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编号:11258956
Relationship between MIC and Minimum Sterol 14-Demethylation-Inhibitory Concentration as a Factor in Evaluating Activities of Azoles against
     Division of Oral Infectious Diseases and Immunology, Faculty of Dental Sciences

    Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

    Department of Bioactive Molecules, National Institute of Infectious Diseases

    Department of Infectious Diseases, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan

    ABSTRACT

    The minimum growth-inhibitory concentrations (MICs) of azole antifungals were compared to their minimum sterol 14-demethylation-inhibitory concentrations (MDICs) for clinical fungal isolates. The ascomycetous Candida yeasts tested were clearly divided into two groups: group I, consisting of C. albicans, C. tropicalis, and C. lusitaniae, had MICs that were much higher than the MDICs, whereas group II, comprising C. glabrata, C. parapsilosis, C. guilliermondii, and C. krusei, had MICs that were approximately equal to the MDICs. In the ascomycetous fungi Aspergillus fumigatus and Sporothrix schenckii, the MICs were indistinguishable from the MDICs. In the basidiomycetous fungi Cryptococcus (Filobasidiella) neoformans, C. curvatus, and Trichosporon asahii, the MICs and the MDICs were practically identical. These results support the notion that there are two distinct classes of fungi differing in their degree of tolerance to sterol 14-demethylation deficiency. These findings have significant implications for both fungal physiology and antifungal chemotherapy.

    INTRODUCTION

    Azole antifungal agents are known to inhibit the activity of cytochrome P45014DM, which catalyzes the 14-demethylation of sterols in ergosterol biosynthesis (21). As a consequence, cells treated with those drugs accumulate 14-methylated sterols in their membranes in place of ergosterol. A notable feature of this change in sterol composition is that it may or may not inhibit cell growth depending on the fungus involved. Thus, our previous work has demonstrated that under conditions of 14-demethylation inhibition, cells of Candida albicans, C. tropicalis, C. guilliermondii, and C. kefyr can proliferate in vitro, while those of C. krusei, C. glabrata, and C. parapsilosis cannot (18). Unfortunately, however, it has remained unclear whether this characteristic varies within each species due to the small number of strains examined.

    In vivo data, however, indicate that azoles are therapeutically effective in C. albicans infection due to the inhibition of 14-demethylation (11). This could be because 14-demethylation-deficient cells are more vulnerable than normal cells to killing by reactive oxygen species (16) or phagocytes (3, 4, 5, 7, 20).

    The most important implication of the above-mentioned findings is that the minimum sterol 14-demethylation-inhibitory concentration (MDIC), rather than the minimum growth-inhibitory concentration (MIC), may be of primary importance for the clinical use of azole drugs. Theoretically at least, if the MIC of a drug is higher than its MDIC for a certain fungus, the drug may suppress infections it causes even at a serum concentration below the MIC (but above the MDIC) as long as host defenses are unimpaired. It is therefore necessary to collect more information about the relationship between MIC and MDIC. In the present study, we provide such data for a number of clinical isolates, including not only Candida species but also other ascomycetous and basidiomycetous fungi.

    MATERIALS AND METHODS

    Fungal strains. Ninety-five clinical isolates of yeast-like fungi, recovered from the bloodstream of patients with fungal infections, consisted of 26 strains of C. albicans, 17 strains of C. tropicalis, 8 strains of C. glabrata, 10 strains of C. parapsilosis, 21 strains of C. guilliermondii, 9 strains of C. krusei (of which 4 are laboratory strains), 2 strains of C. lusitaniae (1), 2 strains of Cryptococcus (Filobasidiella) neoformans, 2 strains of Cryptococcus curvatus (8), and 2 strains of Trichosporon asahii (19). These isolates were identified presumptively by colony morphology on CHROMagar Candida (Becton Dickinson), chlamydospore formation on cornmeal agar, and sugar assimilation patterns with the API ID32C system (BioMerieux SA, Marcy-l'Etoile, France). Species identification was confirmed by direct sequencing of the D1-D2 region of 26S rRNA (6). Five strains of Aspergillus fumigatus isolated from animals were provided by Rui Kano of the School of Veterinary Medicine, Nippon University. Sporothrix schenckii IFM41598, originally isolated from a patient with sporothrichosis, was provided by Kazuko Nishimura of Chiba University.

    MIC and MDIC determination. We have previously devised a simple method capable of measuring the MDIC of an azole drug for those fungi whose viability is unaffected by sterol 14-demethylation deficiency, e.g., C. albicans (17, 18). The method is based on the fact that 14-demethylation deficiency, whether caused by an azole or another mechanism, makes the fungal cells susceptible to growth inhibition by acetate added to the growth medium, probably due to an increase in permeability of the cell membrane (17). Thus, for fungi of this class the MIC of an azole as determined in acetate-supplemented medium (MICAc) is lower than its MIC measured in acetate-free medium and is similar to its MDIC, as estimated by sterol profiling with thin-layer chromatography; hence, the relationship was MIC > MICAc = MDIC (18). In contrast, those fungi which are unable to tolerate 14-demethylation deficiency, e.g., C. krusei, are characterized by the formula MIC = MICAc = MDIC (18). Incidentally, acetate has no effect on sterol demethylation or growth rate at the concentration used. In practice, the determination of MIC and MDIC was carried out as follows. Twofold dilutions of the test drug were made in yeast extract-peptone-glucose (YEPG) medium consisting of 1% yeast extract, 2% polypeptone, and 2% glucose (for MICY, which means MIC measured in YEPG) or YEPG supplemented with 0.24 M sodium acetate (YEPG-Ac) (for MDIC). A 0.1-ml portion of each dilution was mixed in a microplate well with 0.1 ml of a cell suspension in the same medium containing approximately 105 CFU. The plates were incubated at 37°C for 2 days with shaking. The lowest drug concentration that gave a prominent decrease in turbidity was taken to represent the MICY or MDIC of the drug. It should be noted that the MICY obtained in this way is not equivalent to the MIC determined by the routine method described by the National Committee for Clinical Laboratory Standards (9). In the case of S. schenckii, the high susceptibility of this fungus to acetate made the use of the above method for MDIC determination inadequate. Hence, direct estimation of MDIC by sterol profiling with thin-layer chromatography was carried out as previously described (17).

