Programe Abstracts - Zygomycota

8-9 April 2013

Emerging Zygomycetes, a new problem in the clinical lab

A workshop of the ECMM/ISHAM Working Group on Zygomycetes Utrecht, The Netherlands

Programe&

Abstracts

Emerging Zygomycetes, a new problem in the clinical labA workshop of the ECMM/ISHAM Working Group on Zygomycetes Utrecht, The Netherlands, April 8-9, 2013

3


Programe

Introduction

Welcome to the ECMM-ISHAM Workshop on Zygomycetes and Zygomycoses. We are happy to announce that we have a fantastic panel of speakers. If you have any questions, please contact Sybren de Hoog ([email protected]). A special issue on phylogeny and biodiversity of Zygomycotina of the journal Persoonia is now in press. The issue will be available as open access. Further information will be sent to you via your membership of the ECMM-ISHAM Working Group on Zygomycetes. We plan to compose a special issue of Mycoses at the occasion of the present workshop.

Venue

CBS-KNAW Fungal Biodiversity Centre, Utrecht, Uppsalalaan 8, The Netherlands. The institute is easily reached from Amsterdam Schiphol Airport by public transportation, please check www.cbs.knaw.nl. The weather in The Netherlands early April is still fresh, mostly around 10 – 15 °C, and rain may be expected. The workshop including lunches and dinners is provided at no cost.

Accreditation

The meeting has been accredited by the UEMS (European Union of Medical Specialists) with 12 CME accreditation points. These points are also recognized and valid in the United States (by the AMA). All participants will receive an obligatory Evaluation form at the end of the workshop, as part of the accreditation procedure. Certificates of attendance will be handed at the registration desk along with your congress information and abstract book.

Sponsors

We are grateful to our sponsors Gilead Inc., Astellas Inc., and the International Society for Human and Animal Mycology (ISHAM) for generous support of this meeting. Do become a member of ISHAM at www.ISHAM.org! For free membership of the Working group on Zygomycoses, contact Sybren de Hoog [email protected] or Anna Skiada [email protected].


4 5

Monday 8 April

• 08.00 – 09.00Registration, congress bags & badges, uploading oral and poster presentations; coffee

• 09.00 – 09.20 Opening and introductionKerstin Voigt, Sybren de Hoog (Leibniz Institute for Natural Product Research and Infection Biology, Friedrich-Schiller-University Jena, Jena, Germany; CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Phylogeny of the Zygomycetes.

• 09.20 – 09.30 Paul M. Kirk (Royal Botanical Garden, Kew, U.K.): Nomenclatural challenges in a genomic era

Session 1: Clinical aspects. Case reports and therapy. Conveners: George Petrikkos (Greece) and Cornelia Lass-Floerl (Austria).

• 09.30 – 09.45 Anna Skiada (Greece): Clinical presentations.• 09.45 – 10.00 Jin Yu (Department of Dermatology, Peking University First Hospital, Beijing, China):

Cutaneous mucormycosis due to Mucor irregularis, successfully cured by sequential therapy with amphotericin B and itraconazole.

• 10.00 – 10.10 Yuping Ran, Daoxian Kang, Huiying Wan, Xian Jiang, Dan Hao (West China Hospital, Sichuan University, Chengdu, China; Sichuan Provincial Institute of Dermatology and Venereology, Chengdu, China): A case of Mucor irregularis infection mimicked as skin tumor.

• 10.10 – 10.30 Georgios Petrikkos (4st Dept. of Internal Medicine, School of Medicine, National and Kapodistrian University of Athens, Greece): General management and adjunctive therapy.

• 10.30 – 10.45 NN Klimko, SN Khostelidi, TS Bogomolova, LS Zubarovskaya, SN Bondarenko, MO Popova, AG Volkova, MB Belogurova, NV Medvedeva, AS Kolbin, EG Boychenko (North-Western State Medical University, Leningrad Regional Clinical Hospital, Saint Petersburg State Medical University, Pediatric City Hospital № 1, City Hospital № 31, St. Petersburg, Russia): Mucormycosis in hematological and oncological patients in Saint Petersburg, Russia.

• 10.45 – 11.00 Miranda Drogari (Greece): Successandchallengesusingparaffinembeddedtissuesforthe diagnosis of Mucormycosis.

• 11.00 – 11.20 Break• 11.20 – 11.35 Birgit Willinger (Klinisches Institut für Labormedizin, Abteilung für Klinische Mikrobiologie,

Medizinische Universität Wien, Vienna, Austria): Mucormycosis acquired from herbal tea.• 11.35 – 11.50 Alexandro Bonifaz (Hospital Narvarte, Mexico City, Mexico): Mucormycosis in the General

Hospital of Mexico. Experience of 158 cases.• 11.50 – 12.10 Cornelia Lass-Flörl (Division of Hygiene and Medical Microbiology, Innsbruck Medical

University, Innsbruck, Austria): Prevention of infection.• 12.10 – 12.30 Theoklis Zaoutis (University of Pennsylvania School of Medicine, Division of Infectious

Diseases, The Children’s Hospital of Philadelphia, USA): Epidemiology of pediatric zygomycosis.• 12.30 – 12.45 Alessandro Busca (Dipartimento di Oncologia ed Ematologia, SSCVD Trapianto allogenico di

Cellule Staminal, Torino, Italy): Combination therapy.

• 13.00 – 14.00 Lunch at the Botanical Garden

• 14.00 – 17.00


5


Session 2: Infection models, diagnostic tools, epidemiology. Conveners: Dimitrios Kontoyannis (USA) and Ilse Jacobsen (Germany).

• 14.00 – 14.30 Thomas Walsh (Medical Mycology Research Laboratory, Weill Cornell University Medical Center, New York, USA): Advances in detection and diagnosis of zygomycosis: from laboratory to bedside.

• 14.30 – 14.50 Ana Alastruey-Izqueirdo (Spain): Mucoralean diversity in Fungiscope.• 14.50 – 15.10 Grit Walther (Germany): Main ecological trends in the Mucorales.• 15.10 – 15.30 Russell Lewis (Division of Infectious Diseases, S. Orsola Malpighi Hospital, University of

Bologna, Bologna, Italy): Conventional and alternative animal models of mucormycosis.

• 15.30 – 15.50 Break

• 15.50 – 16.10 Ilse Jacobsen (Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany): Lichtheimia infections in mice and alternative hosts.

• 16.10 – 16.30 Alberto Stgichel (Mycology Unit, Medical School, Universitat Rovira i Virgili, Reus, Spain): Unusual agents of mucormycosis.

• 16.30 – 16.45 Somayeh Dolatabadi (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Rhizopus, humans and food.

• 16.45 – 17.00 Ulrike Binder (Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria): Galleria as infection model.

• 17.00 – 19.00 Poster session, drinks and excursion to CBS collection

• 19.30 – 22.00 Dinner at restaurant Oudaen at Oude Gracht, Utrecht (bus 11, stop Neude)•


6 7

Tuesday 9 April

• 08.00 – 09.00Uploading oral presentations; coffee

• 09.00 – 12.50

Session 3: Virulence factors and host response. Conveners: Ashraf Ibrahim (USA) and Emmanuel Roilides (Greece).

• 09.00 – 09.20 Emmanuel Roilides (3rd Department of Pediatrics, Aristotle University School of Medicine and Hippokration General Hospital, Thessaloniki, Greece): Pathogenesis and host defense against Zygomycetes.

• 09.20 – 09.50 Thomas Lehrnbecher (Germany): T-cell adoptive immunotherapeutic strategies against mucormycosis in hematopoietic stem cell transplantation.

• 09.50 – 10.10 Susan Perkhofer (University of Applied Sciences Tyrol, Innsbruck, Austria): Cellular im-mune responses to Zygomycetes.

• 10.10 – 10.30 Hea Reung Park (Leibniz Institute for Natural Prodcut Research and Infection Biology - Hans Knoell Institute, Jena, University of Jena, Germany): Survival and interaction of mucoralean fungi with macrophages.

• 10.30 – 10.50 Kirstin Scherlach (Hans Knöll Institut, Jena, Germany; Friedrich Schiller University, Jena, Germany): Toxins and Mucorales.

• 10.50 – 11.10 Break

• 11.10 – 11.30 Ashraf Ibrahim (David Geffen School of Medicine at UCLA, Los Angeles Biomedical Re-search Institute, Harbor-UCLA Medical Center, Torrance, USA): Iron modulation of host-pathogen interactions during mucormycosis.

• 11.30 – 11.50 Antachopoulos Charalampos (3rd Department of Pediatrics, Aristotle University School of Medicine and Hippokration General Hospital, Thessaloniki, Greece): Cytokines and mucormycosis.

• 11.50 – 12.10 Liu Mingfu (David Geffen School of Medicine at UCLA, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, USA): Contribution of the ferrioxamine receptors to the pathogenesis of mucormycosis.

• 12.10 – 12.30 Maria Simitsopoulou (3rd Department of Pediatrics, Aristotle University School of Medicine and Hippokration General Hospital, Thessaloniki, Greece): Interactions of antifungal drugs and HD against Zygomycetes.

• 12.30 – 12.50 Francisco Nicolas (Departamento de Genética y Microbiología, Facultad de Biología, Uni-versidad de Murcia, Murcia, Spain): Exploring the connection between endogenous sRNAs and Mucor pathogenesis.

