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Antimicrobial Agents and Chemotherapy, January 2008, p. 357-360, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00856-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Differential Activities of Newer Antifungal Agents against Candida albicans and Candida parapsilosis Biofilms{triangledown}

Aspasia Katragkou,1 Athanasios Chatzimoschou,1 Maria Simitsopoulou,1 Maria Dalakiouridou,1 Eudoxia Diza-Mataftsi,2 Chaido Tsantali,3 and Emmanuel Roilides1*

Laboratory of Infectious Diseases, Third Department of Pediatrics, Aristotle University, Hippokration Hospital, 54642 Thessaloniki, Greece,1 First Department of Microbiology, Aristotle University, University Campus, 54124 Thessaloniki, Greece,2 First Department of Pediatrics, Aristotle University, Hippokration Hospital, 54642 Thessaloniki, Greece3

Received 29 June 2007/ Returned for modification 21 August 2007/ Accepted 6 October 2007


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ABSTRACT
 
The activities of voriconazole, posaconazole, caspofungin, and anidulafungin against Candida albicans and Candida parapsilosis biofilms were evaluated. In contrast to planktonic cells, the MICs for voriconazole and posaconazole for the biofilms of the two species were high (≥256 and >64 mg/liter, respectively) but relatively low for the echinocandins caspofungin and anidulafungin (≤1 and ≤2 mg/liter, respectively).


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TEXT
 
Candida spp. have frequently been associated with the formation of biofilms on biological and inert surfaces, such as intravascular catheters (15). Candida albicans and Candida parapsilosis are the most prevalent species related to the biofilm mode of growth (12). Resistance of Candida biofilms to conventional antifungal agents has been previously documented (2, 10, 16). Given that biofilm-associated infections are very difficult to cure without device removal, the demand for newer, more effective therapies has been developed.

The goal of the present study was to examine the activities of newer antifungal agents against C. albicans and C. parapsilosis biofilms and compare them to their corresponding planktonic cells. Voriconazole (VRC; Pfizer, Groton, CT), posaconazole (PSC; Schering-Plough, Kenilworth, NJ), caspofungin (CAS; Merck, Whitehouse Station, NJ), and anidulafungin (AND; Pfizer), were examined.

Documented biofilm-producing strains were used, including C. albicans M61, C. albicans GDH2346, and C. parapsilosis PA/71 (4, 5). Aliquots were maintained in 25% glycerol and 75% peptone at –35°C.

Planktonic MICs were determined according to the Clinical and Laboratory Standards Institute M27-A2 method. MICs were determined as the lowest drug concentrations at which a prominent decrease in turbidity was observed, corresponding to ca. 50% inhibition in growth (8-10, 13). The MICs were recorded after incubation for 24 h.

Biofilm MIC determination was based on modifications of methods previously described (5, 10, 16). Biofilms were grown on the surface of silicone elastomer disks (Bioplexus Corp., Saticoy, CA), pretreated with fetal bovine serum (Gibco, Paisley, Scotland), in 12- or 96-well plates for 48 h for C. albicans strains and 72 h for C. parapsilosis. In an effort to make our adopted model resemble in vivo conditions, organisms were grown on the surface of a silicone substrate coated with a fetal bovine serum-conditioning film under constant linear shaking (4). Mature biofilms were then incubated in RPMI 1640 containing VRC, PSC, CAS, or AND at doubling dilutions (Fig. 1 and 2) for 24 h. Drug-free biofilms containing only RPMI 1640 served as controls. Four replicate biofilms were used for each condition. Biofilm MICs were determined as the minimum antifungal drug concentrations that caused ≥50% reduction in the metabolic activity of the biofilms compared to controls (10). Biofilm formation and antifungal activities were assessed by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT; 0.25 mg/ml) and coenzyme Q (40 µg/ml) assay spectrophotometrically at 450 nm with a reference wavelength of 690 nm (7). Antifungal activities were expressed as a percentage of metabolic activity of drug-treated biofilms compared to drug-free biofilms (controls, considered to be 100%).


Figure 1
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  		FIG. 1. In vitro activity of VRC (A), PSC (B), CAS (C), and AND (D) on biofilms of C. albicans ({blacksquare}, M61; {blacklozenge}, GDH2346) as evidenced by decrease of Candida metabolic activity in XTT colorimetric assay. Values on the y axis represent percentages of the mean absorbance of all drug-treated biofilms to untreated controls (100% survival). The results are from at least six experiments performed on different days. The planktonic MICs of VRC were 0.01 and 4 mg/liter for C. albicans M61 and GDH2346, respectively; the planktonic MICs of PSC were ≤0.001 mg/liter for both strains; the planktonic MICs of CAS were 0.06 and 0.03 mg/liter, respectively; and the planktonic MIC of AND was 0.003 mg/liter. The dotted horizontal line denotes the defined biofilm MIC (see the text).