    RESULTS

    Candida species. Our previous study demonstrated that the strains of Candida species examined were divided into two groups with respect to the relationship between MIC and MDIC (18). However, the small number of test strains made it uncertain whether this characteristic was species specific. In the present work, we studied this dichotomy with a much larger number of strains (Table 1). For strains of C. albicans and C. tropicalis, the MICY (>200 μg/ml for fluconazole) was much higher than the MDIC (0.2 to 3.1 μg/ml), as previously reported. In addition, strains of C. lusitaniae were found to belong to this group. On the other hand, the MICY was essentially equal to the MDIC for strains of C. glabrata, C. parapsilosis, and C. krusei, in agreement with the previous results (18). Only C. guilliermondii exhibited an intraspecific heterogeneity: strain IFO0454, used in the previous studies, is a member of the former group, while the 21 strains tested in this work were found to belong to the latter group. These results strongly suggest that intraspecies variation in this MIC-MDIC characteristic is rare. In other words, this characteristic can be regarded as species specific for Candida species.

    Basidiomycetous yeast species. For all strains of the basidiomycetous yeasts C. neoformans, C. curvatus, and Trichosporon asahii tested, the MICY was found to be comparable to the MDIC (Table 1).

    Filamentous fungi. We successfully applied the YEPG-Ac method for MDIC determination to five strains of the ascomycetous mold A. fumigatus, and we found for all of them that the MICY was comparable to the MDIC (Table 1). The ascomycetous fungus S. schenckii is dimorphic and grows either as filaments at 25°C or as yeasts at 37°C (10). Because the growth of this fungus turned out to be severely inhibited by acetate even in the absence of azoles, the use of YEPG-Ac medium for MDIC determination was considered inadequate. We therefore studied growth and sterol composition of cells of this fungus cultured aerobically in YEPG medium at 25°C in the presence of various concentrations of ketoconazole. The azole drug inhibited by more than 80% both cell growth and sterol 14-demethylation at a concentration of 1.25 μg/ml and higher compared to the drug-free control cells (Fig. 1). The same results were obtained with yeast cells grown at 37°C (data not shown). Thus, the MICY and MDIC of ketoconazole were shown to be similar for this organism. In addition, the MICY estimated by optical density was compatible with morphological observations. While cells grew in typically filamentous form without ketoconazole, the presence of the drug at 1.25 μg/ml completely inhibited their growth.

    DISCUSSION

    The results of the present study are consistent with the notion that fungi can be divided into two types based on their tolerance to deficiency in sterol 14-demethylation. Most, if not all, species appear to be homogeneous with respect to this trait, and the species for which the drug MIC and MDIC are comparable to each other appear to be more common than those for which the in vitro MIC is higher than the MDIC. The mechanism underlying the difference in the degree of tolerance to 14-methylated sterols is unknown at present. One possibility is that different degrees of tolerance may be due to the effect of the 14-methyl group on the function of one or more membrane proteins that interact with sterols in membranes.

    C. albicans and C. tropicalis, for which the MICY is higher than the MDIC, are both important opportunistic pathogens, frequently causing systemic mycoses in compromised hosts. Our previous studies have shown that C. albicans cells exhibit increased sensitivity to various antifungal chemicals when their membranes contain 14-methylated sterols in place of ergosterol, probably due to enhanced membrane permeability (13, 15). This observation immediately suggests the possibility that a combination of an azole and an antifungal from another class could act synergistically on fungal cells at least of this type. It is hoped that this may open up a new avenue of antifungal chemotherapy.

    The values of MDICs for Candida species are similar to MICs obtained by the standard NCCLS RPMI method (9, 18) and correlate with the effectiveness of antifungal agents in vivo. We are therefore currently investigating the wider application of the simple MDIC methodology to the azole susceptibility testing of filamentous fungi, such as Aspergillus spp.

    ACKNOWLEDGMENTS

    This work was supported by a grant from the Japan Health Sciences Foundation to O.S. and M.N. and Health Science Research Grants for Research on Emerging and Re-emerging Infectious Diseases, Ministry of Health, Labor and Welfare of Japan to M.N. and K.K.

    Gifts of azole drugs from Pfizer Pharmaceuticals and Janssen Research Foundation are gratefully acknowledged. We thank Kazuko Nishimura of Chiba University and Rui Kano of Nippon University for providing us with fungal strains. We would also like to thank Hiroaki Nakayama, emeritus professor of Kyushu University, and Richard D. Cannon of Otago University for scientific discussion and critical reading of the manuscript.

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