• 13.00 – 14.00 Lunch at the Botanical Garden


7


Session 4: The genomic approach to understanding virulence. Conveners: Axel Brakhage (Germany) and Joseph Heitman (USA).

• 14.00 – 14.20 Luis Corrochano (University of Seville, Spain): Phycomyces and light.• 14.20 – 14.40 Scott E. Baker (Environmental Molecular Science Laboratory, Richland, USA): Genomic

and post genomic studies on zygomycetes and other fungi. • 14.40 – 15.00 Marina Marcet-Houben and Toni Gabaldón (CRG-Centre for Genomic Regulation Barcelona,

Spain): Zygomycetes seen from a phylome.• 15.00 – 15.20 Christina Cuomo (Fungal Genome Sequencing & Analysis Broad Institute, Cambridge,

USA): Microsporidian genomic analysis: strategies for obligate intracellular growth.• 15.20 – 15.40 Victor Garre (Department of Genetics and Microbiology, University of Murcia, Murcia,

Spain): Genome-wide analysis of light responses in Mucor circinelloides.

• 15.40 – 16.00 Break

• 16.00 – 16.20 Joseph Heitman (Department of Molecular Genetics and Microbiology James B. Duke, Duke University Medical Center, Durham, USA): Comparison of sex locus structure in the Mucorales.

• 16.20 – 16.40 Volker U. Schwartze (Germany): The genome of Lichtheimia corymbifera.

• 16.40 – 17.00 Soo Chan Lee (Department of Molecular Genetics and Microbiology James B. Duke, Duke University Medical Center, Durham, USA): Mucor dimorphism and virulence.

• 17.00 – 17.20 Tamás Papp (Department of Microbiology, University of Szeged, Hungary): Functional characterization of HMG-CoA reductase genes in Mucor circinelloides.

• 17.20 – 17.40 Ekaterina Shelest, Volker U. Schwartze, Kerstin Voigt (Leibniz Institute for Natural Prod-uct Research and Infection Biology): Predicting potential virulence factors in the de novo sequenced genome of Conidiobolus coronatus.

• 17.40 – 18.00 Jean-Paul Latgé (Institut Pasteur, Paris, France): Comparative cell wall studies.

• 18.30 Drinks and dinner at restaurant with performance at De Beleving at Oude Gracht, Utrecht (bus 11, stop Neude)


9


Cytokines and mucormycosis

Charalampos Antachopoulos

Specific pro-inflammatory cytokines such as G-CSF, GM-CSF and IFN-γ are critical components of host defenses. These cytokines promote up-regulation of chemotaxis, phagocytosis, respiratory burst and/or degranulation of neutrophils, monocytes and macrophages. In vitro immunopharmacological studies and experiments in animal models have shown that neutrophil antifungal activity against

different Zygomycetes could be augmented by IFN-γ or GM-CSF alone or in combination with liposomal amphotericin B or voriconazole. In the clinical setting these promising pre-clinical results have been verified only in case reports and small uncontrolled series. In a literature review of 925 reported mucormycosis cases, 15 (83%) of 18 patients who received adjunctive treatment with G-CSF showed favorable clinical response. In this presentation, data from in vitro studies and animal models as well as clinical evidence on the role of adjunctive cytokine treatment in patients with mucormycosis will be discussed.

Oral


10 11

Galleria mellonella as a host model to study invasive fungal infectionsdue to Mucorales

Ulrike Binder, Elisabeth Maurer and Cornelia Lass-Flörl

Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Austria

Invasive fungal infections caused by members of the Mucorales (mucormycosis) have increased remarkably in the last years, with 500-1000 cases per year alone in Europe, making it the third most common invasive fungal infection after aspergillosis and candidiasis. Mucormycosis occurs mainly in immunocompromised or diabetic patients and results in unacceptably high mortality rates (50-90%), even with antifungal

therapy. The great heterogeneity of the Mucorales is reflected in a wide antifungal susceptibility profile, differing at family, genus and species level. Many Mucorales exhibit an inherent resistance to most systemic antifungals, which limits antifungal therapy to the use of amphotericin B and posaconazole. A key tool to a better understanding of infections due to Mucorales is the use of animal models. Since the use of rodents for in vivo studies is very time and cost intense, alternative systems that could provide comparable data are of great advantage. The larvae of the greater wax moth, Galleria (G.) mellonella have been proofed to be an ideal alternative to mammals in studying Aspergillus and Candida infections. Our aim is to adapt the Galleria model as a quick in vivo screening system for antifungal susceptibility and difference in virulence of various members of the Mucorales. In preliminary assays we determined the suitable inoculum size for the Mucorales, and compared our results with those from other infection models, which correlate well. Similar to data obtained in other models, we saw differences in virulence between strains of the same species. Furthermore, we are currently analyzing the effect of amphotericin B and posaconazole treatment on the survival of G.mellonella infected with different members of the Mucorales.

Oral


11


Mucormycosis in the General Hospital of Mexico.Experience of 158 cases.

Bonifaz A, Vázquez-González D, Araiza J, Hernández MA, González F, Chavolla R, Durán MA, Kassack JJ, Rivera C, Ponce RM.

General Hospital of Mexico, “Dr. Eduardo Liceaga”

Mucormycosis is an opportunistic disease caused by different fungus of the subphylum Mucoromycotina (formerly Zygomycetes), produce acute rhinocerebral and pulmonary diseases with thromboses, infarction and blood vessel invasion. Objective. was to present our clinical &mycological experience of mucormycosis about 20 years in the General Hospital of Mexico, tertiary hospital care, located

in Mexico City.

Material and methods. We present a retrospective, linear and non-comparative study of cases of mucormy-cosis in 20 years in the General Hospital of Mexico (1992-2012). Each of the patients was diagnosed by direct examination with KOH, serial cultures in Sabouraud dextrose agar and yeast extract agar; and subsequent micromorphological identification of fungal isolates. Some patients underwent biopsies with special stains (PAS and Grocott) and supplemented with imaging and resonance.

Results. We studied 158 patients with proven mucormycosis, the minimum age was 8 months, with a high of 72, with an average of 38.5 years. The evolution of the disease was at least 5 days, maximum 48 days, with an average of 12.5 days. Presented by gender: males 96 (60.7%), female 62 (39.3%). cases were divided by clinical as follows: Rhinocerebral 128 (81.0%); disseminated 13 (8.2%), primary- cutaneous 10 (6.3%), pul-monary 4 (2.5%) and gastric 3 (1.8%). Associated factors were: uncontrolled diabetes mellitus 107 (67.7%), Leukemia 35 (22.1%), lymphoma 6 (3.7%); malnutrition 4 (2.5%); without factor (immunocompetent) 3 (1.8%) deferoxamine treatment 1 (0.63%), Lupus erythematosus 1 (0.63%), HIV-AIDS 1 (0.63%). Etiologic agents were isolated from 142/158 patients (89.8%). Morphologically identified the etiological agents were: Rhizopus oryzae 101/142 (71.1%); Mucor sp 22/142 (15.4%); Lichteimia corymbifera 8/142 (5.6%), Rhizomucor 5/142 (3.5%); Cunninghamella sp 3/142 (2.1%); Syncephalastrum racemosum 2/142 (1.4%) and Apophysomyces el-egans 1/142 (0.7%).

Conclusions. The mucormycosis is an opportunistic fungal mycosis relatively common in our environment, most cases caused by Mucorales fungi, with the main etiological agent of Rhizopus oryzae, its main clinical form was the rhinocerebral and associated predisposing factor was diabetes ketoacidotic or uncontrolled fol-lowed leukemia.

Poster


12 13

COMBINATION THERAPY FOR TREATMENT OF MUCORMYCOSIS

Alessandro Busca 1, Livio Pagano 2

1 AO Citta della Salute e della Scienza, Turin-Italy, 2 Istituto di Ematologia Universita’ Cattolica del Sacro Cuore, Rome, Italy

Mucormycosis is an increasingly emerging life-threatening infection occurring mostly in immunocompromised hosts. Studies in animal models have shown a synergistic interaction between compounds with a different mechanism of action, providing evidence for testing the clinical relevance of combination therapy Combination therapy with lipid polyenes and posaconazole:

a retrospective study including patients recruited in the SEIFEM and Fungiscope registries, described 32 patients with hematological disorders who were treated with lipid formulations of Amphotericin B (AmB) and posaconazole, in the majority of the cases after failing primary therapy. Posaconazole was administered at the dose of 800 mg/d in 88% of the cases, and Liposomal AmB (L-AmB) was given at high-dose (≥ 5 mg/Kg/d) in 53% of the cases. Overall, a favourable response was observed in 18 patients (56%), including 11 (34%) complete and 7 (22%) partial responses. Van Burik et al. (1) investigated the activity of salvage therapy with posaconazole in 91 patients with mucormycosis: 13 out of the 91 patients received a combination of posaconazole and lipid formulation of AmB with an overall response rate of 46%.

