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Journal of Clinical Microbiology, June 2002, p. 2228-2230, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2228-2230.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vitro Activity of Caspofungin (MK-0991) against Candida albicans Clinical Isolates Displaying Different Mechanisms of Azole Resistance

Stefano P. Bachmann, Thomas F. Patterson, and José L. López-Ribot^*

Department of Medicine, Division of Infectious Diseases, University of Texas Health Science Center at San Antonio, South Texas Centers for Biology in Medicine, San Antonio, Texas 78245

Received 26 December 2001/ Returned for modification 7 March 2002/ Accepted 17 March 2002

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Caspofungin inhibits the synthesis of 1,3-ß-D-glucan, a key step in fungal cell wall biosynthesis. Here we report on its potent in vitro activity (MIC at which 90% of the isolates tested are inhibited = 1 µg per ml of RPMI medium) against 32 Candida albicans fluconazole-susceptible and -resistant clinical isolates irrespective of the underlying resistance mechanism (alterations in ERG11 and/or upregulation of MDR and CDR genes encoding efflux pumps) and provide further evidence that caspofungin is not a substrate for multidrug transporters.

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The increase in occurrence of fungal infections, their changing epidemiology, the emergence of resistance, and the toxicity displayed by some of the presently used antifungal therapies have resulted in the need for an expanded arsenal of antifungal drugs. Antifungal agents at different stages in the development pipeline include new-generation azole derivatives and echinocandins. Azole agents target ergosterol biosynthesis, whereas echinocandins represent a new class of antifungal agents that act by inhibiting synthesis of 1,3-ß-D-glucan, a key step in fungal cell wall biosynthesis (2). Because of their different mode of action and molecular structure, it is unlikely that cross-resistance between azole antifungal agents (targeting ergosterol synthesis) and echinocandins occurs. Thus, echinocandins, which are fungicidal against yeasts and have a different mode of action, may constitute effective prophylactic and therapeutic options for the management of a variety of fungal infections, including those that are refractory to azoles. Caspofungin acetate (Cancidas, formerly reported as MK-0991 and L-743,872) is a water-soluble, potent echinocandin with activity against a number of clinically important fungi (2).

Fluconazole has proven effective in treating mucosal candidiasis even in individuals with advanced immunodeficiency. However, resistance to fluconazole and other azole antifungal drugs has become an important clinical problem in the management of candidiasis. Mechanisms of azole resistance are multifactorial and include alterations in the target enzyme (lanosterol demethylase, encoded by the ERG11 gene), including overexpression and point mutations and increased extrusion of drug mediated by two types of multidrug efflux transporters, the ABC transporters (encoded by CDR genes) and the major facilitators (encoded by MDR genes) (12). Due to the different mechanisms of action, alterations in the lanosterol demethylase are unlikely to affect susceptibility to caspofungin. However, multidrug efflux pumps display affinity for a wide variety of compounds (including different types of antifungals), and thus the possibility exists that their overexpression may affect susceptibility to caspofungin. Previous studies have demonstrated that caspofungin is highly active against Candida species, including some azole-resistant isolates (1, 3, 11), but the molecular mechanisms responsible for azole resistance in these isolates were not known.

(This work was partially presented at the 40th Interscience Conference of Antimicrobial Agents and Chemotherapy held in Toronto, Canada, September 2000.)

The Candida albicans clinical isolates included in this study have been described before (8). Briefly, they were obtained by direct swab or by oral saline rinses from 12 human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis enrolled in a longitudinal study to assess the significance of fluconazole resistance (Table 1). The identity of the isolates as C. albicans was confirmed by standard biochemical and microbiological procedures (7). Initial susceptibility testing against fluconazole was performed following NCCLS broth macrodilution methodology (5). For each patient, matched sets (as determined by DNA-typing methods) of fluconazole-susceptible and -resistant (MIC 64 µg/ml) C. albicans isolates were included in this study. The molecular mechanisms responsible for azole resistance in this series of isolates have been previously reported (8). Briefly, a Northern blot technique was used to study levels of expression of ERG11 (encoding lanosterol demethylase, the target enzyme for azole derivatives) and MDR1 and CDR genes (encoding efflux pumps) implicated in the development of azole resistance. We also obtained the nucleotide sequences of ERG11 genes from all isolates after PCR amplification from genomic DNA. Sequence data were compared to a published ERG11 sequence in search of nucleotide changes resulting in amino acid substitutions affecting the affinity of the enzyme for azole derivatives. Fluconazole MICs and a summary of the molecular resistance mechanisms detected for each of the different isolates are included in Table 1.

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TABLE 1. C. albicans isolates included in this studya

Testing of antifungal susceptibility to caspofungin was determined following NCCLS procedures using a broth microdilution method (5). Caspofungin was provided by Merck Research Laboratories (Rahway, N.J.) as a standard powder and was tested at final concentrations of 0.015 to 16 µg/ml. Both the reference RPMI 1640 and Antibiotic Medium 3 with 2% glucose (AM3) were used as test media. We used the spectrophotometric method of inoculum preparation corresponding to a concentration of 0.5 x 10³ to 2.5 x 10³ cells per ml for each of the isolates prepared in the test medium. Yeast inocula (100 µl) were added to each well of microdilution trays containing 100 µl of antifungal drug solution (prepared at twice the final concentration). MICs were read visually as the lowest concentration at which complete growth inhibition was observed after both 24 and 48 h of incubation.

