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ABSTRACTAspergillussection Fumigaticontains 12 clinically relevant species. Among these Aspergillusspecies, A. fumigatusis the most frequent agent of invasive aspergillosis, followed by A. lentulusand A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigatirevealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelisand Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2isolates of both A. parafelisand A. pseudofelis. The MAT1-2isolate of A. parafeliswas fertile when paired with the MAT1-1isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensisbut was not fertile with the MAT1-1isolate of A. lentulus. The MAT1-2isolates of A. pseudofeliswere fertile when paired with the MAT1-1isolate of A. felisbut not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2isolates of A. parafelisand A. pseudofelisbeing the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management. more...

The incidence of fungal infections due to C. parapsilosisand closely related cryptic species (-psilosiscomplex) has increased in the last few years, but differences in virulence among these species have not been widely studied. Fifteen clinical isolates of C. parapsilosis, C. orthopsilosis, and C. metapsilosis, including the type strains, were used to evaluate their virulence in Galleria mellonellalarvae. Fluctuations in the hemocyte density and in the phagocytic activity were also tested. Differences in the median survival for these species were demonstrated at 37 °C (2.6 ± 1.02, 2.3 ± 0.92, and 4.53 ± 1.65 d for C. parapsilosis, C. orthopsilosis, and C. metapsilosis, respectively). Galleria mellonellahemocytes phagocytosed C. metapsilosisstrains more effectively than did for C. orthopsilosisand C. parapsilosis(P< 0.05). The phagocytosis rate was lower for C. parapsilosisthan for C. orthopsilosis(P< 0.05). The hemocyte density was increased in larvae infected with C. metapsilosiscompared with those infected with C. parapsilosisor C. orthopsilosis(P< 0.05). Moreover, in vitro studies of virulence factors such as pseudohyphae production and hydrolytic enzyme secretion showed that the capability of C. metapsilosisstrains to produce those virulence factors was reduced. Infections due to -psilosiscomplex species produced tissue damage in G. mellonellaand pseudohyphae could be also observed during infection with C. parapsilosis. more...

ABSTRACTA voriconazole-resistant isolate of Aspergillus fumigatuswas recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acid substitution in the azole target (Cyp51A) was identified as the cause of the resistance phenotype. This article describes the first isolation of a voriconazole-resistant A. fumigatusisolate from an immunocompetent patient in Spain. more...

Natural products are an inexhaustible source for drug discovery. However, the validation and selection of primary screening assays are vital to guarantee a selection of extracts or molecules with relevant pharmacological action and worthy of following up. The assay must be rapid, simple, easy to implement, and produce quick results and preferably at a low cost. In this work, we developed and validated a colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using natural extracts against Aspergillus fumigatuswere optimized and set up. The extracts plus RPMI-1640 modified medium containing the spores and 0.002% resazurin were added per well. The fluorescence was read after 24 to 30 h of incubation. The resazurin proved to be as suitable as Alamar Blue for determining the minimal inhibitory concentration of different antifungals against A. fumigatusand effective to analyze fungicidal and fungistatic compounds. An HTS of 12 000 microbial extracts was carried out against two A. fumigatusstrains, and 2.7% of the extracts displayed antifungal activity. Our group has been the first to use this methodology for screening a collection of natural extracts to identify compounds with antifungal activity against the medically important human pathogen A. fumigatus. more...

ABSTRACTRecent studies have demonstrated that some morphologically atypical Aspergillus fumigatusstrains are different species belonging to the section Fumigati. Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentuluscytochrome P450 14-α sterol demethylases, encoded by A. lentuluscyp51A (Alcyp51A) and Alcyp51Bgenes, were identified. Targeted cyp51Agene knockout in A. lentulusshowed that the intrinsic azole resistance of this species is cyp51Adependent. The Δcyp51Astrain was morphologically indistinguishable from the A. lentuluswild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentuluscyp51Awas performed in an A. fumigatuscyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction. more...

ABSTRACTRespiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatusisolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51Agene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillusspp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatusisolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatusisolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatusisolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment. more...

This article identifies novel stress-protective proteins that belong to the family of intrinsically unstructured proteins, DprA and DprB, which are associated with the cytosol and the peroxisomes in Aspergillus fumigatus. more...

ABSTRACTA single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatusisolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatusand other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51Agene were sequenced to identify azole-resistant A. fumigatusisolates. A. fumigatuswas recovered in 144 out of 185 samples (77.8%). Four A. fumigatusisolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulusisolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreuswas present in seven samples from Austria. Multi-azole-resistant A. fumigatusis present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillusspecies with intrinsic resistance to either azoles or amphotericin B were found. more...

ABSTRACTA phylogenetic analysis was performed for 34 Aspergillusstrains belonging to section Nigri. Molecular methods allowed for the correct classification into three different clades (A. niger, A. tubingensis, and A. foetidus). Correlation with in vitro itraconazole susceptibility distinguished the following three profiles: susceptible, resistant, and showing a paradoxical effect. A number of different species whose morphological features resemble those of A. nigershowed unusual MICs to itraconazole that have never been described for the Aspergillusgenus. more...