Combination of polyenes and echinocandins: Reed et al (2) described 6 of 41 patients with rhino-orbital-cerebral mucormycosis who received combination polyene-caspofungin therapy and were evaluable for the response: treatment was successful for all evaluable patients, and in multivariate analysis, only receipt of combination therapy was associated with significantly improved outcomes

Combination of lipid polyenes and Deferasirox (DFX): Spelberg et al. (3) described 8 patients with proven mucormycosis who were treated with DFX at doses ranging between 5 and 20 mg/Kg in combination with other antifungal therapy: 5 patients were cured and off all antifungal therapy. In order to explore the efficacy and safety of DFX combined to L-AmB for the treatment of mucormycosis, a multicenter, placebo-controlled clinical trial has recently conducted (4). Patients with proven or probable mucomycosis were randomized to receive L-AmB and DFX (n=11) or L-AmB plus placebo (n=9). A significant higher proportion of patients in the DFX arm had active malignancy (64% vs 33%), while they were less likely to receive concomitant, non-study antifungal therapy (55% vs 78%). The number of patients with serious adverse events was not significantly different between the two arms, however the global success, defined as patient alive, clinically stable and radiographically improved, tended to be lower in the DFX arm (18% vs 67% at day 30, p=0.06), and mortal-ity was significantly higher (p=0.02) with the use of DFX, although the consistent imbalance in diagnosis and disease status at the time of randomization may have had a remarkable impact on the final outcome.

In conclusion, combination therapy may be considered as an attractive strategy for the treatment of pa-tients with mucormycosis, although owing to the small sample size and the heterogeneity of the patients included into the studies, it is very difficult to dissect the relative contribution of each single component of the antifungal combination to the final outcome.

(1) Van Burik JH. Clin Infect Dis 2006;42:e61(2) Reed C et al. Clin Infect Dis 2008;47:364(3) Spellberg B et al. Antimicrob Agents Chemother 2009; 53:3122(4) Spellberg B et al. JAC 2011;67:715

Oral


13


Siderophore Biosynthesis in Pathogenic Mucorales

C. Carroll, I. Murugathasan, M. Moore

Department of Biological Sciences, Simon Fraser University, Burnaby, Canada

Iron is essential for growth, and in low iron environments such as in serum, some microorganisms secrete ferric iron-chelating molecules called siderophores. A few species of Mucorales fungi have been shown previously to secrete the polycarboxylate siderophore, rhizoferrin. The overall aim of our research is to investigate the role of rhizoferrin in survival of pathogenic Mucorales in vitro and in vivo. Using HPLC, we

examined the supernatants of 6 common Mucorales pathogens after growth in low iron medium containing human serum, and showed that all 6 species secreted rhizoferrin. Iron replete conditions suppressed production of rhizoferrin. Rhizoferrin levels in serum-containing media were quantified using the CAS assay: the maximum amount of rhizoferrin detected was 32 ± 3µM after 15 h at 30°C. In vitro data indicated that rhizoferrin is stable in serum and urine for at least six hours at room temperature. Stability in serum can be maintained for up to four weeks if samples are frozen at -20°C. Together, these data suggest that detection of rhizoferrin has the potential to be diagnostic for mucormycosis. To identify the gene(s) responsible for rhizoferrin biosynthesis, we examined the genome of Rhizopus delemar 99-880 for a homologue to the bacterial gene, sfnaD, shown to be responsible for biosynthesis of staphyloferrin A, a carboxylate siderophore produced by Staphylococcus aureus. Recently, it was shown that SfnaD, a protein belonging to the non-ribosomal peptide synthetase-independent synthase (NIS) family catalyzes the first step in the biosynthesis of staphyloferrin A. A homologue of sfnaD was identified called rfn. Blastp showed a 22% identity and a 37% similarity between Rfn and SfnaD. The 2.1kb rfn gene has 6 exons and 5 introns. The predicted protein is 634 amino acids and contains an N-terminal IucA/IucC family domain, responsible for biosynthesis of the carboxylate siderophore, aerobactin. There is also a C-terminal conserved ferric iron reductase FhuF-like transporter domain. We have cloned and expressed the putative rfn gene from R. delemar in a bacterial expression host. An IPTG-inducible protein was detected that corresponds to the predicted molecular weight of 72kDa. The protein was purified using affinity chromatography and activity assays to confirm rhizoferrin biosynthesis from citrate and diaminobutane are underway.

Poster


14 15

Invasive mucormycosis: single centre experience 2005 – 2012

Vanda Chrenkova 1, Petr Hubacek 1,2, Petr Sedlacek 2, Lucie Sramkova 2, David Summerauer 2, Jan Simonek 3, Jan Bures 4, Petr Hamal 5, Dana Michalska 1, Eliska Bebrova 1, Pavel Drevinek 1

Department of Medical Microbiology, Charles University 2nd Faculty of Medicine and University Hospital Motol, Prague 1, Department of Paediatric Haematology and Oncology, Charles University 2nd Faculty of Medicine and University Hospital Motol, Prague 2, Department of 3rd Surgery, Charles University 2nd Faculty of Medicine and University Hospital Motol, Prague 3, Department of Department of Anaesthesiology and ICM, Charles University 2nd Faculty of Medicine and University Hospital Motol, Prague 4, Department of Microbiology,

Palacky University and University Hospital Olomouc 5, Czech Republic

Objectives: Invasive mucormycosis (IM) is known to be a severe complication in immunocompromised patients. Since we observed an increase in the incidence of IM at our hospital in recent years, we aimed to study this phenomenon further.

Methods: All detected cases of IM in University Hospital Motol between I/2005 and XII/2012 were included in the analysis. We evaluated risk factors, clinical and laboratory signs of the infection and therapy.

Results: We detected 20 cases of IM (11F/9M, age at dg. 0.75 – 74 yrs) in our cohort (17 proven/1 probable/2 possible IM). The mean incidence of IM in our hospital during selected period is 2.5 per year. At the time of IM diagnosis, patients were treated for acute leukaemia (7 ALL/2 AML), CNS tumour (2), non-Hodgkin lym-phoma (1), MDS RAEB (1) or were at risk as lung transplant recipients (3), patients with diabetes mellitus (1), Good-Pasteur syndrome (1), major surgery (1). One patient was without any apparent known risk factor. Until 2011, the most frequently affected patients were those with the haematooncological disorder, three of them were allogeneic HSCT recipients treated for GvHD, but since that time we detected IM more frequently in lung transplant recipients too. Infection was detected in lungs (11 pts), gut (3 pts), paranasal sinuses (2 pts), skin (2 pts), liver (1) and CNS (1). Dissemination occurred in 7 cases. Diagnosis of IM was evident in 18 cases with microscopy; in 17 cases the cultures grew mucormycetes (12 Rhizopus microsporus, 2 Rhizopus oryzae, 2 Rhizopus sp., 1 Absidia sp. and 3 non-identified mucormycetes). Amphotericin B application was started in 13 patients, 8 of them were treated with the combination of amphotericin B and extensive surgery. Diagno-sis post-mortem was made in 4 cases. Nine of 20 patients survived the IM. The combination of surgery and amphotericin B administration was found to be associated with the survival (p=0.0001; Fisher’s exact test).

Conclusion: We observe an increase in the incidence during last 5 years and the change in patients’ distri-bution. Mortality in the presented cohort is 55 % and most frequent underlying disease is acute leukaemia which is in agreement with the literature. We confirmed combination of extensive surgery and amphotericin B therapy to be significant for increased survival of patients.

Supported by the project of Ministry of Health of Czech Republic for conceptual development of research organization 00064203 and by Palacky University Olomouc, Czech Republic (grant no. LF_2013_012).

Poster


15


Microsporidian genomic analysis: strategies for obligate intracellular growth

Christina Cuomo1, Christopher Desjardins1, Malina Bakowski2, Jon Goldberg1, David Heiman1, Sarah Young1, Qiandong Zeng1, Elizabeth Didier3, James Becnel4, Emily Troemel2

1Broad Institute of MIT and Harvard, Cambridge, MA USA 2University of California, San Diego, La Jolla, CA USA 3Division of Microbiology, Tulane National Primate Research Center, Covington, LA USA 4USDA, ARS, CMAVE Gainesville, FL USA

Microsporidia are obligate intracellular parasites that infect virtually every animal phylum. Nematocida species were found to be natural pathogens of C. elegans and other Caenorhabditis species and are part of an early diverging clade of Microsporidia. By comparing the genomes of three Nematocida strains, we found that Nematocida sp 1 appears to be diploid and heterozygous,

with large homozygous regions, suggestive of a rare sexual cycle. Recent analysis has found evidence that additional species of microsporidia are diploid and heterozygous. To examine transcript structure and investigate regulation of gene expression, we sequenced RNA collected from an infection timecourse. This RNA-Seq data provided evidence for short UTRs and a complete absence of introns in N. parisii protein-coding genes and also illustrated the dramatic takeover of the host cell by microsporidia infection. We defined a set of candidate virulence factors, including a Nematocida-specific gene family of predicted secreted proteins that has undergone extensive expansion. Sequencing additional species of microsporidia illustrated the variation in genome size and allowed definition of genes required across microspordia that may be important for an obligate lifestyle. Comparative analysis between Nematocida and other microsporidian and fungal genomes revealed core properties of the divergent microsporidia, and has provided further evidence for the relationship of microsporidia with respect to the fungal kingdom.

This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272200900018C.

