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Antimicrobial Agents and Chemotherapy, August 2009, p. 3347-3352, Vol. 53, No. 8
0066-4804/09/$08.00+0     doi:10.1128/AAC.01480-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Anidulafungin Is Fungicidal and Exerts a Variety of Postantifungal Effects against Candida albicans, C. glabrata, C. parapsilosis, and C. krusei isolates

Katherine T. Nguyen,¹ Philip Ta,¹ Bich Thu Hoang,¹ Shaoji Cheng,^1,4 Binghua Hao,^1,4 M. Hong Nguyen,^1,2,3,4 and Cornelius J. Clancy^1,3,4,5*

Departments of Medicine,¹ Molecular Genetics and Microbiology, University of Florida College of Medicine,² the North Florida/South Georgia Veterans Health System, Gainesville, Florida,³ the Department of Medicine, University of Pittsburgh,⁴ the V.A. Pittsburgh Healthcare System, Pittsburgh, Pennsylvania⁵

Received 5 November 2008/ Returned for modification 2 February 2009/ Accepted 3 April 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anidulafungin targets the cell walls of Candida species by inhibiting β-1,3-glucan synthase, thereby killing isolates and exerting prolonged postantifungal effects (PAFEs). We performed time-kill and PAFE experiments on Candida albicans (n = 4), C. glabrata (n = 3), C. parapsilosis (n = 3), and C. krusei (n = 2) isolates and characterized the PAFEs in greater detail. MICs were 0.008 to 0.125 µg/ml against C. albicans, C. glabrata, and C. krusei and 1.0 to 2.0 µg/ml against C. parapsilosis. During time-kill experiments, anidulafungin caused significant kills at 16x MIC (range, log 2.68 to 3.89) and 4x MIC (log 1.87 to 3.19), achieving fungicidal levels (log 3) against nine isolates. A 1-hour drug exposure during PAFE experiments resulted in kills ranging from log 1.55 to 3.47 and log 1.18 to 2.89 (16x and 4x MIC, respectively), achieving fungicidal levels against four isolates. Regrowth of all 12 isolates was inhibited for 12 h after drug washout. Isolates of each species collected 8 h after a 1-hour exposure to anidulafungin (16x and 4x MIC) were hypersusceptible to sodium dodecyl sulfate (0.01 to 0.04%) and calcofluor white (40 µg/ml). Moreover, PAFEs were associated with major cell wall disturbances, as evident in electron micrographs of viable cells, and significant reductions in adherence to buccal epithelial cells (P 0.01). Finally, three of four PAFE isolates tested were hypersusceptible to killing by J774 macrophages (P 0.007). Our data suggest that the efficacy of anidulafungin in the treatment of candidiasis might stem from both direct fungicidal activity and indirect PAFEs that lessen the ability of Candida cells to establish invasive disease and to persist within infected hosts.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anidulafungin is an echinocandin agent that disrupts the cell walls of Candida species by inhibiting β-1,3-D-glucan synthase. In recent studies of treatment of invasive candidiasis, the agent was shown to be at least as effective as the frontline azole agent fluconazole (12, 22). Additional clinical trial data demonstrating the efficacy of caspofungin and micafungin in the treatment of diverse types of candidiasis make it clear that the echinocandins are significant additions to the antifungal armamentarium (13, 17).

In general, MIC₉₀s of anidulafungin are low against the common pathogens Candida albicans, C. glabrata, C. tropicalis, and C. krusei (0.06 to 0.12 µg/ml), including isolates that are resistant to azole agents (20, 21). MIC₉₀s against C. parapsilosis and C. guilliermondii isolates are higher (2 µg/ml), as also noted for other echinocandins (20, 21). Diminished susceptibility to anidulafungin might reflect changes in the glucan synthase subunit Fks1p (2, 18). Regardless of the mechanism, the clinical significance of elevated anidulafungin MICs remains unclear (22). To date, only a few studies have assessed the anticandidal activity of anidulafungin by time-kill or postantifungal effect (PAFE) methods. Similar to other echinocandins, anidulafungin exhibited concentration-dependent fungicidal activity against C. albicans, C. glabrata, C. tropicalis, and C. krusei isolates during time-kill experiments at concentrations of 4x and 16x MIC (10, 23). In PAFE experiments, a 1-hour exposure to anidulafungin at 4x MIC resulted in prolonged growth inhibition of C. albicans (9). To our knowledge, time-kill or PAFE data have not been published for anidulafungin against C. parapsilosis. Caspofungin, however, is fungicidal and causes prolonged PAFE growth inhibition of C. parapsilosis at concentrations of 4x MIC (7).

