Hoechst Marion Roussel, 102 Route de Noisy, 93235 Romainville Cedex, France
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Abstract |
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Introduction |
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In view of the potential role of this dimorphism, an understanding of the action of antifungal agents on the two forms may help in the development of better antifungal agents. MIC methodologies, including those under standardization by the NCCLS, have greatly assisted the evaluation of antifungal agents against different yeast species. 3 Such methodology takes into account only the inhibition of growth of cells, and does not permit one to assess the effects of drugs on the transformation from the yeast form to the hyphal form. We recently described a modified version of the NCCLS procedure which can be used to study the effects of antifungal agents on the morphogenetic transformation from yeast to hyphal forms. 4
In this study we have used various clinical and laboratory strains to study the effects of a wide range of antifungal agents on the morphogenetic transformation of C. albicans. Our results suggest that antifungal agents that are more fungicidal, for example, amphotericin B and echinocandins, are much more likely to inhibit the transformation than are the less fungicidal agents, for example, azoles and flucytosine.
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Materials and methods |
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Synchronized yeast-phase C. albicans cells were used in all experiments and were prepared as described previously. 4 C. albicans reference strains ATCC 90028 and 90029 3 and clinical isolates 200/175, 352, 94/03, 94/14 and 94/57 were used throughout. Stationary phase synchronized yeast cells were harvested by centrifugation, washed three times in 0.1 M phosphate-buffered saline and used immediately in standard MIC assays 3 and morphogenetic transformation experiments. 4 The effects of different antifungal agents on the morphogenetic transformation of C. albicans were assessed by examining the contents of 96-well microtitre plates after 3 h incubation at 35°C using phase-contrast microscopy (using an Olympus CK microscope and Zeiss phase-contrast microscope) as described previously. 4
Mimimum fungicidal concentration
Samples (10 µL) were removed from all wells of the standard MIC plates and spotted on to rectangular dishes containing Sabouraud dextrose agar. The plates were incubated for 24-48 h at 35°C. The minimal fungicidal concentration (MFC) was defined as the concentration of antifungal agent at which the number of colony forming units was zero.
Antifungal agents and chemicals
Itraconazole, ketoconazole and miconazole were purchased from Janssen Pharmaceutica, Beerse, Belguim. Fluconazole was purified (95% purity) from Diflucan capsules (Pfizer Central Research, Sandwich, UK). Amphotericin B, tunicamycin and flucytosine were purchased from Sigma Chemical Co. (St Louis, MO, USA). Terbinafine and amorolfine were from Sandoz, Vienna, Austria. Mulundocandin and aculeacin were synthesized at Hoechst AG, Frankfurt, Germany. MOPS and dimethyl sulphoxide (DMSO) were from Sigma. Antifungal agents were dissolved in DMSO with the exception of tunicamycin and flucytosine which were dissolved in sterile distilled water. DMSO (final concentration of <2% v/v) did not affect the MIC or morphogenetic transformation.
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Results and discussion |
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The inhibition of growth by the different antifungal agents, and the MFCs of these agents for several clinical isolates and laboratory strains of C. albicans, were examined. All strains were highly sensitive to amphotericin B (MIC 0.06-0.12 mg/L), tunicamycin (MIC 0.12-0.25 mg/L) and aculeacin (MIC 0.25-0.5 mg/L) but less sensitive to mulundocandin (Table). Amphotericin B, mulundocandin and aculeacin were generally fungicidal at concentrations close to the MIC (2-4 x MIC) while tunicamycin exhibited a more moderate fungicidal ability in that it was fungicidal at 8-16 x MIC (Table). Of the azole antifungal agents tested, itraconazole and ketoconazole showed good activity against all of the C. albicans isolates (Table) while fluconazole and miconazole showed good activity against four of these C. albicans strains (ATCC 90028, ATCC 90029, 352 and 200/175) with MIC ranges of 0.25-0.5 mg/L and 0.06-0.25 mg/L, respectively. However, when tested against strains 94/03, 94/14 and 94/57, fluconazole and miconazole were considerably less active with MIC ranges of 8 mg/L and 8-16 mg/L, respectively. Flucytosine exhibited good activity against six of the strains and was inactive against C. albicans ATCC 90029 as described previously. 3 By contrast, terbinafine and amorolfine showed moderate activity against the seven strains with MIC ranges of 2-8 mg/L and 2-4 mg/L, respectively (Table). The azoles and other agents exhibited a fungicidal ability only at high concentrations (32-128 x MIC).
