. The MICs of the two echinocandins for planktonic cells were deﬁned as the lowest concentrations resulting in the prominent inhibition of growth as determined after 24 h of incubation (11).ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. 10 Candida tropicalis. Mailing address: Department of Laboratory Medicine.1 Soon Pal Suh.5 1 2 4 8 16 32 64 1.06 g/ml. have recently been demonstrated to be active against Candida albicans bioﬁlms (3.kr.ac.2 Chonnam National University Medical School. and 9 Candida glabrata isolates. 32 to 0. 15). tropicalis or C. 1520–1523 0066-4804/07/$08. glabrata 12 12 10 9 MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells 3 4 1 1 4 1 3 4 2 3 1 1 1 2 2 1 7 12 10 12 8 10 1 6 2 1 2 1 5 1 8 9 a MIC50s and MIC80s were determined by measuring XTT activity reduction. Gwangju. of isolates for which indicated MIC ( g/ml) was: 0.3. 16 to 0.25 0. Although fungal bioﬁlm-associated infections are frequently refractory to conventional antifungal therapy. 14). 16). which constitute a new class of antifungals that inhibit 1.asm. the echinocandins. 10. and amphotericin B against bioﬁlms formed by BSI isolates of four different Candida species. Chonnam National University Medical School.024 C.org/ on March 1. The MICs for planktonic cells were determined by the standard CLSI (Clinical and Laboratory Standards Institute) M27-A2 broth microdilution method (5). All of the isolates were from blood cultures acquired at Chonnam National University Hospital. 2012 by guest Bioﬁlm-mediated antifungal resistance is a well-documented phenomenon for Candida species and probably contributes to Candida pathogenicity in catheter-related bloodstream infections (BSIs) (6. caspofungin and micafungin were active against bioﬁlms formed by Candida albicans or C. p. parapsilosis. All Rights Reserved.
Downloaded from http://aac. The median mica- TABLE 3. 2007 NOTES 1521 TABLE 2. while the median MIC80 for sessile cells ranged from 2 to 32 g/ml. All isolates were tested at least twice. whereas ﬂuconazole is ineffective.03 No. glabrata 12 12 10 9 MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells 3 3 2 2 1 4 1 1 1 2 9 3 8 2 9 2 6 4 1 6 4 5 4 1 1 1 1 4 5 2 1 1 2 2 2 1 1 1 1 1 1 4 7 4 2 a MIC50s and MIC80s were determined by measuring XTT activity reduction. The MIC50 and MIC80 of each drug for sessile cells were determined and compared to the controls (15). parapsilosis C.org/ on March 1. The median caspofungin MIC80s for sessile cells of C.03 0.5 1 2 4 8 16 32 32 C. . The median MIC50s and MIC80s of ﬂuconazole for sessile cells of all Candida species were 1. of isolates for which indicated MIC ( g/ml) was: 0.05.5 to 1 g/ml. albicans C.25 0. glabrata 12 12 10 9 12 2 6 1 3 2 3 3 2 2 10 1 9 2 6 3 2 1 2 1 1 1 1 1 1 1 1 4 9 5 1 1 4 11 a MIC50s and MIC80s were determined by measuring XTT activity reduction. Distributions of amphotericin B MICs for different Candida species strains under planktonic or bioﬁlm growth conditions Species No. The median MIC50 of amphotericin B for sessile cells of each of the four Candida species ranged from 0. albicans sessile cells ranged from 0.VOL. C.5 1 2 4 8 16 16 Caspofungin C.06 No. 16. which was similar to the median MIC for planktonic cells (0.05. tropicalis C.5 g/ml. parapsilosis C. tropicalis C.12 0. of isolates tested Type of MICa 0.024 g/ml.0 program. of isolates for which indicated MIC ( g/ml) was: 0.06 to 0.asm. glabrata 12 12 10 9 MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells MIC for planktonic cells MIC50 for sessile cells MIC80 for sessile cells 6 4 1 2 1 1 6 9 9 5 3 1 1 6 2 2 2 2 5 12 2 10 4 2 5 5 1 2 1 1 6 3 1 6 7 1 6 Micafungin C. albicans. The distributions of antifungal MICs for planktonic and sessile cells of the different Candida species are shown in Tables 1. These data show that amphotericin B is moderately effective against the bioﬁlms of all four species. tropicalis C.12 0.25 0. Distribution of caspofungin and micafungin MICs for different Candida species strains under planktonic or bioﬁlm growth conditions Drug Species No.5 g/ml). Alpha was set at 0. 2. C. and 1 g/ml. and 3.5. The caspofungin MIC50s for C. of isolates tested Type of MICa 0. tropicalis. 16. 51. parapsilosis. and all the P values were two tailed. similar to the values reported previously (3). glabrata were 0. albicans C.06 0. Differences between the species were considered to be signiﬁcant for P of 0. albicans C. Correlations between the MICs for planktonic cells and those for sessile cells and between the MICs of caspofungin and micafungin for sessile cells were examined by the least-squares method (13). respectively. parapsilosis C. and C. 2012 by guest in the XTT assays and are expressed as percentages of the values for control wells. The ODs of the different Candida species were compared by the Mann-Whitney U test by using the SPSS Win 10.