We hypothesized that anidulafungin would demonstrate significant PAFEs against Candida isolates of diverse species, as measured by growth inhibition following brief drug exposure in vitro. In addition, we hypothesized that anidulafungin's PAFEs would cause changes to the candidal cell wall that would result in decreased cell integrity and adherence to host cells and in increased susceptibility to killing by phagocytes. In this study, we assessed the fungicidal activity of anidulafungin against 12 Candida isolates (4 C. albicans, 3 C. glabrata, 3 C. parapsilosis, and 2 C. krusei isolates) by time-kill and PAFE methods. We then tested PAFE-inhibited cells for susceptibility to cell wall-active drugs and visualized cell walls by electron microscopy. Finally, we assessed adherence to human epithelial cells and killing by macrophages.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anidulafungin, Candida isolates, and measurement of growth rates. Anidulafungin was provided by Pfizer, Inc. (Groton, CT). Ten Candida isolates (3 C. albicans, 3 C. glabrata, 3 C. parapsilosis, and 1 C. krusei isolate) were recovered from the bloodstreams of patients with candidemia. C. parapsilosis isolates were confirmed as belonging to group I (C. parapsilosis sensu stricto) by their ITS1 sequences, using previously described methods (15). C. albicans ATCC 90028 and C. krusei ATCC 6258 were included as quality controls. The in vitro growth rates were determined in yeast peptone dextrose (YPD) and RPMI 1640 liquid media at 30 and 37°C in microtiter plates, as described in our previous publication (5).

Antifungal susceptibility testing. MICs of anidulafungin were determined using a broth microdilution technique (M27-A3) in RPMI 1640 medium buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS), as recommended by the Clinical Laboratory Standards Institute Antifungal Subcommittee (8). The starting inoculum was 0.5 x 10³ to 2.5 x 10³ cells/ml, and incubation was done at 35°C. The MIC was read as the lowest concentration of drug that caused a significant diminution (>50%) of growth below control levels (19). The range of anidulafungin concentrations was 0.008 to 32 µg/µl. Each isolate was tested on at least three occasions.

Antifungal carryover. Antifungal carryover was excluded using standard methods (11). A fungal suspension of approximately 5 x 10³ CFU/ml was prepared, and 100 µl was added to 900 µl of sterile water or sterile water plus anidulafungin. Immediately after the addition of the fungal suspension to the aqueous solution, the test tube was vortexed and a 30-µl aliquot was plated on a Sabouraud dextrose agar (SDA) plate. Following 48 h of incubation at 35°C, the number of CFU was determined. The mean colony count at each multiple of the MIC tested was compared with the data for the control. Significant antifungal carryover was defined as a reduction of CFU of >25% of the control level.

Time-kill and PAFE studies. We measured time-kill data and PAFEs simultaneously for each isolate, using methods previously described by our lab (7, 14). Prior to being tested, isolates were subcultured twice on SDA plates. Colonies from a 24- to 48-hour culture were suspended in 9 ml of sterile water and adjusted to a 0.5 McFarland standard. One milliliter of the suspension was then added to 9 ml of either RPMI 1640 buffered to pH 7.0 with MOPS or a solution of medium plus anidulafungin. These methods resulted in a starting inoculum of approximately 1 x 10⁵ to 5 x 10⁵ CFU/ml. Test solutions were placed on an orbital shaker and incubated at 35°C with agitation. At the desired time points, 100 µl was obtained from each solution and serially diluted 10-fold in sterile water, and 10 µl was plated on SDA plates. Following incubation at 35°C for 48 h, the number of CFU was determined.

