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Antimicrobial Agents and Chemotherapy, February 2007, p. 748-751, Vol. 51, No. 2
0066-4804/07/$08.00+0 doi:10.1128/AAC.01177-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain
Received 21 September 2006/ Returned for modification 21 October 2006/ Accepted 4 November 2006
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ABSTRACT |
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Recently, it has been demonstrated that high genetic variation exists in the P. boydii species complex (2, 4, 8). Two new species, Pseudallescheria minutispora and Scedosporium aurantiacum, are phylogenetically and morphologically separated from P. boydii. In addition, P. angusta, P. ellipsoidea, and P. fusoidea seem to present genetic differences and could be proposed as new species of Pseudallescheria (4).
We have analyzed the prevalence of these new species and their antifungal susceptibility profiles in a collection of clinical isolates of P. boydii.
Strains. A total of 46 clinical isolates of P. boydii and S. apiospermum were included in this study. Twenty-four strains were isolated from respiratory sites, five from biopsies, six from ear samples, five from skin, four from ocular samples, one from blood culture, and one from an abscess.
Table 1 displays the identification of the 42 sequences obtained from the GenBank database that were used as controls.
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PCR and DNA sequencing of the internal transcribed spacer (ITS) region. Molds were cultured in GYEP medium (0.3% yeast extract, 1% peptone [Difco, Madrid, Spain], 2% glucose [Sigma Aldrich Quimica, Madrid, Spain]) for 24 to 48 h at 30°C. Genomic DNA was isolated using a previously described extraction procedure (5).
DNA segments comprising the ITS1 and ITS2 regions were amplified with primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (1) in a GeneAmp 9700 PCR system (Applied Biosystems) (10). The reaction products were analyzed in a 0.8% agarose gel.
Sequencing reactions were done with 2 µl of reaction mixture from a sequencing kit (BigDye terminator cycle sequencing kit, Ready Reaction mixture; Applied Biosystems), 1 µM of the primers (ITS1 and ITS4), and 3 µl of the PCR product in a final volume of 10 µl.
Sequence analysis. Sequences were assembled and edited using the SeqMan II and EditSeq software packages (Lasergene; DNAstar, Inc., Madison, WI). Sequence analysis was performed by comparison of the DNA sequences with 42 ITS sequences of Scedosporium and Pseudallescheria strains obtained from the GenBank database (http://www.ncbi.nih.gov/GenBank/). Full information about these strains is displayed in Table 1.
Phylogenetic analysis. All phylogenetic analyses were conducted with Fingerprinting II Informatix software, version 3.0 (Bio-Rad Laboratories, Madrid, Spain). The methodology used was maximum parsimony clustering. Phylogram stability was assessed by parsimony bootstrapping with 2,000 simulations. The ITS sequence of Scedosporium prolificans CNM-CM-3571 (Mold Collection of the Spanish National Center for Microbiology) was used as the outgroup.
Antifungal susceptibility testing. Microdilution testing was performed following the CLSI (formerly NCCLS) reference method (6) with the following minor modifications. (i) RPMI 1640 was supplemented with glucose to reach a 2% concentration. (ii) Inoculum size was between 1 x 105 to 5 x 105 CFU/ml. Inoculum preparations were performed by means of counting spores in a hematocytometer (1, 7, 9). Aspergillus fumigatus ATCC 2004305 and Aspergillus flavus ATCC 2004304 were used as quality control strains (6).
The antifungal agents used in the study were amphotericin B (range, 16 to 0.03 mg/liter) (Sigma Aldrich Química), itraconazole (range, 8 to 0.015 mg/liter) (Janssen S.A., Madrid, Spain), and voriconazole (range, 8 to 0.015 mg/liter) (Pfizer S.A.). The endpoint was the antifungal concentration that produced a complete inhibition of visual growth at 48 h.
Morphological identification. All strains were identified as Scedosporium apiospermum by conventional methods (3). We were not able to identify by morphological characteristics the new proposed species (4, 8).
Molecular identification.
Figure 1 shows the phylogenetic analysis by means of maximum parsimony of a subset of the strains included in the study. Among 46 clinical strains analyzed, four S. aurantiacum strains were identified with a bootstrap value of 
99. They were isolated from sputum, bronchial aspirate, a corneal sample, and an ear swab. In this collection of clinical isolates, we did not found any strain of P. minutispora or P. angusta, but the sequences obtained from GenBank were supported by bootstrap values of 73 and 65, respectively. On the other hand, the analyses of ITS sequences indicate that Pseudallescheria ellipsoidea and P. fusoidea are indistinguishable from S. apiospermum isolates (Fig. 1).
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We examined the prevalence of these new species in a collection of clinical isolates. ITS sequence analyses revealed the presence of four S. aurantiacum strains, obtained from an ocular sample, an ear swab, bronchial aspirate, and sputum, and no strains of P. minutispora. Although Gilgado et al. (4) have defined morphological characteristics to identify S. aurantiacum and P. minutispora, great experience in classical taxonomy is required to perform this task. Therefore, it is not expected that many clinical microbiology laboratories are able to identify those new species unless they use molecular methodology. Regarding susceptibility to antifungal drugs, S. aurantiacum seems slightly more resistant than S. apiospermum to amphotericin B and itraconazole, although a higher number of isolates should be analyzed before any conclusion is drawn (Table 2).
Because little is know about the prevalence of these new species and therefore there are no studies regarding epidemiology, pattern of disease, risk factors, antifungal susceptibility testing, etc., we strongly recommend sending all strains of Scedosporium species involved in human infections to reference laboratories where those isolates can be properly identified to the species level and antifungal susceptibility testing can be performed. In this way, the importance of these new species can be ascertained. Meanwhile, and especially for clinical use, it would be better to maintain the use of the S. apiospermum name. From a practical point of view and for clinical microbiology laboratories, we suggest performing antifungal susceptibility testing rather than applying molecular methods for the identification of these species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 13 November 2006.
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