JAC Advance Access published online on May 19, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn216
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Research letter |
Antifungal activity against Candida albicans of nikkomycin Z in combination with caspofungin, voriconazole or amphotericin B
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
* Corresponding author. Tel: +972-3-6409870; Fax: +972-3-6422494; E-mail: segale{at}post.tau.ac.il
Key Words: antifungal combinations , in vitro activity
Until the last decade, antifungal therapy was based mostly on drugs acting on the fungal membrane, such as amphotericin B and azoles, and the rationale for the use of combination therapy remained questionable.1 Thus, the only drug combination of two antifungals with two modes of activity used clinically, primarily in cryptococcosis, was amphotericin B and 5-fluorocytosine.2 The introduction of echinocandins, which act on the fungal cell wall by inhibiting glucan synthesis, opened the approach to explore different drug combinations, such as echinocandins and polyenes, or echinocandins and azoles,3,4 for various mycoses.
Nikkomycin Z inhibits chitin synthesis, by acting as a competitive analogue of chitin synthase substrate UDP-N-acetylglucosamine.3 Since chitin is found in most fungal cell walls, inhibition of its synthesis may be considered as a potential means for antifungal therapy. Although nikkomycin Z is still a compound under investigation, not yet approved clinically, combinations involving this compound may open additional possible avenues for useful therapy.
This study reports on the assessment of in vitro activity of nikkomycin Z in combination with either amphotericin B, voriconazole or caspofungin against Candida albicans.
C. albicans CBS 562, a laboratory strain, and a clinical strain, 58919, were used throughout the study. For the susceptibility tests, the Candida strains were grown on Sabouraud agar for 24 h and suspended in PBS to a concentration of 0.5–2.5 x 106 cells/mL using haemocytometer counts. This stock suspension was further diluted in yeast nitrogen base (YNB) to a final concentration of 0.5–2.5 x 103 cells/mL.
Caspofungin, voriconazole, amphotericin B and nikkomycin Z stock solutions were prepared at concentrations of 5 and 1 mg/mL, respectively, and kept at –70°C. At the initial stage of the study, the sensitivity of Candida was tested for each drug using a broth microdilution technique,5 determining minimal inhibitory concentration (MIC—the lowest drug concentration inhibiting yeast growth) and minimal fungicidal concentration (MFC—the lowest drug concentration killing the yeasts). The MIC and MFC values of amphotericin B, caspofungin and voriconazole from three to four experiments (data not shown) were compatible with the data reported in the literature, with no significant differences between the test strains. The nikkomycin Z values were high for both C. albicans strains, with differences between the strains. Drug combinations were assessed by chequerboard assays using the broth microdilution methodology.6 The experiment was performed in 96-well microtitre plates using YNB as a medium. The results presented are MIC values and are those recorded after 48 h of incubation at 37°C. Each plate included rows with combinations of drugs and control rows of the drugs alone. The results were used to determine the fractional inhibitory concentration (FIC; in mg/L) of the combination of nikkomycin Z and either caspofungin, voriconazole or amphotericin B. FICs were calculated for MIC endpoint measurements taken from the micro-well with the lowest concentration of the drug combination needed to achieve the respective endpoints. The FIC of a drug for an individual isolate was calculated as the MIC of the drug when used in combination with another drug divided by the MIC of the drug when used alone. The FIC index (FICI) value was calculated by adding the FIC of nikkomycin Z to that of each of the other drugs. FICI values were interpreted as follows: FICI 
0.5, synergistic; 0.5 < FICI 4, no interaction; FICI > 4, antagonistic. Table 1 shows the susceptibility of the C. albicans strains for three different drug combinations: amphotericin B + nikkomycin Z, voriconazole + nikkomycin Z and caspofungin + nikkomycin Z. For each combination, three separate tests were carried out for each C. albicans strain. The assays showed that the MIC values of nikkomycin Z in combination with voriconazole or caspofungin were lower in comparison with those of the drug alone. The analysis of the FICI data indicates that the effect of the three combinations is similar for both C. albicans strains. In two of the combinations (caspofungin + nikkomycin Z and voriconazole + nikkomycin Z), the result is synergy, whereas in the third combination (amphotericin B + nikkomycin Z), there was no interaction.
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This study explored the in vitro anti-Candida activity of drug combinations from four classes: the polyenes, second-generation triazoles, echinocandins and nikkomycins. While both the polyenes and triazoles act on the fungal cell membrane, although through different mechanisms, the echinocandins and nikkomycins affect the cell wall, interfering with the synthesis of glucan and chitin, respectively.1,3 Our data show that combining voriconazole or caspofungin with nikkomycin Z resulted in a synergistic effect. To the best of our knowledge, the specific drug combinations used in our study against C. albicans have not been described before. Whether combinations of antifungals with different modes of activity could enhance the therapeutic efficacy beyond that of monotherapy should be corroborated in animal models, leading eventually to use in humans.
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None to declare.
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Two of the co-others (R. S. and Y. L.) were graduate students, hence the funding was internal from Tel Aviv University.
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