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Antimicrobial Agents and Chemotherapy, June 2006, p. 2214-2216, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01610-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Caspofungin Susceptibility in Aspergillus and Non-Aspergillus Molds: Inhibition of Glucan Synthase and Reduction of ß-D-1,3 Glucan Levels in Culture

Jennifer Nielsen Kahn,* Ming-Jo Hsu, Fred Racine, Robert Giacobbe, and Mary Motyl

Infectious Disease Research, Merck and Co., P.O. Box 2000, Rahway, New Jersey 07065-0900

Received 19 December 2005/ Returned for modification 24 January 2006/ Accepted 12 March 2006


   ABSTRACT
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Caspofungin inhibits synthesis of ß-D-1,3 glucan, essential to cell walls in Candida and Aspergillus spp., but activity against less common molds is largely uncharacterized. We demonstrate that caspofungin inhibits ß-D-1,3 glucan synthesis and reduces in vitro growth of clinical isolates from the genera Alternaria, Curvularia, Scedosporium, Acremonium, Bipolaris, and Trichoderma.


   TEXT
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Serious fungal infections caused by pathogens other than Candida and Aspergillus spp. are receiving increasing attention, but evaluation of susceptibility is often limited to MIC and occasionally minimum effective concentration (MEC) measurements (1, 4, 6). We report two additional methods (drug-induced restriction of radial growth on agar and reduction in 1,3-ß-D-glucan accumulation in liquid culture) that can be applied to growing filamentous fungi to assess their potential susceptibilities to echinocandin antifungal agents. Results from these assays correlate with the in vitro sensitivity of 1,3-ß-D-glucan synthase (GS), the echinocandin target.

Susceptibility testing. Caspofungin MICs were determined using method M38-A from the CLSI (formerly NCCLS) (3). The end point was either the lowest concentration fully inhibiting growth (MIC100) or the lowest concentration producing a significant change in hyphal morphology visible to the eye (MEC).

Sensitivity of radial spreading. Conidia (1 x106 to 5 x106/ml) were applied at a single point on the surface of a potato dextrose agar (PDA; Difco) plate with or without 1 µg/ml caspofungin, and radial growth was compared.

ß-D-1,3 Glucan assay. Microcentrifuge tubes containing 0.5 ml YME medium (4 g/liter yeast extract, 10 g/liter malt extract, 4 g/liter glucose) were inoculated with 106 conidia/ml, and cultures were grown at 30°C for 18 h. The mycelium was harvested by centrifugation, washed, treated with 0.25 ml of 1 M NaOH, sonicated with a microprobe, and incubated at 52°C for 30 min. ß-D-1,3 Glucan levels were determined by aniline blue fluorescence (7).

Isolation of GS from filamentous fungi. YME medium was inoculated with 1 x 106 to 5 x 106 conidia/ml and incubated for 24 to 36 h at 30°C. Mycelia were harvested and disrupted as described previously (2). GS was isolated and assayed as described for Candida albicans (5).

Validation of susceptibility methods using Aspergillus fumigatus. The radial growth of A. fumigatus following application of conidia to the surface of PDA was arrested by inclusion of 1 µg/ml of caspofungin in the agar (Fig. 1A). There was no filamentous growth beyond a flat mat of deformed mycelium around the edges of the original application on plates incubated for as long as 7 days at 30°C or 37°C. 1,3-ß-D-Glucan levels in the cell walls of growing fungi were measured using a microtiter plate assay (7) adapted to accommodate filamentous fungi. A titration curve with A. fumigatus (Fig. 1A) shows a 50% inhibitory concentration (IC50) for fluorescence of approximately 100 ng/ml of caspofungin. The profound caspofungin sensitivity of crude or enriched GS derived from this isolate is shown in Fig. 1B. Crude microsomes are less sensitive, illustrating the necessity of purification, as described recently for Candida albicans (5). A panel of 15 additional clinical Aspergillus isolates, including representatives from the species Aspergillus flavus, Aspergillus terreus, Aspergillus niger, and Aspergillus nidulans, was examined by all three procedures. For all isolates, caspofungin restricted growth on agar, depleted whole-cell 1,3-ß-D-glucan accumulation by more than 75%, and inhibited GS with an IC50 of <1.0 ng/ml (results not shown). The simpler whole-cell procedures were predictive of profound sensitivity at the caspofungin target.


