Identifier: TDX:511
Authors: Gilgado Barbadilla, Felix
Abstract:
Pseudallescheria boydii is a ubiquitous ascomycetous fungus that causes a wide array of human infections that can affect practically all the organs of the body. These infections have been known for a long time, but in recent years, a marked increase in severe invasive infections has been noticed, mainly in immunocompromised hosts. They present a high mortality rate and are difficult to treat. One of the most typical features of this species, which is very rare in other pathogenic fungi, is its ability to develop sexual structures on routine culture media. Until the present thesis Scedosporium apiospermum was considered the anamorph of P. boydii. <br/> Recently, it had been demonstrated that high genetic variation exists within P. boydii and other authors had reported considerable differences with respect to growth and sporulation. All these data seemed to suggest that P. boydii was probably a species complex. For this reason we performed a morphological and molecular study involving numerous strains of clinical and environmental origins and from different countries. The analysis of partial sequences of the β-tubulin (two loci) and calmodulin genes and the internal transcribed spacer region of the rDNA gene demonstrated that P. boydii is a species complex. A combined analysis of the sequences of the four loci of 60 strains from different origins showed 8 phylogenetic species within this species, grouped in 5 different clades. The clades 1 and 2 were described as the new species Scedosporium aurantiacum and Pseudallescheria minutispora, respectively. Clade 5 consisted of four subgroups incorporating the ex-type strains of P. boydii, Pseudallescheria angusta, Pseudallescheria ellipsoidea and Pseudallescheria fusoidea, respectively, and clades 3 and 4 remained unnamed. Later, in order to facilitate their identification in the clinical microbiology laboratories we tried to characterize phenotypically those 8 phylogenetic species. In order to increase the robustness of the different clades, we included in this study numerous fresh isolates identified by sequencing the TUB region of the β-tubulin gene, which previously was revealed as the most informative molecular marker of the four evaluated. The most representative macro- and microscopic morphological features of all them were studied and their responses to 59 physiological tests were evaluated. The combination of these studies allowed us to distinguish phenetically the mentioned phylogenetic species. The new species Scedosporium dehoogii (previously clade 3) was described and Scedoporium apiospermum (previously clade 3) and P. boydii were considered two different species The new name Scedosporium boydii was proposed for the anamorph of the latter species. Scedosporium dehoogii can be separated from the rest because it does not growth at 40ºC and present unbranched conidiophores. Although S. apiospermum were morphologically indistinguishable from the anamorph of Pseudallescheria boydii both species can be separated by the response to D-ribose test and by the presence or absence of a teleomorph. <br/> During the study of fresh isolates recovered from different geographical areas we recovered an interesting Scedosporium-like fungus from Argentinean soil samples which was proven to be genetically and morphologically different from the known species of Scedosporium and relatives. Diverse morphological and molecular studies confirmed the uniqueness of such fungus and was proposed as the new genus Parascedosporium.. This genus is mainly characterized by producing sympodial conidia from denticulate conidiogenous cells. Further studies demonstrated that such isolate was identical to Graphium tectonae and thus the new combination Parascedosporium tectonae was proposed. <br/> In this thesis we also evaluated the in vitro activities of 11 drugs (amphotericin B, albaconazole, fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, voriconazole, terbinafine, micafungin and flucytosine) by using the microdilution method following the CLSI guidelines against 84 isolates belonging to the eight species that constitute the Pseudallescheria boydii complex. We found significant differences among the species, with Scedosporium aurantiacum being the most resistant. In general, voriconazole was the most active drug, showing a total geometric mean MIC of 0.61 g/ml, followed by posaconazole. <br/> Another objective of the present thesis was to compare the virulence of the different species of the complex using a murine model of disseminated infection by these species. We used two different inocula, i.e., 5x104 conidia/ml and 1x106 conidia/ml, for each fungal strain tested. When mice were infected with the first inoculum animals were immunosuppressed while that those infected with the second inoculum were inmunocompetent. No significant differences in mortality rates were observed (p>0.05) among the species in the immunosupressed animals. In the case of inmunocompetent animals, S. aurantiacum and S. dehoogii were clearly the most virulent species of the complex both showed the highest mean mortality rate (80% and 70%, respectively, versus 0%-20% for the other species). <br/> Finally, since none of the strains studied of the most common species of the complex, S. apiospermum, never developed the sexual state we considered of interest to determine if it was a heterothallic species. For this purpose, 15 strains of this species were paired in all possible combinations, including self-pairings to corroborate this hypothesis. All strains were self-sterile and several combinations of strains produced fertile ascomata. The strains could separate in two groups of different mating types. We corroborated the bi-allelic heterothallic mating system suggested by these results with crosses among F1 progeny ascospores from one positive mating test. All these data confirmed that S. apiospermum is a heterothallic species.
Repositori URV