Resumen de Filogènia molecular i caracterització fenotípica del complex d'espècies de sporothrix schenckii
Rita Marimón Picó
Sporothrix schenckii és el fong dimòrfic responsable de la esporotricosi, una important infecció crònica de distribució mundial. En la present tesi a partir de la seqüenciació de tres gens estructurals (quitina sintasa, ß-tubulina i calmodulina) es demostra que S. schenckii és un complex despècies filogenètiques.
Sha realitzat un exhaustiu estudi fenotípic daquestes espècies i, combinant alguns dels diferents caràcters morfològics i fisiològics estudiats [presència/absència i morfologia de conidis sèssils pigmentats, taxes de creixement de les colònies (30, 35 i 37ºC) i assimilació de sacarosa, rafinosa i ribitol], es descriuen tres noves espècies per a la ciència: S. globosa, S. brasiliensis i S. mexicana. A més es demostra que S. albicans, fins ara considerada sinònima dS. schenckii, i S. schenckii var. luriei són espècies diferents dS. schenckii.
En lestudi de sensibilitat in vitro als antifúngics realitzat amb les diferents espècies del complex, es demostra que lantifúngic més eficaç és la terbinafina, seguit del ketoconazol i el posaconazol. Lespècie més sensible ha resultat ser S. brasiliensis i les més resistents S. globosa i S. mexicana.
En lestudi de la patogènia en ratolins shan trobat diferències en la virulència de les espècies del complex, essent S. brasiliensis i S. schenckii les més patògenes.
Summary PhD Thesis Molecular and phenotypic characterization of Sporothrix schenckii Sporothrix schenckii is the pathogenic dimorphic fungus responsible for sporothrichosis, an important fungal infection with a worldwide distribution. Recently, molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. 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 quitin synthase, ß-tubulin and calmodulin genes demonstrated that S. schenckii is a species complex. A combined analysis of the sequences of the three loci of 60 strains from different origins showed 6 phylogenetic species within this species demonstrating that most of these species prevail in different geographical regions. Since the delineation of of such species could be of extreme importance from a clinical point of view, we studied a total of 127 isolates, most of which were received as S. schenckii, including the available type strain of species currently considered synonyms, and also some close morphological species. We have phenotypically characterized all these isolates using different culture media, growth rates at different temperatures, and numerous nutritional tests and compared their calmodulin gene sequences, which previously was revealed as the most informative molecular marker of the three evaluated. The molecular analysis revealed that Sporothrix albicans, S. inflate and S. schenckii var. luriei are species that are clearly different from S. schenckii. The combination of these phonetic and genetic approaches allowed us to propose the new species Sporothrix brasiliensis, S. globosa, S. mexicana and S. luriei. The key phenotypic featudes for recognizing these species are the morphology of the sessile pigmented conidia, growth at 30, 35, and 37ºC, and the assimilation of sucrose, raffinose, and ribitol.
In this thesis we also evaluated the in vitro activities of 12 drugs (amphotericin B, albaconazole, fluconazole, eberconazole itraconazole, ketoconazole, posaconazole, ravuconazole, voriconazole, terbinafine, micafungin and flucytosine) by using the microdilution method following the CLSI guidelines against 92 isolates belonging to the six species that constitute the sporotrhix schenckii complex. There were significant differences among the species, Sporothrix brasiliensis was the species that showed the best response to antifungals and S. mexicana was the worst. In general, terbinafine was the most active drug, followed by ketoconazole and posaconazole.
Another objective of the present thesis was to perform a comparative study on the experimental pathogenicity of six species of Sporothrix of clinical interest, i.e. S. albicans, S. brasiliensis, S. globosa, S. mexicana, S. luriei and S. schenckii sensu stricto, it was performed using a murine model with immunocompetent mice. We used two different inocula, i.e., 1x108 conidia/ml and 1x105 conidia/ml, for each fungal strain tested. Two isolates of each species were used. Two inocula of each isolate, i.e. 1 x 108 and 1 x 105 conidia/ml, were tested, the animals being infected intravenously. Only the low inoculum of one isolate of S. brasiliensis and the high inoculum of the isolates of S. brasiliensis and S. schenckii were able to cause 100% mortality. The other inocula and species tested did not kill any animal.
