bAspergillus fumigatus intrinsic fluconazole resistance has been demonstrated to be linked to the CYP51A gene, although the precise molecular mechanism has not been elucidated yet. Comparisons between A. fumigatus Cyp51Ap and Candida albicans Erg11p sequences showed differences in amino acid residues already associated with fluconazole resistance in C. albicans. The aim of this study was to analyze the role of the natural polymorphism I301 in Aspergillus fumigatus Cyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue in A. fumigatus Cyp51Ap was replaced with a threonine (analogue to T315 at Candida albicans fluconazole-susceptible Erg11p) by changing one single nucleotide in the CYP51A gene. Also, a CYP51A knockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 g/ml) than the parental strain (MIC, 640 g/ ml), while no changes in MIC were observed for other azole-and non-azole-based drugs. These data strongly implicate the A. fumigatus Cyp51Ap I301 residue in the intrinsic resistance to fluconazole. Aspergillus fumigatus is the most common hyphomycete to cause disease in humans (1-3). It is intrinsically resistant to ketoconazole and fluconazole but normally susceptible to the other available azole antifungal agents (itraconazole, posaconazole, voriconazole, and isavuconazole) (4-8). The molecular mechanism for fluconazole intrinsic resistance has not been described yet. However, a hypothetical molecular mechanism has been proposed by Edlind et al., who linked A. fumigatus fluconazole intrinsic resistance with a naturally occurring amino acid substitution in Cyp51Ap (14-␣ sterol demethylase A) (9). These authors carried out an in silico comparison of the Candida albicans Erg11p and A. fumigatus Cyp51Ap sequences and found that among the residues most commonly implicated in fluconazole resistance in C. albicans (Y132, T315, S405, G464, and R467) (10, 11), only the T315 residue is not conserved in A. fumigatus Cyp51Ap and is naturally replaced by a nonpolar isoleucine (I301). In C. albicans, the replacement of the polar T315 residue by the nonpolar alanine (T315A) is enough to confer fluconazole resistance on the yeast (10).The aim of this study was to molecularly confirm that the natural polymorphism I301 in the Cyp51Ap is necessary and sufficient to explain the intrinsic reduced fluconazole susceptibility of A. fumigatus. An A. fumigatus mutant harboring the I301T substitution was generated, and susceptibilities to fluconazole and other antifungals were tested. Also, a CYP51A-defective mutant was obtained using the same parental strain in order to compare their antifungal susceptibility patterns. MATERIALS AND METHODS Strains.Aspergillus fumigatus akuB KU80 (12) was considered the wild-type strain, and its DNA was used as the template for all PCRs. It was the recipient strain for electroporation assays. Escherichia coli TOP10 (Promega) wa...

Background Candida auris is an emerging MDR pathogen. It shows reduced susceptibility to azole drugs and, in some strains, high amphotericin B MICs have been described. For these reasons, echinocandins were proposed as first-line treatment for C. auris infections. However, information on how echinocandins and amphotericin B act against this species is lacking. Objectives Our aim was to establish the killing kinetics of anidulafungin, caspofungin and amphotericin B against C. auris by time–kill methodology and to determine if these antifungals behave as fungicidal or fungistatic agents against this species. Methods The susceptibility of 50 C. auris strains was studied. Nine strains were selected (based on echinocandin MICs) to be further studied. Minimal fungicidal concentrations, in vitro dose–response and time–kill patterns were determined. Results Echinocandins showed lower MIC values than amphotericin B (geometric mean of 0.12 and 0.94 mg/L, respectively). Anidulafungin and caspofungin showed no fungicidal activity at any concentration (maximum log decreases in cfu/mL between 1.34 and 2.22). On the other hand, amphotericin B showed fungicidal activity, but at high concentrations (≥2.00 mg/L). In addition, the tested polyene was faster than echinocandins at killing 50% of the initial inoculum (0.92 versus >8.00 h, respectively). Conclusions Amphotericin B was the only agent regarded as fungicidal against C. auris. Moreover, C. auris should be considered tolerant to caspofungin and anidulafungin considering that their MFC:MIC ratios were mostly ≥32 and that after 6 h of incubation the starting inoculum was not reduced in >90%.

Mucormycosis is an emerging disease with high mortality rates. Few antifungal drugs are active against Mucorales. Considering the low efficacy of monotherapy, combination-therapy strategies have been described. It is known that fungi are susceptible to zinc deprivation, so we tested the in vitro effect of the zinc chelators clioquinol, phenanthroline, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine combined with amphotericin B or posaconazole against 25 strains of Mucorales. Clioquinol-posaconazole was the most active combination, although results were strain dependent.