but a positive correlation between the caspofungin and micafungin MICs for sessile cells was noted (R. The inhibitory effect of each concentration of antifungal was measured as the average OD of all antifungal-treated wells and expressed as a percentage of the OD of control (antifungal-free) wells (considered to be 100%) as determined in XTT reduction assays. 8). respectively. . tropicalis bioﬁlms. mainly due to the low ODs of the control wells. Therefore. 0. SMIC80.4 for MIC50 for sessile cells. there were signiﬁcant speciesspeciﬁc differences in the echinocandin activities against the Candida bioﬁlms (P 0. glabrata. Figure 1 shows the inhibition curves for bioﬁlm-grown Candida species in the presence of different concentrations of the four antifungal agents.3). C. P 0. These data suggest different drug resistance mechanisms for different bioﬁlm-forming Candida species. The observed differences in echinocandin susceptibilities among different Candida species suggest the involvement of novel biochemical and genetic mechanisms in bioﬁlm formation. In vitro activities of different concentrations of drugs against bioﬁlms formed by four individual bloodstream isolates representing four different Candida species. These interstudy differences may be due to the differences in the Candida bioﬁlm models used or to the bioﬁlm-forming abilities of the Candida isolates tested (8. metabolic activity (12). the criterion that we used for isolate selection may have introduced a bias. glabrata. There was no correlation between the MICs for planktonic cells and those for sessile cells of a given strain. In contrast to that in the C. since the bioﬁlms with lower turbidities gave nonreproducible MIC results for sessile cells. MIC80 for sessile cells. and the rate of drug diffusion through the bioﬁlm (2) have been reported for different Candida species. Downloaded from http://aac. C. glabrata bioﬁlms was observed following exposure to relatively low concentrations (0. .01). R. AGENTS CHEMOTHER. Species-speciﬁc differences are evident for the activities of caspofungin and micafungin against Candida bioﬁlms (P 0. SMIC50. 17). The mechanisms of echinocandin activity against bioﬁlms formed by different Candida species are unknown (7). 0. However. We selected 43 out of the 95 isolates from preliminary experiments in which the bioﬁlms had high turbidities at 48 h (OD 0.014. C. 16. (9) have shown that both echinocandins inhibit one of two C. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005E00082). ‚. }.5 g/ml and 0. tropicalis. in contrast to the activities of ﬂuconazole and amphotericin B. parapsilosis and C.25 g/ml for C. and 16 g/ml for both C. tropicalis isolates) and echinocandin susceptibilities. Clinical signiﬁcance remains to be veriﬁed. The inhibitory effects of ﬂuconazole and amphotericin B showed no signiﬁcant differences among the Candida species.01).05).25 to 1 g/ml) of caspofungin and micafungin.1522 NOTES ANTIMICROB.asm. In the present study.05). Symbols: . P 0.org/ on March 1. tropicalis (P 0. albicans. given the variations in testing methods and isolate selection. parapsilosis. parapsilosis and C. parapsilosis strains while exhibiting high MICs for the other strain. 2012 by guest FIG. differences related to the composition of the Candida bioﬁlm matrix (1. Kuhn et al. parapsilosis (12 strains) were less susceptible to both echinocandins in vitro. C. Further studies are needed on the potential associations between the densities of bioﬁlms (particularly those formed by C. a reduction in XTT activity of up to 80% in the C. albicans and C. MIC50 for sessile cells. albicans and C. the bioﬁlms formed by C. fungin MIC80s for sessile cells were 0. parapsilosis and C. 1.9 for MIC80 for sessile cells. However.
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