Using this protocol, we set up time-kill and PAFE experiments for control (no drug) and 0.25x, 1x, 4x, and 16x MIC tubes in duplicate tubes (labeled "time-kill" and "PAFE"). We plated all tubes at time zero. After an incubation period of 1 hour, we removed the respective PAFE tubes of the set, washed and centrifuged the cells at 1,400 x g for 10 min (3 cycles), and resuspended the fungal pellets in warm RPMI medium (9 ml) prior to reincubation with the time-kill tubes. Both sets of tubes were plated on SDA plates after 2, 4, 8, 12, 24, and 48 h, and CFU were enumerated after incubation at 35°C for 48 h. Time-kill and PAFE experiments were conducted twice for each isolate, and data presented are mean values.

"Persister" Candida isolates were not killed by anidulafungin after 12 h in time-kill and PAFE experiments. For each isolate, three colonies growing on SDA plates at 48 h were selected, and their in vitro growth rates and susceptibility to anidulafungin were tested using the methods described above.

Sensitivities to cell wall agents SDS and calcofluor white. Each of the following experiments was performed in triplicate. Candida cells were recovered 8 h after a 1-hour exposure to anidulafungin at 0x, 1x, and 4x MIC. Cells were subcultured in YPD liquid medium with 1% glucose until exponential growth phase and then diluted to an optical density at 599 nm of 0.1. Four-microliter samples of undiluted culture and serial 10-fold dilutions of each culture were spotted onto YPD plates containing calcofluor white (40 µg/ml) (6) or sodium dodecyl sulfate (SDS) (0.01 to 0.04%). The plates were incubated at 30°C for 72 h. In preliminary experiments, Candida isolates not exposed to anidulafungin were only slightly susceptible to these concentrations of calcofluor white and SDS. For this reason, we hypothesized that the concentrations would be useful for showing increased susceptibility among Candida isolates exposed to anidulafungin.

Electron microscopy. Transmission electron microscopy was performed by the Electron Microscopy Laboratory at the North Florida/South Georgia Veterans Health System, in accordance with their established protocol. Briefly, Candida cells recovered 8 h after a 1-hour exposure to anidulafungin at 0x, 1x, and 4x MIC were cultured on SDA plates for 24 h at 35°C. Cells selected from colonies on the plates were fixed at 4°C in 0.1 M sodium cacodylate buffer (pH 7.2) containing 2% glutaraldehyde and 2% paraformaldehyde. The samples were then dehydrated through a graded series of ethanol and embedded in Lowicryl K4M (Electron Microscopy Sciences, Hatfield, PA). Thin sections were imaged with a Zeiss EM902 electron microscope.

Adherence of C. albicans to human BECs. Adherence of C. albicans to human buccal epithelial cells (BECs) was determined as described previously (6). BECs were collected from three investigators by gently scraping the cheek mucosa with a cotton swab and were dispensed into 10 ml phosphate-buffered saline (PBS). The pooled BECs were then washed four times with PBS and counted using a hemocytometer. A final concentration of 1 x 10⁵ epithelial cells/ml was obtained by adjustment with PBS. Candida cells recovered 8 h after a 1-hour exposure to anidulafungin at 4x MIC were cultured on SDA for 24 h at 35°C. For the adherence assay, 0.5 ml of the washed epithelial cells was incubated in a glass tube with 0.5 ml of washed Candida cells in PBS at a concentration of 1 x 10⁶ per ml in a shaking incubator at 37°C for 1 hour; as a control, 0.5 ml of BECs was mixed with 0.5 ml of PBS. Following incubation, the cells were vacuum filtered through prewet 20-mm-diameter polycarbonate filters with a 12-mm pore size (Costar, MA) mounted on a filter manifold (Millipore, Bedford, MA). Each filter was washed 10 times with PBS to remove unattached candidal cells. The washed filters were then removed and pressed gently onto glass slides. The slides were air dried, heat fixed for 1 min, and Gram stained. The slides were examined by light microscopy in 1-mm intervals, and the number of candidal cells attached to 100 BECs was counted. Each experiment was performed on at least two separate occasions, using BECs harvested from the same individuals at the same time on successive days; on each occasion, the experiments were performed in duplicate. Differences between isolates exposed to anidulafungin and the corresponding controls were determined using Student's t test.