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The effect of the antifungal agents on the morphogenetic transformation of the various C. albicans strains was also examined. The fungicidal agents amphotericin B, mulundocandin and aculeacin all blocked the morphogenetic transformation at concentrations below their corresponding MIC values (Table). Tunicamycin also inhibited the morphogenetic transformation but at concentrations higher than the MIC (morphogenetic transformation of 0.25-0.5 mg/L; MICs of 0.12-0.25 mg/L). Fluconazole and miconazole had low MICs against the four azole-susceptible strains and affected the morphogenetic transformation only at higher concentrations (Table). The effect on morphogenetic transformation was observed at even higher concentrations on the three strains with reduced susceptibility to azole agents (data not shown). Similarly, itraconazole and ketoconazole, which were active against all strains by MIC determinations, only affected morphogenetic transformation at high concentrations (Table). Of the remaining antifungal agents, flucytosine was a poor inhibitor of the morphogenetic transformation despite its good activity against six of the strains as measured by MIC. Terbinafine and amorolfine showed some activity as judged by MIC but had little effect on morphogenetic transformation (Table).
Comparison of MIC, morphogenetic transformation and MFC
In order to describe the differences better, the morphogenetic transformation/MIC ratios were calculated for each antifungal agent (Table). These ratios can be used to determine the relative potency of each agent to inhibit growth and transformation: (i) ratio < 1 (agents which preferentially inhibit morphogenetic transformation): amphotericin B, mulundocandin and aculeacin; (ii) ratio of 1-2 (those with approximately equal effects on these two processes): tunicamycin; and (iii) ratio > 2 (agents with a lower ability to inhibit the hyphal form and which exert their activity by inhibiting the yeast form): azoles, terbinafine, flucytosine and amorolfine. The MFC/MIC ratio was also calculated (Table). The data suggest that agents with a high fungicidal potential, for example amphotericin B, also have a high potential to block morphogenetic transformation. Agents with a low fungicidal potential, for example fluconazole, are strikingly less able to inhibit morphogenetic transformation.
The combination of studying the effects of antifungal agents on morphogenetic transformation and MIC provides additional information concerning the action of antifungal agents against both the yeast and hyphal forms of C. albicansand may be related to their fungicidal potential. Our data suggest that fungicidal agents act against both morphogenetic transformation and the budding process. The fungicidal agents, amphotericin B, mulundocandin and aculeacin, disrupt membrane 5 or cell wall integrity, 6 and consequently inhibit the hyphal form at low concentrations. The less fungicidal agents (azoles, terbinafine and flucytosine), which exert their antifungal action through inhibition of cytochrome P450 demethylase, 7 squalene epoxidase, 8 and RNA and DNA synthesis, 5 respectively, tend to be less effective against morphogenetic transformation, suggesting that they preferentially inhibit the budding process. It is tempting to suggest that the ability of amphotericin B and echinocandins to reduce the fungal load efficiently in vivo, as compared with the azoles, may in part be related to their ability to affect morphogenetic transformation.
In conclusion, the morphogenetic transformation and MIC assays used in this study, combined with the calculation of morphogenetic transformation/MIC ratios, may assist in preliminary screening for new antifungal agents. Indeed, certain types of antifungal agents have previously been demonstrated to show little or no activity by MIC methodology though they do show efficacy in vivoechinocandins generally show weak or no activity against Aspergillus spp. as judged by MIC but are efficacious in vivo and are known to reduce hyphal formation. 6 The morphogenetic transformation assay would greatly assist in the characterization of the activity of new antifungal agents and may distinguish fungicidal from fungistatic compounds. The assay is rapid: data are generated within 3 h, as compared with 48 h for standard MIC procedures.
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Notes |
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References |
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2 . Lo, H. J., Kohler, J. R., Didomenico, B., Loebenberg, D., Cacciapuoti, A. & Fink, G. R. (1997). Nonfilamentous C. albicans mutants are avirulent. Cell 90,939 -49.[ISI][Medline]
3 . National Committee for Clinical Laboratory Standards. (1995). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Tentative Standard M27-T. NCCLS, Villanova, PA.
4 . Hawser, S., Francolini, M. & Islam, K. (1996). The effects of antifungal agents on the morphogenetic transformation by Candida albicansin vitro. Journal of Antimicrobial Chemotherapy 38, 579-87.[Abstract]
5 . Kerridge, D. (1986). Mode of action of clinically important antifungal drugs. Advances in Microbial Physiology 27, 1 -72.[ISI][Medline]
6 . Kurtz, M. B., Heath, I. B., Marrinan, J., Dreikhorn, S., Onishi, J. & Douglas, C. (1994). Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-ß-D-glucan synthase. Antimicrobial Agents and Chemotherapy 38, 1480-9.[Abstract]
7 . Vanden Bossche, H. (1988). Mode of action of pyridine, pyrimidine and azole antifungals. In Sterol Biosynthesis Inhibitors: Pharmaceutical and Agrochemical Aspects (Berg, D. & Plempel, M. Eds), pp. 79- 119. VCH Publishers Inc., New York.
8 . Ryder, N. S. (1988). Mechanisms of action and biochemical selectivity of allylamine antimycotic agents. Annals of the New York Academy of Sciences 544, 208-20.[Medline]
Received 7 April 1998; returned 17 June 1998; revised 28 July 1998; accepted 19 October 1998