Figure 1
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  		FIG. 1. Susceptibility of A. fumigatus to caspofungin. (A) Aniline blue fluorescence from 1,3-ß-D-glucan in the mycelium of Aspergillus fumigatus is inhibited by caspofungin. (Inset) Radial growth on the surface of PDA without caspofungin and with 1 µg/ml caspofungin. (B) Caspofungin titration curves for GS in crude microsomal membranes (triangles) or in an enriched enzyme preparation (squares).

 
Susceptibilities of less common (non-Aspergillus) molds to caspofungin. The effects of caspofungin on the accumulation of whole-cell 1,3-ß-D-glucan and the GS enzymatic activity of Curvularia geniculata and Curvularia lunata are shown in Fig. 2. While C. lunata showed restriction of radial spreading by 1 µg/ml of caspofungin, it was less sensitive than C. geniculata in the assay of whole-cell 1,3-ß-D-glucan synthesis (IC50, ca. 1 µg/ml), reflecting a GS with reduced sensitivity (IC50, 4.6 ng/ml). Results for a panel of fungi are presented in Table 1. All isolates from the genus Curvularia or Alternaria, and single examples of strains from other genera, were sensitive to caspofungin at low levels. The Phialophora richardsiae isolate has GS activity with substantially reduced caspofungin sensitivity, but this strain retains some susceptibility in assays of whole-cell glucan accumulation. Radial growth of some rare molds was not fully restricted by caspofungin (e.g., Paecilomyces variotii and Penicillium chrysogenum) (data not shown), and these isolates were not characterized further.


Figure 2
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  		FIG. 2. Inhibition of glucan synthesis by caspofungin, measured by aniline blue fluorescence for C. geniculata MF2541and C. lunata MF5573. Bottom graph shows the in vitro sensitivity of isolated GS activity in the presence of caspofungin for C. lunata (triangles) and C. geniculata (squares).

 

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  		TABLE 1. In vitro caspofungin activity against less common molds

 
In summary, alternative methods for evaluating the susceptibilities of filamentous fungi to echinocandins are described in this work. These assays are generally adaptable to a wide range of non-Aspergillus molds. The GS echinocandin target has not been examined previously in any of the strains described here. We show that the glucan synthase of selected isolates from the genera Curvularia, Alternaria, Bipolaris, Trichoderma, and Scedosporium is as sensitive to echinocandins as the enzyme from Aspergillus fumigatus, for which there is now extensive preclinical and clinical experience. Based on these results, evaluation of the efficacy of caspofungin against infections caused by these fungi is warranted.


   ACKNOWLEDGMENTS
 
We thank Nicholas Kartsonis for thoughtful input into this study and Cameron Douglas, Rosemarie Kelly, and Paul Liberator for helpful discussions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Disease Research, Merck and Co., 80Y-210, P.O. Box 2000, Rahway, NJ 07065-0900. Phone: (732) 594-6799. Fax: (732) 594-1399. E-mail: jennifer_nielsen_kahn{at}merck.com. Back


   REFERENCES
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  2. Kelly, R., E. Register, M.-J. Hsu, M. Kurtz, and J. Nielsen. 1996. Isolation of a gene involved in 1,3-ß-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein. J. Bacteriol. 178:4381-4391.[Abstract/Free Full Text]
  3. NCCLS. 2002. Reference method for broth microdilution susceptibility testing of yeasts. M38-A. Approved standard, 2nd ed. NCCLS, Wayne, Pa.
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  5. Park, S., R. Kelly, J. N. Kahn, J. Robles, M.-J. Hsu, E. Register, W. Li, V. Vyas, H. Fan, G. Abruzzo, A. Flattery, C. Gill, G. Chrebet, S. A. Parent, M. Kurtz, H. Teppler, C. M. Douglas, and D. S. Perlin. 2005. Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob. Agents Chemother. 49:3264-3273.[Abstract/Free Full Text]
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Antimicrobial Agents and Chemotherapy, June 2006, p. 2214-2216, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01610-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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