Macrophage phagocytosis assay. Macrophage phagocytosis assays were performed as described previously (4). Candida cells were recovered as described for adherence assays, opsonized with 50% human serum for 30 min at 37°C, washed with PBS, counted in a hemocytometer chamber, and transferred to liquid RPMI medium. The murine macrophage-like tumor cell line J774A.1 (ECACC 85011428, from a female BALB/c mouse) was obtained from the American Type Culture Collection (ATCC), Manassas, VA. Cells were cultured at 37°C and 5% CO₂ in complete medium (RPMI supplemented with 10% [vol/vol] heat-inactivated fetal calf serum, 5 mM L-glutamine, 100 mg/liter streptomycin, and 50 mg/liter penicillin). Viability was assessed by trypan blue exclusion (Gibco) and was >95%. For quantification experiments, J774A.1 monolayer cells were challenged with Candida cells at a ratio of 1:10 and then incubated for 2 h at 37°C. The medium was then removed and the monolayers were washed thoroughly 10 times with cold PBS, after which the mixtures were resuspended in 1 ml of sterile distilled water at 37°C for 5 min and agitated vigorously with a micropipette until the macrophages were completely lysed. Serial 10-fold dilutions were made, and a colony count was performed following incubation at 30°C for 48 h. The percentage of surviving Candida cells was calculated in comparison with the CFU of Candida grown under the same conditions without macrophages. The assays were performed in triplicate and repeated at least twice. Differences between the null mutant and strain CAI12 were determined using Student's t test.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anidulafungin is fungicidal and exerts prolonged PAFEs against diverse Candida spp. MICs of anidulafungin are shown in Table 1. The ranges of MICs are consistent with values for the given species reported in large surveillance studies of anidulafungin activity in vitro (20). As anticipated, MICs against C. parapsilosis isolates were higher than those for other species.

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TABLE 1. Anidulafungin MICs

The results of time-kill and PAFE experiments are summarized in Table 2. Representative kill and PAFE curves are shown in Fig. 1 for three Candida isolates. In the time-kill experiments at 16x MIC, the range of log time-kill data was 2.68 to 3.89. Anidulafungin was fungicidal against nine isolates (defined as a kill of log 3), including all C. parapsilosis, C. glabrata, and C. krusei isolates. At 4x MIC, the range of log time-kill data was 1.87 to 3.19, and anidulafungin was fungicidal against two of the C. parapsilosis isolates. A 1-hour exposure to anidulafungin during PAFE experiments also resulted in significant kills, ranging from log 1.55 to 3.47 at 16x MIC and from log 1.18 to 2.89 at 4x MIC. Indeed, a 1-hour exposure to 16x MIC was fungicidal against four isolates, including all three C. parapsilosis isolates. Moreover, regrowth of all 12 isolates was inhibited for at least 12 h after anidulafungin was washed out, consistent with sustained PAFEs.

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TABLE 2. Reductions in starting inocula of Candida isolates during time-kill and PAFE experimentsa

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FIG. 1. Anidulafungin causes significant kills of Candida isolates during both time-kill and PAFE experiments. (Left) Time-kill experiments were conducted for 48 h in the presence of anidulafungin. (Right) In PAFE experiments, the isolates were exposed to anidulafungin for 1 hour, followed by washout and growth in the absence of drug for 48 h. Anidulafungin at 0.25x MIC did not result in significant killing of strains, and data for this concentration are not included in these curves. Time-kill and PAFE experiments were conducted twice for each isolate, and representative curves for three isolates are shown in the figure.

It is notable that persister Candida isolates that were not killed by anidulafungin in our study did not exhibit elevated MICs upon retesting. Moreover, growth of the persisters in liquid media after recovery from time-kill and PAFE experiments was not reduced.

PAFEs of anidulafungin increase susceptibility to cell wall-active drugs and result in cell wall damage. To characterize the PAFEs on the cell walls of isolates, we recovered cells of seven isolates 8 hours after a 1-hour exposure to anidulafungin at 0x, 1x, 4x, and 16x MIC. The isolates collected were C. albicans 1, C. albicans ATCC 90028, C. parapsilosis A, C. parapsilosis 1, C. glabrata 1, C. glabrata 3, and C. krusei ATCC 6258, all of which exhibited significant 8-h growth inhibition at both 4x and 16x MIC. Using standard in vitro assays of susceptibility to cell wall-active agents previously developed in our lab (6), we demonstrated that viable cells of each isolate recovered after exposure to anidulafungin at 4x and 16x MIC were significantly more susceptible to increasing concentrations of SDS and calcofluor white than were control cells (i.e., no drug). In addition, cells of C. albicans 1 (Fig. 2) and C. krusei ATCC 6258 exposed to 1x MIC for 1 hour were also significantly more susceptible than control cells to these agents.

