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Antimicrob. Agents Chemother.-2007-Choi-1520-3[2]

Antimicrob. Agents Chemother.-2007-Choi-1520-3[2]

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Species-Specific Differences in the Susceptibilities of Biofilms Formed by Candida Bloodstream Isolates to Echinocandin Antifungals

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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Within the therapeutic range of concentrations of each drug. Apr.ac.1128/AAC. the echinocandins. 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. including 12 C. of isolates for which indicated MIC ( g/ml) was: 0. 12 Candida parapsilosis. albicans. Downloaded from http://aac. In this system. 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 1.06 g/ml.asm. Chonnam National University Medical School. Gwangju. of isolates tested Type of MICa 0. p.12 0. other non-C.1* Sook In Jung. Fax: 82 (62) 224-2518. However. amphotericin B. 15). and the two echinocandins. 16 to 0.01141-06 Copyright © 2007. 16).1 Seung Jung Kee.org/ on March 1. The drugs were prepared in a series of twofold dilutions as follows: fluconazole.1 Soon Pal Suh. 32 to 0.3.25 0. fluconazole. tropicalis C. 8 Hakdong Dongku. 1520 .024 C.ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. Gwangju 501-757. Although fungal biofilm-associated infections are frequently refractory to conventional antifungal therapy. * Corresponding author. between January 1999 and December 2003. have recently been demonstrated to be active against Candida albicans biofilms (3. South Korea. 51. albicans C.1 and Dong Wook Ryang1 Departments of Laboratory Medicine1 and Internal Medicine.03 0. 14). We examined 43 Candida species isolates. 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. Vol.024 to 2 g/ml.2 Duck Cho. Published ahead of print on 5 February 2007. 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. E-mail: shinjh@chonnam.06 No. All of the isolates were from blood cultures acquired at Chonnam National University Hospital. All Rights Reserved. The MICs for planktonic cells were determined by the standard CLSI (Clinical and Laboratory Standards Institute) M27-A2 broth microdilution method (5).-D-glucan synthase.1 Myung Geun Shin. 2007. While C. American Society for Microbiology.03 g/ml. 1520–1523 0066-4804/07/$08. parapsilosis C. glabrata but not those formed by C. there have been few comparisons of the activities of echinocandins against biofilms formed by different Candida species. and 9 Candida glabrata isolates.2 Chonnam National University Medical School. albicans is the most commonly isolated Candida species. South Korea. tropicalis or C. 10. caspofungin and micafungin were active against biofilms formed by Candida albicans or C. 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 2.00 0 doi:10. Distributions of fluconazole MICs for different Candida species strains under planktonic or biofilm (sessile-cell) growth conditions Species No. Phone: 82 (62) 2205342. 4 Species-Specific Differences in the Susceptibilities of Biofilms Formed by Candida Bloodstream Isolates to Echinocandin Antifungals Hyun Woo Choi. which constitute a new class of antifungals that inhibit 1. We compared the in vitro activities of caspofungin. Gwangju. Mailing address: Department of Laboratory Medicine.1 Jong Hee Shin. albicans species have been increasingly recognized as catheter-related BSI pathogens (4. micafungin. parapsilosis.kr.5 1 2 4 8 16 32 64 1. No.3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrozolium5-carboxanilide (XTT) absorbance assay. The MICs for sessile cells (biofilms) were determined using a microtiter-based assay (3.2 Kyung Hwa Park. 1. 10 Candida tropicalis. and amphotericin B against biofilms formed by BSI isolates of four different Candida species. 16). 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).