Figure 2
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  		FIG. 2. In vitro activity of the triazoles VRC ({blacktriangleup}) and PSC ({blacksquare}) (A), as well as the echinocandins CAS (•) and AND ({blacksquare}) (B) on biofilms of C. parapsilosis strain PA/71, as evidenced by a decrease of Candida metabolic activity in an XTT colorimetric assay. Values on the y axis represent the percentages of the mean absorbance of all drug-treated biofilms compared to untreated controls (100% survival). The results are from at least six experiments performed on different days. The planktonic MICs of VRC, PSC, CAS, and AND were 0.03, 0.01, 0.06, and 0.125 mg/liter, respectively, for C. parapsilosis PA/71. The dotted horizontal line denotes the defined biofilm MIC (see the text).

To evaluate the differences among drug and control group, statistical analysis included test of homogeneity of variance, analysis of variance, and post hoc analysis (version 11.5; SPSS, Inc., Chicago, IL).

Planktonic MICs of all Candida isolates were highly susceptible to the antifungal agents tested (see the figure legends), except that C. albicans GDH2346 tended to be less susceptible to VRC (MIC, 4 mg/liter).

As shown in Fig. 1A and B, 50% reduction in the metabolic activities of biofilms of both C. albicans strains (BF MICs) was not achieved at antifungal concentrations up to 256 and 64 mg/liter for VRC and PSC, respectively. In contrast, BF MICs of CAS (Fig. 1C) were comparable to those of the planktonically grown C. albicans estimated at 0.06 and 0.03 mg/liter for M61 and GDH2346, respectively. Similarly, the BF MICs of AND (Fig. 1D) were determined to be 0.5 and 0.12 mg/liter, falling within the susceptibility range, even though they were >100 times higher than the planktonic MICs of this echinocandin, and breakpoints for the candins are not yet established (14).

The maximum inhibitory effect of CAS against C. albicans biofilms was noted for both strains at 2 mg/liter and was ca. 30% of the control metabolic activity, while the respective values for AND were 40% at concentrations of 0.5 and 8 mg/liter, depending on the strain (Fig. 1C and D). However, at higher concentrations (>2 mg/liter), CAS exhibited a reduced antifungal activity against C. albicans biofilms. Such a paradoxical effect has been observed for both biofilm and planktonic cells of C. albicans isolates (3, 17). Taken together, both CAS and AND showed enhanced potency against C. albicans biofilms at clinically relevant concentrations. Whether this different in vitro activity of CAS versus AND (30 versus 40%, P < 0.01) confers enhanced in vivo efficacy against C. albicans biofilms remains to be determined.

Similarly, with C. albicans biofilms, increased resistance to azoles was demonstrated by C. parapsilosis biofilms. As depicted in Fig. 2A, BF MICs for VRC was 256 and for PSC was >256 mg/liter. In contrast, echinocandins were more active in inhibiting metabolic activity of C. parapsilosis biofilms. BF MICs for echinocandins, although 16 times higher than the corresponding planktonic MICs, remained within the therapeutic range, increasing to 1 and 2 mg/liter for CAS and AND, respectively. Notably, no antifungal agent appeared to achieve complete sterility of C. albicans and C. parapsilosis biofilms formed on silicone substrate.

Overall, our results extend previously published data showing complete resistance of C. albicans biofilms to VRC and susceptibility to CAS (1, 10). However, there are reports demonstrating that CAS used at planktonic MIC concentrations was insufficient against C. albicans biofilms, whereas, when used at therapeutic concentrations (2 mg/liter), it caused a significant reduction in biofilm metabolic activity (6). The data on C. parapsilosis biofilms are rather scant and concern mainly the effect of CAS and VRC (10). To the best of our knowledge, ours is the first publication of data on the efficacy of PSC and AND against Candida spp. biofilms.

While the planktonic MICs of AND for C. albicans are much lower than those of CAS, the biofilm MIC appears to be similar for both drugs. This may be a reflection of the biofilm complex nature, which affects the drug resistance. Using various biofilm model systems, a number of resistance mechanisms have been investigated with no single mechanism providing a sufficient explanation (11, 12).

In conclusion, while newer azoles have no adequate activity against both C. albicans and C. parapsilosis biofilms, echinocandins retain their activity against biofilms. Validation of our results in animal models and subsequent clinical trials could establish echinocandin therapy as an effective strategy against catheter-related candidiasis.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from the European Society of Pediatric Infectious Diseases and a grant from Pfizer.

We thank Thomai Konstantinou for technical assistance and M. Ghannoum and L. J. Douglas for kindly providing the isolates used in this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Third Department of Pediatrics, Hippokration Hospital, Konstantinoupoleos 49, GR-546 42 Thessaloniki, Greece. Phone: 30-2310-892444. Fax: 30-2310-992981. E-mail: roilides{at}med.auth.gr Back

{triangledown} Published ahead of print on 15 October 2007. Back


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Antimicrobial Agents and Chemotherapy, January 2008, p. 357-360, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00856-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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