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FIG. 2. Exposure of Candida isolates to anidulafungin for 1 hour results in increased susceptibility to the cell wall-active agents SDS and calcofluor white. Representative images are presented for C. albicans 1 in the presence of SDS, to which viable cells recovered after exposure to anidulafungin at 1x, 4x, and 16x MIC were increasingly susceptible. C. krusei 6258 showed similar results (data not shown). For the other five strains tested, cells recovered after exposure to anidulafungin at 1x MIC resembled controls. Similar susceptibility results were obtained in the presence of calcofluor white (20 µg/ml) for all strains (data not shown).

We next examined the cell walls of four strains (C. albicans 1, C. parapsilosis A, C. glabrata 1, and C. krusei 1) by electron microscopy. Cells recovered 8 h after a 1-hour exposure to anidulafungin at 0x, 1x, and 4x MIC were cultured on SDA for 24 h at 35°C, and colonies were selected from the plates for electron microscopy. All control cells (no drug) exhibited intact cell walls with normal inner and outer layers (Fig. 3). At 4x MIC, the cell walls were markedly disturbed, with a loss of distinct inner and outer layers (Fig. 3). At 1x MIC, the cell wall of C. albicans 1 more closely resembled those of cells recovered after exposure to 4x MIC than it did those of controls. For the other isolates, the cells exposed to 1x MIC more closely resembled the control cells.

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FIG. 3. Electron micrographs of viable C. albicans 1 (top) and C. glabrata 1 (bottom) cells recovered 8 h after a 1-hour incubation with anidulafungin at 0x MIC (control), 1x MIC, and 4x MIC. Cell wall damage was also noted for C. parapsilosis A and C. krusei 1 (data not shown).

PAFEs of anidulafungin result in reduced adherence of Candida isolates to epithelial cells and in increased susceptibility to killing by macrophages. We hypothesized that the PAFEs of anidulafungin would impair the ability of Candida cells to adhere to host epithelial cells. We tested this hypothesis for C. albicans 1, C. parapsilosis A, and C. glabrata 1 cells recovered as described for the electron microscopy experiments. Using standard assays of adherence to BECs (6), we demonstrated that the relative adherence of C. albicans 1, C. parapsilosis A, and C. glabrata 1 cells was 58% ± 18%, 69% ± 28%, and 62% ± 21%, respectively (versus 100% for corresponding control cells; all P values were 0.01).

Finally, we hypothesized that the cellular changes induced by PAFEs would make cells more susceptible to killing by macrophages. We tested this hypothesis by coincubating the macrophage cell line J774 with C. albicans 1, C. parapsilosis A, C. parapsilosis 1, and C. glabrata 1 cells that had been recovered after exposure to anidulafungin at 4x MIC as in the previous experiments. In fact, C. albicans 1, C. parapsilosis A, and C. glabrata 1 cells exposed to anidulafungin were significantly more sensitive to killing by macrophages than were corresponding control cells (Table 3). For C. parapsilosis 1, there were no differences between cells exposed to anidulafungin and controls (Table 3).

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TABLE 3. Killing during phagocytosis assays

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anidulafungin, an echinocandin agent that exerts antifungal effects against diverse Candida species by disrupting the cell wall through inhibition of β-1,3-D-glucan synthase, has been shown to be at least as effective as previous frontline agents in the treatment of candidemia and other types of systemic and mucosal candidiasis (12, 22). Our in vitro findings are consistent with this clinical efficacy. We demonstrated that anidulafungin killed 12 Candida isolates during time-kill experiments at concentrations above the MIC, achieving fungicidal levels against nine isolates. Moreover, we showed that anidulafungin exerted profound PAFEs on Candida isolates, resulting in prolonged growth inhibition, disruptions of cell wall structure, decreased adherence to epithelial cells, and increased susceptibility to cell wall-active agents and killing by macrophages. Our data suggest that the efficacy of anidulafungin among patients with candidiasis might stem from both direct fungicidal effects and indirect effects that lessen the ability of Candida cells to establish invasive disease and to persist within infected hosts.