Oral


16 17

Mucorales, food and humans

Somayeh Dolatabadi1,2, Grit Walther1,3, G. Sybren de Hoog1,2,4,5

1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; 2Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands; 3Institute of Microbiology, Department of Microbiology and Molecular Biology, University of Jena, Jena, Germany; 4Peking University Health Science Center, Research Center for Medical Mycology, Beijing;5Sun Yat-sen Hospital,

Sun Yat-sen University, Guangzhou, China

The genus Rhizopus was first described in 1821 by Ehrenberg and belongs to the order Mucorales in the Zygomycotina. Since ancient time this group of fungi has been used for production of fermented food, especially in Far East Asian countries. They are highly potent for industrial enzyme production. Rhizopus arrhizus (R. oryzae) and R. microsporus are widely used for production of oriental fermented

foods, but the species are also causative agents for severe human infections known as mucormycosis; at present this is the third most frequent, potentially fatal opportunistic infection in human. Many taxonomic studies have been performed, mostly based on morphology. With this parameter, many novel species have been introduced in literature. To find out a better resolved taxonomy for these fungi and to reveal pathogenicity traits and physiological properties, a phylogenetic study was conducted. Strains including different sources, and from different continents were considered. MLST studies based on the internal transcribed spacer (ITS), parts of the actin (ACT1), and the elongation factor 1-α (TEF) gene were included and were compared with AFLP results. Physiological properties as well as thermotolerance, mating tests, and MALDI-TOF profiles were determined. Obviously two different groups could be distinguished in the Rhizopus arrhizus complex based on molecular studies, namely Rhizopus oryzae and Rhizopus delemar. Conversely, all type strains of older species in the literature were reduced to synonymy.

Also all varieties defined within Rhizopus microsporus were shown to be similar and members of the differ-ent varieties proved able to mate successfully, so that they were reduced to synonymy. Based on this data, Rhizopus microsporus was redefined. In contrast to ascomycetes, the Mucorales exhibit a process of reduction of the number of species, and many names were proven to be synonymous.

Oral


17


Genome-wide analysis of light responses in Mucor circinelloides

Victoriano Garre, Sergio López-García, Eusebio Navarro, Santiago Torres-Martínez

Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain

Light regulates developmental and physiological processes in a wide range of fungi. Particularly, Zygomycete fungi have developed complex mechanisms to control the responses to light that await detailed characterization at molecular level. The zygomycete Mucor circinelloides is a good model for this purpose because its genome has been sequenced and several molecular tools are available.

These tools include a high efficient transformation procedure and techniques for generations of knockout strains, overexpression of genes and silencing of genes (RNAi). Mucor, like other Zygomycetes, has three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) that code for proteins which present characteristics of photoreceptors. Each mcwc-1 gene controls a specific response to light. Thus, mcwc-1a and mcwc-1c control phototropism and photocarotenogenesis, respectively, whereas the mcwc-1b function in regulation by light has not been proved. Interestingly, white collar-1 genes promote virulence in two divergently related pathogenic species, the basidiomycete Cryptococcus neoformans and the ascomycete Fusarium oxysporum, suggesting that these genes may be conservatively linked to fungal pathogenesis. An involvement of mcwc-1 genes in Mucor pathogenesis needs further analyses. In order to deepen in the regulation by light in Mucor and the functions of mcwc-1 genes, a systematic approach using microarrays was followed to characterize white light-inducible transcriptional changes in wild-type and knockout mutants for each mcwc-1 gene.

Analysis of microarray data revealed that light is mainly a positive signal for transcription in Mucor, as in other fungi, since 130 genes were up-regulated in the wild-type strain in response to light, whereas only 30 were down-regulated, considering a threshold of 2.8-fold change. Genes strongly induced by light included genes known to be up-regulated by light, like the carotenogenic gene carB (74-fold), cryptochrome (45-fold) and mcwc-1c (22-fold), supporting reliability of the microarray data. Although many of up-regulated genes code for proteins implicated in protection against light-induced damage, several of them code for protein involved in signal transduction that could be involved in light responses or other physiological responses.

Transcriptomic analysis of mcwc-1 mutants showed that induction by light of nearly all genes is mediated by mcwc-1a, whereas mcwc-1c and mcwc-1b play accessories roles in regulation by light, suggesting that mcwc-1a is the main photoreceptor. Searching for cis-acting regulatory motifs upstream of genes regulated by mcwc-1a identified consensus sequences similar to those found in light regulated genes of Neurospora crassa. Interestingly, the identification of a small group of genes regulated by the three mcwc-1 genes independently of light points out that the three proteins form complexes to regulate gene expression. This result supports that mcwc-1 genes could control physiological responses other than responses to light, which could include pathogenesis.

Funded by MINECO (BFU2012-32246), Spain.

Oral


18 19

Iron modulation of host-pathogen interactions during mucormycosis.

Ashraf S. Ibrahim

Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Mucormycosis is a fungal infection that causes 40% mortality rate despite first-line therapy. Because angioinvasion is a hallmark of mucormycosis infections, we sought to define the interaction of Rhizopus oryzae (the most common cause of mucormycosis) and endothelial cells. We previously reported Glucose Regulated Protein 78 (GRP78) as a novel host cell receptor for Mucorales during

host cell invasion. GRP78 was found to be unregulated in elevated glucose and iron concentrations often seen in diabetic ketoacidotic (DKA) patients, thereby elucidate the unique susceptibility of these patients to mucormycosis. Here we discover CotH genes which are unique to Mucorales and predicted to encode cell surface proteins. CotH genes are also upregulated in iron and glucose concentrations seen in DKA patients. Heterologous expression of CotH in the normally non invasive Saccharomyces cerevisiae renders them invasive to host cells via binding to GRP78. Further, CotH proteins co-localize with surface GRP78 during invasion of endothelial cells by R. oryzae. Anti-CotH antibodies abolish the ability of R. oryzae to invade host cells. Similarly, gene silencing of CotH genes reduces R. oryzae ability to: 1) invade and damage endothelial cells; 2) damage Chinese Hamster Ovarian (CHO) cells overexpressing GRP78; and 3) cause disease in DKA mice. Our results further explain the unique susceptibility of DKA patients to mucormycosis in the sense that CotH surface proteins are unique ligands present only in Mucorales which bind to the novel receptor of GRP78. The CotH/GRP78 interactions may provide a foundation for novel therapeutic interventions against deadly mucormycosis.

Oral


19


Fungal mating types and sexual reproduction

Joseph Heitman

Department of Molecular Genetics and Microbiology, Duke University, USA

Our studies focus on the pathogens Cryptococcus neoformans and Cryptococcus gattii. C. neoformans is globally distributed and responsible for >1,000,000 infections annually, >620,000 attributable deaths, and up to one-third of all AIDS associated deaths. C. gattii is more geographically restricted, but has recently emerged to cause an outbreak in largely immunocompetent hosts in the Pacific

Northwest that has spread now into the US. Mating is linked to virulence and the generation of infectious spores and yet the population is largely one mating type, raising a conundrum as to how infectious spores might be produced in nature.

We have defined the structure and evolution of the unusually large, complex mating type locus of Cryp-tococcus neoformans and Cryptococcus gattii that is linked to sexual identity, sexual reproduction and viru-lence. Our studies on closely aligned saprobes (Cryptococcus amylolentus, Cryptococcus heveanensis) reveal extant tetrapolar sexual cycles, and support an evolutionary model for tetrapolar-bipolar transitions in these and other pathogenic species that may contribute to promote inbreeding. In the pathogenic species a further transition has occurred leading to an unusual unipolar mode of sexual reproduction involving only one mating type (same-sex mating/unisexual reproduction) that generates infectious spores and both admixing and de novo diversity within unisexual populations. The discovery by Richard Bennett and colleagues that Candida albicans also undergoes unisexual reproduction illustrates convergent evolution in two of the most successful systemic fungal pathogens of humans. Further investigation of these cryptic sexual cycles promises to reveal insight on microbial pathogen evolution and virulence.

Oral


20 21

Sex in basal fungi: Phycomyces, Mucor, and Rhizopus

Joseph Heitman1, Soo Chan Lee1, Charles H. Li1, Alexander Idnurm2, Maria Cervantes3, Rosa M. Ruiz-Vazquez3, Santiago R. Torres-Martinez3, Andrii P. Gryganskyi4, and Rytas Vilgalys4

1Department of Molecular Genetics and Microbiology, 4Department of Biology, Duke University, 2Division of Cell Biology and Biophysics, University of Missouri, Kansas City, and 3Departamento de Genetica y Microbiologia, Universidad de Murcia, Spain

How sexual identity is defined and the roles of sexual reproduction in the generation of diversity and evolution are of general interest. Given its ubiquity, sex is thought to have arisen once early in the evolution of eukaryotes. The fungal kingdom provides a broad window on the evolution of sex and its myriad diversifications, and the close alignment of the fungal and animal kingdoms within the

opisthokont supergroup of eukaryotes suggests that principles that emerge from studies of fungi will further advance our understanding of both kingdoms and beyond. Many fungi once thought to be asexual have been revealed by genomics to retain the machinery for sex, and laboratory studies are uncovering extant sexual cycles. In turn, the genomes of fungi with well-established sexual cycles provide insights on the mechanisms of sex determination and sexual reproduction. Molecular and genetic studies of the sex/mating type locus of model and pathogenic zygomycete fungi (Phycomyces, Mucor, Rhizopus) reveal divergent HMG domain proteins (SexM, SexP) define sexual identity and an interesting example of conidia spore size dimorphism linked to virulence. Comparisons of these species highlight the evolutionary trajectory of the sex locus, and suggest that HMG factors may be the ancestral sex determinant given their roles in sex determination in the Mucorales, hemi- and euascomycete species, and as the mammalian sex determinant Sry. Alternatively, there may have been two ancestral sex determining systems, one based on HMG factors and the other on homeodomain (HD) proteins, which have vied for pre-eminence during fungal evolution. Interestingly, in some extant fungal species both HMG and HD factors are encoded by the mating type locus. An RNA helicase gene linked to the sex locus may play novel roles in RNAi mediated silencing processes, based on the functions of the N. crassa ortholog SAD-3 that functions in meiotic silencing of unpaired DNA. Taken together, these studies of sex and its determination, evolution, and impact throughout the fungal kingdom illustrate general principles by which genetic diversity is generated and maintained in eukaryotic microbes, with implications for both other fungal phyla and metazoans.