The results of the MIC readings for caspofungin against C. albicans fluconazole-susceptible and -resistant clinical isolates are shown in Table 1. When RPMI 1640 was used as the test medium, the caspofungin MICs measured at 24 h ranged from 0.25 to 1 µg/ml; 48-h readings ranged from 0.25 to 2 µg/ml. The caspofungin MICs at which 90% of isolates were inhibited at 24 and 48 h were 0.5 and 1 µg/ml, respectively, and these values were the same for both azole-susceptible and -resistant isolates. With AM3, the MICs were all < 0.015 µg/ml at 24 h and ranged from < 0.015 to 0.03 µg/ml at 48 h. Again, caspofungin was equally active against fluconazole-susceptible and fluconazole-resistant isolates (the MIC at which 90% of isolates tested were inhibited was < 0.015 µg/ml at both 24 and 48 h) when tested using AM3. Our results confirmed previously published data showing remarkably lower MICs of caspofungin when AM3 instead of RPMI 1640 was used for the assay (6). The reason for this finding is still not clear. Although interpretative criteria have not yet been defined for caspofungin, the MICs obtained here were in the range of recently reported achievable levels of caspofungin in serum in humans that are approximately 1 µg/ml for a 50-mg caspofungin daily dose (J. A. Stone, J. B. McCrea, P. J. Wickersham, S. D. Holland, P. J. Deutsch, S. Bi, T. Cicero, H. Greenberg, and S. A. Waldman, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 854, p. 26, 2000) and thus should be considered susceptible.

To further assess the potential role of multidrug efflux pumps in the susceptibility to caspofungin, we also performed susceptibility testing of the parental strain C. albicans SC5314 and the following mutants with knockouts in genes encoding multidrug efflux pumps: DS 448 (cdr1), DS 653 (cdr2), DS 465 (mdr1), DS 654 (cdr1/cdr2), and DS 468 (cdr1/mdr1). These strains were a kind gift from D. Sanglard (9, 10). The caspofungin susceptibility profiles of all multidrug transporter mutants tested were unchanged from that of strain SC5314. This was in contrast to their susceptibility to fluconazole, for which these mutants have been shown to be hypersusceptible (9, 10), and in our assay they demonstrated up to a three- or twofold-dilution decrease in fluconazole MICs compared to their parental strain. These strains were tested only against caspofungin in RPMI medium. At 24 h, the caspofungin MICs were between 0.5 and 1 µg/ml, whereas at 48 h, all MICs were the same, 1 µg/ml.

Azoles target ergosterol synthesis by blocking lanosterol demethylase, an enzyme that is encoded by the ERG11 gene. Caspofungin, on the other hand, affects fungal cell wall synthesis by inhibiting the production of 1,3-ß-D-glucan (2). It is therefore unlikely that cross-resistance between these two classes of antifungals could be explained by an overexpression of ERG11 or a point mutation within the ERG11 gene. Our results supported this theory. It was not yet clear if overexpression of genes encoding efflux pumps, like CDR or the fluconazole-specific MDR1, could affect susceptibility to caspofungin. Our clinical isolates demonstrated low-stable caspofungin MICs, independent of fluconazole resistance and overexpression of genes encoding these efflux pumps. Thus, these results are indicative that caspofungin is not likely to be a substrate for these types of transporters. The observation that a series of multidrug transporter mutants did not exhibit a hypersusceptible phenotype against caspofungin compared to their parental strain corroborated that caspofungin is not a substrate for these pumps that have been widely implicated in resistance to different classes of antifungal drugs and other compounds. Of note, it has been previously shown that the majority of fluconazole-resistant isolates included in this study also exhibit decreased susceptibility against other triazoles, including the new investigational azole agents to which our patients have not had any previous exposure (8). This is in line with observations indicating that cross-resistance between different azoles, even investigational substances, can occur, as demonstrated by other authors (4).

In conclusion, caspofungin seems to be highly active against azole-susceptible and -resistant isolates, regardless of the underlying molecular mechanism(s) of azole resistance. It may therefore constitute an effective therapeutic option for the treatment of C. albicans infections, including those refractory to conventional treatment with triazole agents.

 
This work was supported by a grant from Merck & Co., Inc., and by Public Health Service grant 1 R01 DE11381.

We also thank Dominique Sanglard for providing us with the efflux pump mutant strains of C. albicans.

 
* Corresponding author. Mailing address: Department of Medicine, Division of Infectious Diseases, University of Texas Health Science Center at San Antonio, South Texas Centers for Biology in Medicine, 15355 Lambda Dr., San Antonio, TX 78245. Phone: (210) 562-5017. Fax: (210) 562-5016. E-mail: ribot{at}uthscsa.edu.

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Journal of Clinical Microbiology, June 2002, p. 2228-2230, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2228-2230.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bachmann, S. P. Articles by López-Ribot, J. L. Search for Related Content PubMed PubMed Citation Articles by Bachmann, S. P. Articles by López-Ribot, J. L.