Our time-kill and PAFE results corroborate and extend those of previous studies (7, 9, 10, 16, 23). Anidulafungin's fungicidal activity against C. albicans, C. glabrata, and C. krusei isolates was previously demonstrated in small time-kill studies (10, 23), and the fungicidal activity against C. parapsilosis isolates was similar to that reported for caspofungin (7). Indeed, we found that anidulafungin killed C. parapsilosis at least as well as other species during time-kill experiments, provided that sufficient drug concentrations above the MIC were used. The fungicidal activity against C. glabrata and C. krusei is important given the acquired and intrinsic resistance of these species to the commonly used antifungal agent fluconazole. Anidulafungin's prolonged postantifungal growth inhibition of C. albicans was consistent with previous reports for this agent and other echinocandins (9, 16). We documented similar prolonged inhibitory effects against C. glabrata, C. krusei, and C. parapsilosis isolates. Overall, a 1-hour exposure to anidulafungin was fungicidal against four isolates and inhibited regrowth of all isolates for 12 h. Interestingly, persister Candida isolates that were not killed by anidulafungin in our study did not exhibit elevated MICs upon retesting, indicating that growth despite exposure to the drug was not due to acquired resistance or preexisting resistant subpopulations.

The cell wall defects of Candida cells recovered during PAFE experiments were profound and long-lasting, as demonstrated by electron micrographs of isolates of all four species tested. The electron micrographs are consistent with descriptions of aberrant cell surface morphology and derangements of the normal layering of the cell wall in C. albicans cells exposed to micafungin (1). Interestingly, the cell wall changes were evident in our study even though the isolates were cultured for 24 h on SDA prior to electron microscopy. We chose this experimental design because it ensured that all cells examined by electron microscopy were viable, whereas examination of isolates recovered directly from liquid media during PAFE experiments might have included both viable and dead cells. The results suggest that anidulafungin PAFEs on the cell wall persist for a time after an isolate begins to regrow.

The cell wall changes were associated with decreased adherence to BECs and with increased susceptibility to killing by macrophages in vitro. Our adherence findings were consistent with earlier reports that micafungin attenuated the adherence of a C. albicans isolate to epithelial cells and extracellular matrix proteins (3, 24). Moreover, recent studies have demonstrated that subinhibitory concentrations of caspofungin unmask cell wall β-glucans, resulting in greater β-glucan receptor-mediated expression of inflammatory cytokines by murine macrophages (25). Under normal growth conditions, β-glucans are not recognized by phagocytic cells because they are buried beneath a mannoprotein coat, a strategy by which Candida might protect itself from immune clearance during the infectious process (25). Although anidulafungin inhibits β-1,3-glucan synthesis, the global disruption of cell wall architecture that results from drug exposure might paradoxically increase β-glucan exposure. The effects of anidulafungin on the distribution of cell wall proteins, chitin, and glucans will be characterized in our labs in follow-up studies, as will the mechanisms by which these disturbances impact adherence to host cells and susceptibility to phagocytosis.

 
Experiments were performed in the laboratories of C. J. Clancy and M. H. Nguyen at the North Florida/South Georgia Veterans Health System, Gainesville, FL.

This project was funded by a research grant to C.J.C. from Pfizer, Inc. C.J.C. and M.H.N. were supported by the Medical Research Service of the Department of Veterans Affairs. Their research was conducted as part of the University of Florida Mycology Research Unit. C.J.C. has received research funding from Astellas Pharmaceuticals, Pfizer, Inc., and Merck & Co., Inc. C.J.C. and M.H.N. have received funds to speak at a symposium organized on behalf of Three Rivers Pharmaceuticals, Inc.

 
* Corresponding author. Present address: University of Pittsburgh, 867 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261. Phone: (412) 624-0309. Fax: (412) 648-8455. E-mail: cjc76{at}pitt.edu

Published ahead of print on 13 April 2009.

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Antimicrobial Agents and Chemotherapy, August 2009, p. 3347-3352, Vol. 53, No. 8
0066-4804/09/$08.00+0     doi:10.1128/AAC.01480-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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