Oral


21


Nomenclatural Challenges in a Genomic Era

Paul M. Kirk

Royal Botanic Gardens, Kew, UK

The major changes to the ICNafp will be briefly mentioned in the context of the challenges of naming organism discovered in the genomic era. In a Code essentially written for macro/mega organisms the solutions implemented to name microorganism in a compliant way will be reviewed. Options for the present and for the future of microorganism nomenclature will be presented

Oral


22 23

Mucormycosis – prevention

Cornelia Lass-Flörl

Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Fritz-Pregl-Straße 3, 6020 Innsbruck, Austria

Broad-spectrum antifungals show promising results in preventing and treating invasive fungal infections (IFIs) in high-risk patients. However, concerns rise over breakthrough IFIs (bIFIs), the emergence of azole-resistant strains and a shift to rare fungi, the mucormycetes. Infections caused by mucormycetes have increased markedly during the last decade. It is estimated that between

500-1000 cases of mucormycosis occur every year in Europe, making it the third most common invasive fungal infection after invasive aspergillosis and candidiasis. The reasons of these epidemiological changes are not yet fully understood but possibly the extensive use of echinocandins and voriconazole led to such situation.

In our centre, since 2009 we use posaconazole prophylaxis, which in turn reduced the incidence of in-vasive aspergillosis from 25% to 5%; however, we also notice a shift to infections (primary and bIFI) due to mucormycetes. The incidence of bIFIs was 13% (27/202), with 11/27 (41%) proven and 16/27 (59%) probable bIFIs. Proven infections were mainly localized in the lungs (85%). Species diagnosis exclusively revealed non-Aspergillus species, i.e. mucormycetes in 55% and yeasts in 45%. The median overall survival for patients with bIFIs was 5.2 months. Sixteen of 27 patients with bIFIs (proven and probable) succumbed. A significant proportion of bIFIs, compared with historical data, with a shift to non-Aspergillus spp. and in particular to mucormycetes was observed in patients at high risk for IFI during posaconazole and echinocandin prophylaxis. All patients were hosted in HEPA-filtered rooms in the hospital. We performed routine investigations to check, whether the main source of infection is/was the hospital environment. Paddle testers display mucormycetes to be detectable in the environment.

Oral


23


Mucor dimorphism and virulence

Soo Chan Lee, Alicia Li, Silvia Calo, and Joseph Heitman

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA

Fungal dimorphism evolved in multiple fungal lineages. Many pathogenic fungi are dimorphic, for example, switching between yeast and filamentous states. This switch alters host-pathogen interactions and is critical for pathogenicity. However, in pathogenic zygomycetes, whether dimorphism contributes to pathogenesis is a central unanswered question. Mucor exhibits multi-

budded yeast growth under anaerobic/high CO2 growth conditions, which Louis Pasteur discovered (Etudes sur la Biere. 1876). Interestingly, we found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits multi-budded yeast growth. We discovered that Mucor encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Disruption of the cnbR gene results in mutants locked in yeast phase growth. These results reveal that the calcineurin pathway governs the dimorphic transition from yeast to hyphae. In virulence tests, we found that the cnbR yeast-locked mutants are less virulent than wild-type in a heterologous host system, providing evidence that hyphae are a more virulent form of this fungus. Protein kinase A activity was elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, indicating a novel connection between PKA and calcineurin.

The cnaA mutants are hypersensitive to calcineurin inhibitors and display a hyphal polarity defect. The mutants produce spores that are larger than wild-type. Notably, we found spore size is linked to virulence in previous studies (Li et al. PLoS Pathogens. 2011). Interestingly, the cnaA mutants were found to be more viru-lent than wild-type. We also observed that the cnaA mutants germinate earlier inside macrophages, providing a possible explanation for the greater virulence of the mutants.

Another pathogenic zygomycete, Rhizopus delemar has three cna genes. Phylogenetic analysis revealed that the triplicated cna genes might result from a whole genome and/or segmental gene duplications.

Our results demonstrate that the calcineurin pathway orchestrates the dimorphic transition, spore size di-morphism, virulence, and hyphal polarity in Mucor, and the calcineurin pathway elements have been adapted in zygomycetes via variation in their evolutionary trajectory.

Oral


24 25

T-Cell Adoptive Immunotherapeutic Strategies against Mucormycosis in Hematopoietic Stem Cell Transplantation

Thomas Lehrnbecher, Lars Tramsen, and Stanislaw Schmidt

Pediatric Hematology and Oncology, Johann Wolfgang Goethe-University, Frankfurt, Germany

Mucormycoses remain a serious complication in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). In these patients, mortality rates of mucormycosis reach up to 90%, which is due, at least in part, to the severe and prolonged immunosuppression after transplantation. Although prolonged neutropenia is one of the most important risk factors for

mucormycosis, other cells, such as CD4+ T-cells, may also provide critical defense mechanisms against this infection. This hypothesis is supported by the clinical observation that mucormycoses often occur several months after allogeneic HSCT, at a time, when neutropenia has already resolved, but adaptive immune responses are still hampered. In analogue to the fact that a clinical benefit of the adoptive transfer of anti-Aspergillus TH1 cells to HSCT recipients with invasive aspergillosis has been demonstrated, the reconstitution of cellular immunity by the administration of ex vivo generated donor-derived antifungal T cells against mucormycetes (“adoptive immunotherapy”) might improve the prognosis of HSCT recipients suffering from mucormycosis. In all healthy individuals analyzed, we detected a low number of anti-Rhizopus oryzae T cells; these cells could be enriched and cultivated. The generated cells were characterized as memory effector TH1 cells, which proliferated upon restimulation, exhibited cross-reactivity to other mucormycetes, and increased the activity of professional phagocytes. Compared to the original cell fraction, the generated cells exhibited a significant lower alloreactive potential. Our results may form the basis for assessing efficacy and side effects of adoptively transferred donor-derived anti-mucormycete TH1 cells in animal studies with the ultimate goal to evaluate this strategy in allogeneic HSCT recipients suffering from mucormycosis.

Oral


25


Animal Models of Mucormycosis

R.E.Lewis

Infectious Diseases Unit, S.Orsola Malpighi Hospital, Department of Medical Sciences and Surgery, Alma Mater Studiorum-University of Bologna, Bologna, Italy

Animal models play a critical role in improving our understanding of the pathogenesis, diagnosis and treatment of human mucormycosis. A review of published primary research in the SCOPUS database (1972-2013, accessed March 11, 2013) identified 60 studies describing an animal model of mucormycosis. The most common animals used, included mice (72%), rabbits (14%), invertebrates

(Drosophila or Galleria, 5%) or Guinea pigs (2%). In terms of murine infection models, most investigators used disseminated (intravenous) infection models (76%) in streptozocine-induced diabetic mice or chemotherapy-induced neutropenic mice for studies of antifungal agents (79%). Sinopulmonary infection models in neutropenic/corticosteroid immunosuppressed mice were less common (17% of cases). The most frequent genera studied were Rhizopus (44%), Absidia (16%), Mucor (5%) or Cunninghamella (5%) alone, or infection models that compared multiple genera (21%). The primary study objective in most animal models were evaluation of antifungal activity (53%) or fungal pathogenesis (41%), with relatively few studies evaluating diagnostics (3%)- the major unmet need in human mucormycosis.

All of heretofore models have relative strengths and weakness depending on the research objectives. Investigators must also be aware of the limitations of each model, as well as critical aspects in inoculum preparation and processing of infected tissue, which can profoundly influence experimental reproducibility. Consequently, consensus protocols and standardized models for mucormycosis research are needed.

Oral


26 27

Zygomycetes as seen from a phylome

Marina Marcet-Houben and Toni Gabaldón

Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain, Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain

The advent of genome sequencing is producing a huge amount of data that need to be processed so that information can be extracted from it. One of the most powerful tools at our disposal at the moment to infer genes from genomic sequences, assign gene function or determine evolutionary events is comparative genomics. It consists in drawing information from the comparison of multiple,

related organisms. A basic resource for these kind of analyses are phylogenetic trees. To match the amount of data currently being processed, the construction of one single tree is not longer enough. With the advances in computational tools and algorithms the reconstruction of phylomes, the complete collection of phylogenetic trees in a genome, is now possible. From the phylome we can derive different kind of information. Primarily we can establish reliable orthology and paralogy relationships based on phylogenetic data. Once this data is available we can use it to transfer functional annotation to a newly sequenced genome. Or we can explore the duplications that have occurred during the lineage of a given species, whether in terms of average rates of duplications or to look for species-specific expansions. Another application is to search for evolutionary events that have shaped the history of a given fungi. Events such as horizontal gene transfer, hybridization, whole genome duplication or incomplete lineage sorting can be detected by carefully analyzing the thousands of trees provided in a phylome.