ABSTRACTThis study analyzed 28 Aspergillusstrains belonging to the section Fumigatithat were isolated from clinical samples in Spain. All isolates sporulated slowly and were unable to grow at 48°C. Phylogenetic analysis based on sequencing of partial sequences of the β-tubulin and rodlet A genes was used to classify the 28 strains into six different clades (Neosartorya hiratsukae, Neosartorya pseudofischeri, Aspergillus viridinutans, Aspergillus lentulus, Aspergillus fumigatiaffinis, and Aspergillus fumisynnematus). Antifungal susceptibility testing showed heterogeneous patterns and grouped the strains together by species. Most A. lentulusand A. fumigatiaffinisisolates showed high MICs of amphotericin B (geometric mean [GM] MICs, ≥4.5 μg/ml), itraconazole (GM MICs, ≥6 μg/ml), voriconazole (GM MICs, ≥3 μg/ml), and ravuconazole (GM MICs, ≥3 μg/ml); N pseudofischeriand A. viridinutansshowed high MICs of itraconazole (GM MICs, ≥8 μg/ml), voriconazole (GM MICs, ≥3.33 μg/ml), and ravuconazole (GM MICs, ≥2 μg/ml); and N. hiratsukaeand A. fumisynnematuswere susceptible to all the antifungals tested. In conclusion, a number of different species whose morphological features resemble those of Aspergillus fumigatuscould succeed in producing invasive infections in the susceptible host. In addition, some of them showed high MICs for most of the antifungals available for the treatment of patients infected with these organisms. The epidemiology and clinical relevance of these species should therefore be addressed. more...

ABSTRACTActivities of 35 combinations of antifungal agents against Scedosporiumspp. were analyzed by a checkerboard microdilution design and the summation of fractional concentration index. An average indifferent effect was detected apart from combinations of azole agents and echinocandins against Scedosporium apiospermum. Antagonism was absent for all antifungal combinations against both species. more...

ABSTRACTTwo erg3genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatuswere characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3Aand erg3Bgenes and a double gene knockout, erg3A−erg3B−, showed that they are not essential for A. fumigatusviability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatusergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3Aand erg3Bwere knocked out alone and in combination. more...

ABSTRACTSeven cases of disseminated infection due to Dipodascus capitatusare reported. Infections occurred in a hematological unit of a tertiary hospital during a period of 5 years. Five cases were refractory to antifungal therapy. Antifungal susceptibility testing of seven isolates was performed, and strains were typed by PCR fingerprinting with the core sequence of phage M13 and by random amplification of polymorphic DNA with two primers, Ap12h and W-80A. A very short range of MICs of each antifungal agent was observed. The MICs of amphotericin B ranged between 0.50 and 2 µg/ml. Strains were susceptible in vitro to flucytosine and susceptible (dose-dependent) to fluconazole and itraconazole. Voriconazole exhibited an activity in vitro comparable to that of itraconazole. Typing techniques allowed seven additional isolates of D. capitatusneither geographically nor temporally related to be classified into two different genomic patterns. The genomic type of the seven strains from the hematological unit was identical regardless of typing technique utilized. It would indicate that the seven cases of disseminated infection could be related epidemiologically. more...

ABSTRACTFive sequential Cryptococcus neoformansisolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-α lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S. more...

ABSTRACTRecently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination. However, the utilization of a spectrophotometer requires studies for standardization. The present work analyzes the following parameters: (i) accuracy of inoculum preparation, (ii) correlation between optical density and CFU per milliliter, (iii) influence of the wavelength on the endpoint determination, and (iv) influence of the dimethyl sulfoxide concentration on the growth kinetics. The main results can be summarized as follows: (i) inoculum preparation following the methodology recommended by the National Committee for Clinical Laboratory Standards is an exact procedure; (ii) the relationship between optical density and CFU per milliliter is linear (coefficient of determination, r2= 0.84); (iii) MICs obtained by means of spectrophotometric readings at different wavelengths are identical (for amphotericin B, an intraclass correlation coefficient of 0.98 was obtained; for fluconazole, the intraclass correlation coefficient was 1); and (iv) a 2% concentration of dimethyl sulfoxide produces a significantly slower and lower growth curve of Candidaspp. than other concentrations. more...

ABSTRACTA major limitation of the National Committee for Clinical Laboratory Standards M27-A methodology is reliable detection of amphotericin B (AMB) resistance. The results obtained by using Iso-Sensitest, a synthetic medium, to detect AMB resistance were analyzed and compared with those obtained with RPMI and antibiotic medium 3 (AM3). The ability to detect AMB resistance with RPMI is not enhanced by using a higher inoculum, glucose supplementation at a final concentration of 20 g/liter, spectrophotometric reading, or 24 h of incubation time. Testing using AM3 and an inoculum of 103CFU/ml detects resistance. Identification of resistant isolates is not improved by glucose supplementation, changes in reading method, or changes in incubation time. However, the use of AM3 as assay medium and an inoculum of 105CFU/ml did not allow detection of AMB resistance. Testing using Iso-Sensitest medium appears to be similar to AM3 in detecting resistance. The most pronounced discrimination is achieved by testing in Iso-Sensitest supplemented with glucose and spectrophotometric reading after 24 h of incubation. The reproducibility of MIC testing was greatest for Iso-Sensitest-based procedures. Use of Iso-Sensitest produces both highly reproducible MICs and reliable identification of AMB-resistant Candidaisolates. more...

ABSTRACTWe report the simultaneous isolation of one Aspergillus flavusstrain from the aortic prosthesis of a heart surgery patient and another two isolates recovered from a dual-reservoir cooler-heater used in the operating room where this patient was operated on. Genetic typing of these three isolates by randomly amplified polymorphic DNA (RAPD) revealed identical genotypes. Eight unrelated control strains of A. flavushad eight different genotypes. These results clearly indicated the nosocomial origin of theA. flavusstrain isolated from the patient. We suggest that the RAPD technique is a rapid and reliable tool to ascertain the epidemiology of infections caused by A. flavus. more...