We compared the activities of antifungal agents against a wide range of yeasts and filamentous fungi. The methodology of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for yeasts and spore-forming molds was applied; and a total of 349 clinical isolates of Candida spp., other yeast species, Aspergillus spp., and nondermatophyte non-Aspergillus spp. were investigated. The average geometric mean (GM) of the MICs of the various drugs for Candida spp. were as follows: amphotericin B (AMB), 0.55 g/ml; liposomal amphotericin B (l-AMB); 0.35 g/ml; itraconazole (ITC), 0.56 g/ml; voriconazole (VRC), 0.45 g/ml; posaconazole (POS), 0.44 g/ml; and caspofungin (CPF), 0.45 g/ml. The data indicated that the majority of Candida spp. were susceptible to the traditional and new antifungal drugs. For Aspergillus spp., the average GM MICs of AMB, l-AMB, ITC, VRC, POS, and CPF were 1.49 g/ml, 1.44 g/ml, 0.65 g/ml, 0.34 g/ml, 0.25 g/ml, and 0.32 g/ml, respectively. For the various zygomycetes, the average GM MICs of AMB, l-AMB, ITC, and POS were 1.36 g/ml, 1.42 g/ml, 4.37 g/ml, and 1.65 g/ml, respectively. Other yeastlike fungi and molds displayed various patterns of susceptibility. In general, the minimal fungicidal concentrations were 1 to 3 dilutions higher than the corresponding MICs. POS, AMB, and l-AMB showed activities against a broader range of fungi than ITC, VRC, and CPF did. Emerging pathogens such as Saccharomyces cerevisiae and Fusarium solani were not killed by any drug. In summary, the EUCAST data showed that the in vitro susceptibilities of yeasts and filamentous fungi are variable, that susceptibility occurs among and within various genera and species, and that susceptibility depends on the antifungal drug tested. AMB, l-AMB, and POS were active against the majority of pathogens, including species that cause rare and difficult-to-treat infections.

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This study investigated the basis for intrinsic amphotericin B (AMB) resistance in Aspergillus terreus. The ergosterol content, cell wall composition, and lipid peroxidation level had no influence on AMB resistance. The level of catalase production in A. terreus was significantly higher than that in A. fumigatus (P < 0.05). This higher-level production may contribute to AMB resistance in A. terreus since oxidative damage has been implicated in AMB action.Invasive aspergillosis has emerged as a leading cause of morbidity and mortality in immunocompromised patients (7). Aspergillus terreus in particular is an amphotericin B (AMB)-resistant mold that has been recognized as a cause of frequently lethal infections (25). The exact mechanism of AMB action is still incompletely understood. Previous work has shown that AMB binds to the ergosterol in the fungal cell membrane; this step introduces aqueous pores into the lipid bilayers, and thus small ions leak out, causing a disruption of the proton gradient (24). Others have demonstrated AMB to cause cell death by oxidative damage (21). AMB is active against a wide range of fungi, yet certain species, such as A. terreus and Pseudallescheria boydii, are intrinsically resistant to AMB (7,17,22,23). Manavathu et al. (14) generated AMBresistant mutants of Aspergillus fumigatus by UV radiation. Alternatively, resistant isolates have been collected by repeated subculturing on agar containing increasing amounts of polyene compounds (24). It has been suggested previously that for Aspergillus flavus and Candida albicans, the ergosterol content, the composition of the fungal cell wall, and the ability to produce catalase may play a role in AMB resistance (15,20,21). So far, the reason for the in vitro and in vivo AMB resistance in A. terreus is not known. This study examined the basis for AMB resistance in A. terreus.Clinical isolates of AMB-sensitive A. fumigatus (n ϭ 10) and AMB-resistant A. terreus (n ϭ 10) were used in this study. AMB susceptibility testing was performed according to the CLSI (formerly NCCLS) M38-A technique (16). In parallel, we prepared cell wall-free Aspergillus protoplasts that were AMB sensitive (MIC Ͻ 1 g/ml) and resistant (MIC Ͼ 2 g/ml); protoplast preparation, including the determination of the regeneration rate, was done according to a protocol of Hearn et al. (10). MIC testing with protoplasts was performed in RPMI 1640 containing 0.55 M sorbitol as a stabilizer. Our results provide evidence that AMB resistance in A. terreus is not associated with the cell wall, as MICs for conidia and cell wall-free fungi did not differ. The mean AMB MICs for conidia and protoplasts of A. fumigatus were 0.21 Ϯ 0.06 and 0.19 Ϯ 0.05 g/mg, and those for conidia and protoplasts of A. terreus were 1.70 Ϯ 0.58 and 1.41 Ϯ 0.38 g/mg, respectively.Fungal ergosterol was analyzed by a previously published method with slight modifications (2, 4). Fifty milliliters of Sabouraud medium (Merck, Vienna, Austria) was inoculated with 10 6 conidia and incubated for 24 h with shaking (...

Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 g/ml; voriconazole MIC, 2 g/ml; ravuconazole MIC, 4 g/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 g/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the ␤-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazoleresistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.