The early diverging fungi are probably the less studied group of fungi and as such present many questions as yet unanswered. The slowly increasing amount of genomes belonging to this group is now giving us the data to find answers to many of these questions. One such example is the phylogenetic position of microsporidia. This group of obligate intracellular pathogens has been placed in numerous points in the Eukaryotic species tree since its discovery. It is now firmly established in the vicinity of fungi but the exact location of it is still heavily discussed. Possible topologies included, but were not limited to placing Microsporidia within the Zygomyco-tina, as sister species to the Chytrid Rozella allomycis, as sister to Blastocladiales and Entomophthoromycota or as sister to all fungi. With the use of phylogenomics data, we have established that its position, regarding the currently completely sequenced fungi, is at the base of all fungi. Whether it should be considered a fungi or just a sister to them all is still a matter of debate.

Oral


27


Fluorescence in situ hybridisation (FISH) targeting the 18S rRNA of mucorales in tissue samples

McCormick Smith I 1, Lee SC 2, Heitman J 2, Fredricks D 3, Rickerts V 1

1 Robert Koch Institut, Berlin, Germany, 2 Duke University, Durham, USA, 3 Fred Hutchinson Cancer Research Center, Seattle, USA

Background: Diagnosis of mucormycosis is difficult due to nonspecific clinical signs and frequently negative culture results. Early diagnosis may enable early antifungal therapy, that has been associated with superior survival. We aimed to develop a FISH probe to detect mucorales in Formalin fixed tissue specimens as a tool for rapid diagnosis.

Methods: Sequences of the 18S rRNA-Gene of mucorales and other pathogenic fungi were downloaded from genbank. An alignment was constructed and visually searched for regions conserved among mucorales show-ing differences to other fungi. Promising probes were evaluated using germlings of fungal pathogens fixed in formalin. Fluorescence intensity of probes was compared to standard probes EUK 516 and non EUB, all labeled with Cy5. Brain tissue from a mouse infected with Rhizopus oryzae was hybridized with the Mucor-probes and a nonsense probe, both labeled with Cy 5. In addition, biopsy samples from one patient with Aspergillosis and one with Mucormycosis (fungal etiology was documented by hyphal morphology and sequencing of a broad-range PCR assay) was hybridized using Mucor and Aspergillus probes.

Results: No single locus allowed for an inclusion of all clinically important mucorales. A combination of two probes of 23 bases each was needed to include the most common agents of mucormycosis, e.g. Rhizo-pus, Lichtheimia, Rhizomucor, Mucor, and Cunninghamella. Other human fungal pathogens displayed 3-5 mismatches, making hybridisation unlikely. Evaluation with germlings showed hybridisation of all mucorales tested. Fluorescence intensity was comparable to the positive control. No hybridisation signal was detected with other human fungal pathogens (e.g. Candida, Aspergillus, Scedosporium). Fungal cytoplasma in mouse brain tissue demonstrated fluorescence after hybridisation with the mucor probes, but not with the nonsense probe. In clinical samples, parts of fungal cytoplasm showed a positive hybridization signal with the specific fungal probes. Differentiation between aspergillosis and mucormycosis was possible using Cy5 labeled fungal probes.

Conclusions: A combination of two probes targeting the 18S rRNA identifies most common agents of mu-cormycosis. Further evaluation with additional clinical samples is ongoing to determine their potential as a diagnostic tool.

Poster


28 29

Contribution of the ferrioxamine receptors to the pathogenesis of mucormycosis

Liu Mingfu1 and Ashraf S. Ibrahim1,2

1Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, CA, USA: 2David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Background: Iron-overload patients treated with deferoxamine are uniquely susceptible to mucormycosis, because Rhizopus spp. can obtain iron from ferrioxamine (deferoxamine + Fe3+). Previously we have identified two closely related, ferrioxamine-inducible R. oryzae genes (FOB1 and FOB2) in which their proteins bound ferrioxamine in vitro. We sought to determine if these two

genes encode the ferrioxamine receptor(s) and their contribution to virulence in vivo.

Methods: RNAi was used to silence FOB1 or FOB2 expression individually or collectively. For individual silencing, two plasmids were generated in plasmid pRNAi-pdc by cloning a 450-bp fragment unique to each gene and its corresponding inverted repeat separated by a 100-bp intron (total of 1 kb). These two constructs are termed pFOB1 and pFOB2, respectively. To silence both genes at the same time, the 1 kb sequence from pFOB1 was cloned downstream of pFOB2, separated by a 50-bp interval to yield pFOB1/2. Individual con-structs were transformed into R. oryzae pyrF mutant and transformants were selected on medium lacking uracil. Transformants were characterized for ferrioxamine gene expression, growth on medium with ferriox-amine as a source of iron, and 55Fe uptake from ferrioxamine. Additionally, the virulence of these transfor-mants was compared to R. oryzae pyrF mutant transformed with empty plasmid in a deferoxamine-treated or diabetic ketoacidotic (DKA) mouse models.

Results: qRT-PCR showed that R. oryzae transformed with either pFOB1 or pFOB2 had 80-85% inhibition in the corresponding gene. These transformants had reduced growth between 20-25% on medium supplemented with 10 mM ferrioxamine and 45-75% inhibition in 55Fe uptake compared to control cells transformed with empty plasmid. In contrast, cells transformed with the dual construct (pFOB1/2), had up to 95% inhibition in expression of both FOB1 and FOB2. Dual inhibition transformants had 45% inhibition in growth on medium with ferrioxamine and up to 85% reduction in 55Fe uptake compared to control cells. Finally, dual inhibition transformant had reduced virulence in the deferoxamine-treated mice but not in the DKA mice.

Conclusion: Our data suggest that the two CBS domain-containing proteins represent the ferrioxamine receptors and they are required for R. oryzae virulence in the deferoxamine-treated mouse model of mucor-mycosis.

Oral


29


Multiple acquisitions of YR retrotransposons by fungi

Anna Muszewska, Kamil Steczkiewicz, Krzysztof Ginalski

Laboratory of Bioinformatics and Systems Biology, CENT, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland

Retrotransposons with a tyrosine recombinase (YR) have been discovered recently [1] and lack thorough annotation in fungi. YR retrotransposons are divided into 3 groups [2]: DIRS, Ngaro and VIPER (known only from Kinetoplastida). We used comparative genomics to investigate the evolutionary patterns of retrotransposons in the fungal kingdom. The identification of both functional and remnant

elements provides a unique view on both recent and past transposition activity. Our searches covering a wide range of fungal genomes allowed us to identify 2249 YR retrotransposons. Based on the clustering of concatenated sequences of the reverse transcriptase, RnaseH and YR protein domains we propose a revised classification of YR elements expanded by two new categories of Ngaro elements. A phylogenetic analysis of 477 representatives supports this observation and additionally demonstrates that DIRS and Ngaro abundance changed independently in Basidiomycota and Blastocladiomycota/Mucoromycotina/Kixellomycotina. Our analysis revealed also that three Pucciniomycotina taxa, known for their overall mobile element abundance and big genome size, encode an elevated number of elements.

Considering the presence of DIRS elements in all analyzed Mucoromycotina, Kickxellomycotina and Blas-tocladiomycota genomes one might assume a common origin of fungal DIRS elements with a loss in Dicarya. Ngaro elements described to date from Opisthokonta [1], seem to have invaded the ancestor of Basidiomy-cota with a consecutive loss in Ustilagomycotina. Yet, most of analyzed genomes are devoid of YR elements and majority of identified retrotransposons are incomplete.

1. Goodwin, T.J., and Poulter, R.T. (2004). A new group of tyrosine recombinase-encoding retrotransposons. Mol Biol Evol 21, 746-759.

2. Lorenzi, H.A., Robledo, G., and Levin, M.J. (2006). The VIPER elements of trypanosomes constitute a novel group of tyrosine recombinase-enconding retrotransposons. Mol Biochem Parasitol 145, 184-194.

3. Piednoel, M., Goncalves, I.R., Higuet, D., and Bonnivard, E. (2011). Eukaryote DIRS1-like retrotransposons: an overview. BMC Genomics 12, 621.

Oral


30 31

Exploring the connection between endogenous sRNAs and Mucor pathogenesis

Francisco E. Nicolas 1,2, Santiago Torres-Martinez 1, Rosa M. Ruiz-Vazquez 1

1 Department of Genetics and Microbiology, University of Murcia, Spain, 2 Regional Campus of International Excellence ‘‘Campus Mare Nostrum’’, Murcia, Spain

Mucor circinelloides is an emerging opportunistic pathogen and a causal agent for the rare but lethal fungal infection mucormycosis. Mucormycosis mainly affects immunocompromised patients, although recently it has been described in otherwise healthy individuals. Besides this emerging pathogenicity, M. circinelloides has also outstand for being one of the first fungi in

which endogenous small RNAs (esRNAs) with putative regulatory functions have been identified. Most of the esRNAs in M. circinelloides derive from exons (ex-siRNAs) and regulate the expression of the protein coding genes from which they are produced. M. circinelloides mutants that are affected in genes involved in the production of these ex-siRNAs present defects in general developmental processes, such as growth and vegetative sporulation, as well as in the response to environmental signals, such as autolysis induced by nutritional stress. This may suggest a role for ex-esRNAs in pathogenesis, since spore size has been identified as a virulence factor in this fungus. Our work is currently focused on finding new ex-siRNAs and target mRNAs that could have a role in fungal pathogenesis.

