Draft Genome Sequence of the Environmental Fungus Scedosporium dehoogii.

Today, the genus Scedosporium comprises at least ten species with four of them, Scedosporium apiospermum, Scedosporium boydii, Scedosporium aurantiacum and Scedosporium minutisporum capable of colonizing the lungs of patients with cystic fibrosis. Scedosporium dehoogii, which is also common in the soil, has never been reported as causing human pulmonary infections. Here we report the first genome sequence for S. dehoogii, an invaluable resource to understand the genetic bases of pathogenesis in the genus Scedosporium.

A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts.

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 mutations on azole resistance. Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae, allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianusCandida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in Candida species, and the equivalent position K151 in K. marxianus, plays a critical role in fluconazole resistance.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex.

PURPOSE: The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. METHODS: Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). RESULTS: Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. CONCLUSIONS: These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF).

Human-impacted areas of France are environmental reservoirs of the Pseudallescheria boydii/Scedosporium apiospermum species complex.

Species of the Pseudallescheria boydii/Scedosporium apiospermum complex (PSC) are emerging fungal pathogens able to chronically colonize the airways of patients with cystic fibrosis (CF). As P. boydii was found more frequently colonizing the lungs of CF patients in France than in other European countries in a previous report, the present study was conducted in order to clarify distribution of PSC species in France and to characterize their natural habitat. The highest densities of PSC isolates were found in human-impacted areas, i.e. agricultural areas, fluids obtained from wastewater treatment plants, playgrounds and industrial areas. PSC was not detected from soil samples collected in forests. Most PSC culture-positive soil samples exhibited a pH range of 6-8. Scedosporium dehoogii, the most abundant species, was detected in all human-impacted area types except vineyards, whereas Scedosporium aurantiacum was mostly found in agricultural areas. Pseudallescheria boydii and S. apiospermum were predominantly isolated from seashores and playgrounds respectively. Pseudallescheria minutispora was found only once from a playground. This study highlights potential sources of contamination of the patients, especially in the CF context.

Scedo-Select III: a new semi-selective culture medium for detection of the Scedosporium apiospermum species complex.

The Scedosporium apiospermum complex is responsible for a large variety of infections in human. Members of this complex have become emerging fungal pathogens with an increasing occurrence in patients with underlying conditions such as immunosuppression or cystic fibrosis. A better knowledge of these fungi and of the sources of contamination of the patients is required and more accurate detection methods from the environment are needed. In this context, a highly selective culture medium was developed in the present study. Thus, various aliphatic, cyclic, or aromatic compounds were tested as the sole carbon source, in combination with some inorganic nitrogen sources and fungicides. The best results were obtained with 4-hydroxy-benzoate combined with ammonium sulfate and the fungicides dichloran and benomyl. This new culture medium called Scedo-Select III was shown to support growth of all species of the S. apiospermum complex. Subsequently, this new culture medium was evaluated successfully on water and soil samples, exhibiting higher sensitivity and selectivity than the previously described SceSel+ culture medium. Therefore, this easy-to-prepare and synthetic semi-selective culture medium may be useful to clarify the ecology of these fungi and to identify their reservoirs in patients' environment.

Distribution of the different species of the Pseudallescheria boydii/Scedosporium apiospermum complex in French patients with cystic fibrosis.

As various new sibling species within the Pseudallescheria boydii/Scedosporium apiospermum complex have been described recently with differences in their susceptibility to antifungals, this study was conducted in order to determine their respective frequency in cystic fibrosis. Results indicated that P. boydii largely predominated (62%), followed by S. apiospermum (24%), Scedosporium aurantiacum (10%) and Pseudallescheria minutispora (4%). Scedosporium dehoogii was not recovered in this study. The multiple correspondence factor analysis highlighted geographical discrepancies within species distribution: P. boydii was rarely encountered in Northern France, while S. apiospermum was less represented in the west of the country. Additionally, we demonstrated that all species encountered in the cystic fibrosis context were capable to chronically colonize the respiratory tract of patients. Molecular typing of a large set of environmental and clinical isolates should be conducted to delineate the epidemiology of each sibling species in the complex.

Enterohemorrhagic Escherichia coli pathogenesis: role of Long polar fimbriae in Peyer's patches interactions.

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.

Saprochaete clavata invasive infection in a patient with severe aplastic anemia: Efficacy of voriconazole and liposomal amphotericin B with adjuvant granulocyte transfusions before neutrophil recovery following allogeneic bone marrow transplantation.

We report a case of a 27-year old man with severe aplastic anemia who developed a Saprochaete clavata (Geotrichum clavatum) disseminated invasive infection shortly prior a scheduled allogeneic bone marrow transplantation. Treatment with a combination of voriconazole, liposomal amphotericin B and adjuvant granulocyte transfusions was successful before neutrophil recovery.

Trichosporon faecale invasive infection in a patient with severe aplastic anemia: Efficacy of voriconazole and liposomal amphotericin B before neutrophil recovery.

We report a case of a 51-year old man with a severe aplastic anemia who developed an invasive trichosporonosis to Trichosporon faecale with fungemia and skin lesions during severe neutropenia. The treatment was successful before neutrophil recovery with a combination of voriconazole and liposomal amphotericin B.