12 0. parapsilosis C.03 No. tropicalis C. 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.5 g/ml. while the median MIC80 for sessile cells ranged from 2 to 32 g/ml. albicans C. of isolates tested Type of MICa 0. The ODs of the different Candida species were compared by the Mann-Whitney U test by using the SPSS Win 10. Downloaded from http://aac. albicans sessile cells ranged from 0.05. similar to the values reported previously (3). Distribution of caspofungin and micafungin MICs for different Candida species strains under planktonic or biofilm growth conditions Drug Species No. albicans C. tropicalis C. tropicalis C. and C. 2007 NOTES 1521 TABLE 2. respectively.0 program.25 0. The median MIC50s and MIC80s of fluconazole for sessile cells of all Candida species were 1.VOL. C.12 0.5 1 2 4 8 16 16 Caspofungin C. glabrata were 0. of isolates tested Type of MICa 0. 16.5 g/ml). 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.03 0. and all the P values were two tailed. The MIC50 and MIC80 of each drug for sessile cells were determined and compared to the controls (15). parapsilosis.org/ on March 1. Differences between the species were considered to be significant for P of 0. albicans. 16. These data show that amphotericin B is moderately effective against the biofilms of all four species. The caspofungin MIC50s for C. Distributions of amphotericin B MICs for different Candida species strains under planktonic or biofilm growth conditions Species No. of isolates for which indicated MIC ( g/ml) was: 0. parapsilosis C. of isolates for which indicated MIC ( g/ml) was: 0. The distributions of antifungal MICs for planktonic and sessile cells of the different Candida species are shown in Tables 1. tropicalis.5. 2. 2012 by guest in the XTT assays and are expressed as percentages of the values for control wells. All isolates were tested at least twice.05.06 to 0.06 0.asm. The median mica- TABLE 3. C. The median caspofungin MIC80s for sessile cells of C. 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. parapsilosis C. which was similar to the median MIC for planktonic cells (0.25 0.5 to 1 g/ml. whereas fluconazole is ineffective. 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).06 No.5 1 2 4 8 16 32 32 C.024 g/ml. The median MIC50 of amphotericin B for sessile cells of each of the four Candida species ranged from 0. 51. and 1 g/ml. . Alpha was set at 0. and 3.

Therefore. 1. parapsilosis strains while exhibiting high MICs for the other strain. 16. albicans and C. 0. In the present study. tropicalis. In contrast to that in the C. mainly due to the low ODs of the control wells. The mechanisms of echinocandin activity against biofilms formed by different Candida species are unknown (7). in contrast to the activities of fluconazole and amphotericin B.05). glabrata. R. a reduction in XTT activity of up to 80% in the C. but a positive correlation between the caspofungin and micafungin MICs for sessile cells was noted (R.01).3). parapsilosis. ‚. 8).org/ on March 1. Kuhn et al. The observed differences in echinocandin susceptibilities among different Candida species suggest the involvement of novel biochemical and genetic mechanisms in biofilm formation. albicans. Further studies are needed on the potential associations between the densities of biofilms (particularly those formed by C. Clinical significance remains to be verified. metabolic activity (12). 0. (9) have shown that both echinocandins inhibit one of two C. However. there were significant speciesspecific differences in the echinocandin activities against the Candida biofilms (P 0. parapsilosis and C. 2012 by guest FIG. . since the biofilms with lower turbidities gave nonreproducible MIC results for sessile cells.014. . albicans and C. P 0. respectively.asm.4 for MIC50 for sessile cells. 17).5 g/ml and 0. 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. The inhibitory effects of fluconazole and amphotericin B showed no significant differences among the Candida species. }.25 to 1 g/ml) of caspofungin and micafungin. glabrata biofilms was observed following exposure to relatively low concentrations (0. parapsilosis and C. and 16 g/ml for both C. and the rate of drug diffusion through the biofilm (2) have been reported for different Candida species. C.1522 NOTES ANTIMICROB. SMIC80. the criterion that we used for isolate selection may have introduced a bias. tropicalis isolates) and echinocandin susceptibilities. C. C. MIC50 for sessile cells. Species-specific differences are evident for the activities of caspofungin and micafungin against Candida biofilms (P 0. Downloaded from http://aac. given the variations in testing methods and isolate selection. glabrata.01). parapsilosis (12 strains) were less susceptible to both echinocandins in vitro.25 g/ml for C. MIC80 for sessile cells. the biofilms formed by C.05). P 0. Symbols: . C. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005E00082). 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. We selected 43 out of the 95 isolates from preliminary experiments in which the biofilms had high turbidities at 48 h (OD 0. However. parapsilosis and C. In vitro activities of different concentrations of drugs against biofilms formed by four individual bloodstream isolates representing four different Candida species. These data suggest different drug resistance mechanisms for different biofilm-forming Candida species.9 for MIC80 for sessile cells. fungin MIC80s for sessile cells were 0. AGENTS CHEMOTHER. differences related to the composition of the Candida biofilm matrix (1. SMIC50. Figure 1 shows the inhibition curves for biofilm-grown Candida species in the presence of different concentrations of the four antifungal agents. tropicalis (P 0. There was no correlation between the MICs for planktonic cells and those for sessile cells of a given strain. tropicalis biofilms.

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