Oral


31


Functional characterization of HMG-CoA reductase genes in Mucor circinelloides

Tamás Papp, Gábor Nagy, Anita Farkas, Orsolya Páll, Árpád Csernetics and Csaba Vágvölgyi

University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Szeged, Hungary

Exploration of new potential targets for antifungal therapy is a constant demand for the mycological studies. HMG-CoA reductase is a key enzyme of the acetate-mevalonate pathway, the early phase of the biosynthesis of terpenoids. This enzyme has a central role in the formation of ergosterol, a cell membrane component, which is the target of many antifungal agents. In the genome of the

zygomycete model organism, Mucor circinelloides, three HMG-CoA reductase genes (hmgR) were found. After the isolation of these genes, we started their functional analysis. Their expression was studied by quantitative real-time PCR under aerobic and anaerobic conditions. Transcription of all three genes was proven. Over-expression of both hmgR2 and hmgR3 affected the ergosterol production as well as sensitivity to statins and they proved to be regulated by the oxygen level of the environment. Silencing of the three genes was also carried out by using the antisense RNA (asRNA) technique. Three different vectors containing the antisense DNA segments of the hmgR genes, between the promoter and terminal sequences of the glyceraldehyde-3-phosphate dehydrogenase gene were constructed and transformed into the fungus by PEG-mediated protoplast transformation. Carotene and ergosterol content, growth rate and micro-morphology of the transformants were analysed. Based on these tests, it seems that hmgR2 has an important role in the ergosterol biosynthesis, while hmgR3 participates in the morphogenesis and the germination of the sporangiospores.

This study was supported by the grant OTKA_A_08-1-2009-0049, the Hungarian–French Intergovernmental S&T Cooperation Programme (TÉT_10-1-2011-0747) and by the European Union co-funded by the European Social Fund (TÁMOP-4.2.2/B-10/1-2010-0012).

Poster


32 33

Survival and interaction of mucoralean fungi with macrophages

H.R. Park, V.U. Schwartze, H.M. Dahse, P.F. Zipfel, A.A. Brakhage, K. Voigt

Leibniz Institute for Natural Prodcut Research and Infection Biology - Hans Knoell Institute, Jena, University of Jena, Germany

Zygomycetes are filamentous fungi, ubiquitously found in nature. The number of cases of zygomycosis has increased over the past few years. The infection is most commonly associated with diabetic patients, immunocompromised patients especially with neutropenic condition. The overall survival rate is approximately 50 % including superficial infections. The main route of the infection is via the

respiratory tract, followed by the skin and less commonly via the gut. Alveolar macrophage (AM) is important for the protection against the zygomycosis as AM is present at the site of infection where it actively engulfs those spores leading to the clearance of the spores and recruiting other immune cells to the foci of infection. Hence, it is crucial to study the interaction between AM and human pathogenic Zygomycetes to have a better understanding of the pathogenesis of the life threatening infection. However, one must also appreciate the interaction between amoeba and zygomycetes to have a better understanding of the mechanisms behind the intracellular survival of the fungi. One study revealed that some organisms exhibited similar survival strategies within the host cells despite of their unrelatedness. This hypotheses that ameba, an organism abundantly found in the environment, being an environmental school for the other organisms including Zygomycetes to train them for survival strategies in host cells such as alveolar macrophage. So in this study, Acanthamoeba castellanii along with macrophage is used for the co-infection model to demonstrate the theory of convergent evolution. This study will consider various methods including killing assay, to allow the broad screening of Zygomycetes to study.

Oral


33


Cellular immune responses to Zygomycetes

Susanne Perkhofer

University of Applied Sciences Tyrol, Innsbruck, Austria

Zygomycosis is a frequently lethal invasive infection in high risk patients such as immunocompromised hosts. Patients with haematologic malignancies or haemato poietic stem cell transplantation are particularly at risk. Zygomycosis has a poor prognosis in these patients with mortality rates >90% in disseminated infection. Neutropenia represents the most important pathogenic mechanism

influencing the onset and outcome of zygomycosis. Therefore, cellular immune response remains an important way to combat this devastating disease. The main line of cellular host response to filamentous fungi as the Zygomycetes consists of circulating polymorphonuclear neutrophils, mononuclear cells and macrophages, particularly pulmonary alveolar macrophages. Also human platelets with specialized recognition receptors may contribute to the engulfment of pathogens with their specific feature of thrombosis. Monocytes and neutrophils are rapidly recruited to the vessel wall and/or are incorporated into growing intravascular clots during thrombus development. Also immunopharmacology of antifungal agents to exert immunomodulatory effects on host innate immune defense against zygomycetes is of importance. The antifungal activity of cells in combination with antimycotic substances against Zygomycetes can have the potential of increasing the host response to Zygomycetes. This would be clinically important as strategies that aim to increase host immune cells could play an important role in the management of this life-threatening infectious complication. Studies on the role of cellular immune response to mucormycosis is of importance for future therapies of zygomycosis that may include agents that accelerate activity of effector cells thereby increasing antifungal host defense. A better understanding of the role of the host/pathogen interaction will support further development of potential novel antifungal therapies.

Oral


34 35

Carbon source utilization patterns in the order Mortierellales used as characters for stochastic mapping

Tamás Petkovits1, Ildikó Nyilasi1, Stella A. Kovács1, Csaba Vágvölgyi1 and Tamás Papp1

1Department of Microbiology, University of Szeged, Faculty of Sciences and Informatics, Közép fasor 52., H-6726 Szeged, Hungary

Members of the Mortierellales belong to one of the largest groups of Zygomycota. Most of the species can be isolated from soil where – like many other organisms - they act as decomposers of various organic materials. Some of them are also known as opportunistic animal pathogens (such as Mortierella wolfii) by causing severe diseases of the respiratory tract or fetal abortion. On the

other hand many species are widely used by the biotechnological industry as producers of polyunsaturated fatty acids (e.g. M. alpina). Formerly we inferred a phylogeny of the order by using a combined data set of two protein coding genes (tef and RPB1) and three ribosomal sequences (the nrSSU and nrLSU genes and the complete ITS region). We also made a study focusing on the carbon source utilization of the fungi. The growing patterns of the isolates – e.g. shape of the colony, colour, appearance of sporangia – had been observed. Carbon sources had been separated into two groups according to their effects on the isolates: inhibiting (0) or utilizable (1). This binary data matrix was documented. We tested 67 different carbon sources from which 39 showed larger differences in their effects. We combined the phylogenetic data and the results of the carbon source utilization tests by mapping the binary characters on the tree using the softwares Mesquite and SIMMAP. Our results show that utilization capabilities appeared independently in many cases. We also found that members of the closely related Mucorales show differences in their carbon source utilization patterns.

Oral


35


General management and adjunctive therapy

George L Petrikkos

The therapeutic approach to mucormycosis is multimodal, including antifungal agents, surgical debridement, and correction of the underlying condition predisposing the patient to the disease. Control of underlying conditions is critical in mucormycosis. Rapid correction of metabolic abnormalities is mandatory in uncontrolled diabetes. Corticosteroids should be discontinued, if

feasible, and other immunosuppressive drugs should be tapered as much as possible. Among the more recent therapeutic developments in mucormycosis treatment are the lipid formulations of amphotericin B, which are now the drugs of first choice.,the new triazole posaconazole, with promising efficacy as salvage treatment; the echinocandins in combination with amphotericin B and recombinant growth factors such as granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) may be active. Another option for the treatment of mucormycosis is the adjunctive use of iron-chelators but clinical trial, testing the combination of deferasirox with liposomal amphotericin B showed a higher mortality in the deferasirox group, probably due to the baseline characteristics of the patients. Adjunctive hyperbaric oxygen therapy, which can directly impact the growth of Zygomycetes, has been used successfully for adjunctive treatment of mucomycosis in a small number of patients without randomization to a control group This treatment has not been studied vigorously for efficacy and cannot be routinely recommended at this time.Despite the progress in the treatment of zygomycosis, many problems are yet unsolved and the mortality of the disease is still high.

Oral


36 37

Interaction of different zygomycete species with the complement system

G. Rambach, M. Hagleitner, C. Lass-Floerl, C. Speth

Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria

Zygomycoses are emerging angioinvasive infections caused by ubiquitous filamentous fungi belonging to the class of Zygomycetes (newly Mucormycetes). In the bloodstream, they come in contact with the complement system, which is a central element of the soluble innate immunity. The multiple antimicrobial effector functions that can be exerted by the complement system include fungal

damage, opsonization and support of phagocytosis, and attraction of immune cells. However, complement-induced exaggerated inflammation may also cause tissue damage and contribute to the pathology of fungal infection. We therefore aim to study in detail this interaction between Zygomycetes and the complement system. Different Zygomycete species (Lichtheimia corymbifera, Lichtheimia ramosa, Rhizopus microsporus, Rhizopus oryzae, Rhizomucor pusillus and Mucor spp) were compared for their capacity to activate the complement system and to be opsonized by the various complement factors. In addition, a putative intra-species variation was assessed by testing up to three different clinical isolates from each species. For comparison, Aspergillus fumigatus and A. terreus were included in all experiments. We found that sporangiospores of all tested species were able to induce complement activation with subsequent opsonization of the conidial surface; deposition of the complement factors C1q, C3 and Terminal Complement Complex (TCC) on the spore surface could be detected. The degree of opsonization considerably varied between the different species, and variations also occurred within the same species. On the average, the zygomycete species Rhizopus microsporus, Rhizopus oryzae, Rhizomucor pusillus and Mucor spp all showed high opsonization intensity on their spore surface in comparison to the Aspergillus species. In contrast, the complement deposition on the two Lichtheimia species L. corymbifera and L. ramosa was weaker than on the other zygomycetes species; depending on the complement factor the deposition corresponded to that on the Aspergillus conidia or was slightly lower. Covering of the zygomycetes spores with complement proteins strongly influences the interaction with neutrophil granulocytes. While virtually no interaction between neutrophils and non-opsonized Rhizopus microsporus and Rhizomucor pusillus spores was detected, opsonization of the spores resulted in strong adherence to or even phagocytosis by granulocytes. These preliminary findings indicate that complement may play an important role in the pathogenesis of zygomycoses and encourage further study efforts in this topic.

Poster


37


A case of Mucor irregularis infection mimicked as skin tumor

RAN Yuping1, KANG Daoxian1, WAN Huiying2, JIANG Xian1, HAO Dan1

1 Department of Dermatoverenology, West China Hospital, Sichuan University, Chengdu, China 2 Sichuan Provincial Institute of Dermatology and Venereology, Chengdu, China

We reported a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 47-year-old farmer presented at our clinic with a 1-year history of progressive red plaque around the inner canthus following dacryocystectomy. She was suspected as a skin tumor and hospitalized in a Tumor Hospital intend for anti-tumor therapy. Broad, no-septated and right-angled branching

hyphae were observed by both direct KOH examination and biopsy. Fungal culture revealed light yellow filamentous colonies which were identified as Mucor irregularis by molecular validation. SEM observations indicated non-apophysate sporangia with pronounced columellae and conspicuous collarette at the base of the columella following sporangiospore dispersal. Increased dose of amphotericin B after pre-application of dexamethasone was used to improve the clinical situation; the maximum dose was 35mg/d. Then the dose of amphotericin B and dexamethasone was decreased gradually, the total course was 43days. The plaque became smooth and her general condition was maintained. Serum potassium and electrocardiogram was monitored closely and treated promptly during the course of treatment. Hypokalemia, liver dysfunction and decline of white blood cell were the main side effects which were reduced by the application of potassium chloride oral liquid, compound glycyrrhizin and burnet root leukopoietic tablets. In this case, we presented not only the morphological characters of Mucor irregularis by KOH examination, SEM and biopsy, but also a successful treatment of primary cutaneous mucormycosis.

Oral


38 39

Pathogenesis and host defense against Zygomycetes

Emmanuel Roilides

Aristotle University of Thessaloniki, Thessaloniki, Greece

Infections caused by Zygomycetes have been reported with increasing frequency in recent years and still have unacceptably high morbidity and mortality. A number of risk factors are known to be associated with invasive mucormycosis, including hematologic malignancies and transplantation, iron overload, diabetes and ketoacidosis, birth prematurity, and possibly prior exposure to certain Aspergillus active antifungal

agents (ie voriconazole and caspofungin). Among clinically relevant Zygomycetes, the most frequent species are Rhizopus oryzae and Rhizopus microsporus. Cunninghamella bertholetiae is less commonly encountered but is associated with more severe infections. By comparison, Lichtheimia corymbifera is a less virulent and infrequent pathogen. The first defense against sporangiospores of Zygomycetes are intact barriers, ie skin and respiratory as well as intestinal mucosa. Innate immune cells such as neutrophils, monocytes/macrophages and dendritic cells are important players in the host defense against these organisms. Immunosuppression is among the most important risk factors for mucormycosis. Adaptive immunity does not appear to play a significant role. Rhizopus oryzae is recognized by Toll-like receptor-2 and upregulates release of a number of cytokines and chemokines from phagocytes, among which are TNF-a and IL-6. Toll receptors in Drosophila play a significant role in innate immune response to R. oryzae. This organism is more resistant to phagocytosis and hyphal damage than Aspergillus fumigatus. Host defense is modulated by a number of factors as evidenced from in vitro and preclinical data but only form few case reports. While in vitro cytokines enhance antifungal activity of phagocytes, augmentation is less evident in vivo. Echinocandins and amphotericin B formulations exert immunomodulatory activity on phagocytes. In a murine mucormycosis model posaconazole and G-CSF do not synergize for more efficacy. However, in another murine mucormycosis model liposomal amphotericin B and GM-CSF synergize for better efficacy. More comprehensive studies of augmentation of host defenses against Zygomycetes in vitro and in vivo are needed especially in conjunction with antifungal agents. In addition, other factors that may augment host defenses (ie acting on TLR2, inhibiting immunosuppressive cytokines) may be important. Adoptive T cell immunity is a promising future method to create and transfer immune response to Zygomycetes.

Oral


39


Biosynthesis of toxins in consortia of zygomycetes and Burkholderiales

K. Scherlach1, B. Busch1, N. Möbius1, C. Ross1, G. Lackner1 & C. Hertweck1,2

1Hans Knöll Institut, Jena, Germany 2Friedrich Schiller University, Jena, Germany

Ilamentous mould fungi of the genus Rhizopus are widespread in the environment. While some species are known to cause serious infections in humans, others are infamous plant pathogens accounting for substantial losses in agriculture. On the other hand, zygomycetes are also industrially important organisms, as certain species are used for food fermentation purposes. The phytopathogenic strain Rhizopus microsporus is the

causative agent of rice seedling blight. Recently, it was shown that this fungus is associated with endobacteria that not only control the vegetative reproduction of their host but also contribute important virulence factors to aid the fungus in colonizing rice plants.[1] We identified an unprecedented case of symbiotic cooperation in the biosynthesis of the pathogenicity factor rhizoxin. We found that the zygomycete specifically tailors the polyketide toxin provided by its endosymbiotic bacteria to increase its phytotoxic potency.[2] This discovery not only facilitates our understanding of complex ecological processes but also opens possibilities for the development of new drug candidates and potential bio-control agents against crop diseases.

In contrast, Rhizopus oligosporus is frequently used for fermentation of soybeans, cereal grains and coco-nuts in order to produce the Southeast-Asian speciality tempe. A common contaminant of the food fermenta-tion fungus is the proteobacterium Burkholderia gladioli which has caused countless health-threatening and even lethal intoxications after consumption of tempe. These bacteria produce bongkrekic acid, a polyketide which acts as a respiratory toxin and efficiently inhibits the adenine nucleotide translocase.[3] Even though it was well known that the toxinogenic bacteria are contaminants of the fungus, the genetic basis for toxin bio-synthesis has remained elusive. Through sequencing of the bacterial genome and functional analyses of the biosynthetic genes, we identified the gene locus and the accessory enzymes involved in the assembly of this unusual polyketide.[4] Our findings may contribute to the identification of toxinogenic B. gladioli pathovars in the context of food processing and agriculture and also set the foundation for engineering specific ligands for the adenine nucleotide translocator, a promising target for apoptosis research and chemotherapy.

1. L. P. Partida-Martinez, C. Hertweck, Nature 2005, 437, 884-888; L. P. Partida-Martinez, S. Monajembashi, K. O. Greulich, C. Hert-weck, Curr. Biol. 2007, 17, 773-777.

2. K. Scherlach, B. Busch, G. Lackner, U. Paszkowski, C. Hertweck, Angew. Chem. Int. Ed. 2012, 51, 9615-9618.3. B. Rohm, K. Scherlach, C. Hertweck, Org. Biomol. Chem. 2010, 8, 1520-1522.4. N. Moebius, C. Ross, K. Scherlach, B. Rohm, M. Roth, C. Hertweck, Chem. Biol. 2012, 19, 1164-1674.

Oral


40 41

Lichtheimia infections in mice and alternative hosts

Volker U. Schwartze, Kerstin Voigt, Ilse D. Jacobsen

Hans Knoll Institut, Jena, Germany

Mucormycosis is caused by a variety of mucoralean genera and species. However, well-characterized infection models for mucormycosis have only been established for few causative agents, mainly Rhizopus spp. and Mucor spp.. Although Lichtheimia species represent the second and third most common cause of mucormycosis in Europe and worldwide, respectively, virulence and pathogenesis

of this genus has not been addressed in detail yet.

To date only three of the five species of the genus Lichtheimia have been found to be involved in mucor-mycosis, namely L. corymbifera, L. ramosa and L. ornata. To clarify whether the clinical situation reflects differences in virulence between the species of Lichtheimia, we used an alternative infection model based on chicken embryos to screen the virulence potential of the different Lichtheimia species. The three clinically relevant Lichtheimia species, L. ramosa, L. corymbifera and L. ornata displayed a comparable virulence po-tential in embryonated eggs. In contrast, the L. sphaerocystis and L. hyalospora complexes were significantly attenuated in comparison to L. corymbifera. Furthermore, we observed differences in virulence between strains of the same species, independent of their origin (environmental vs. clinical) or phylogenetic position within the species.

We selected a L. corymbifera strain of typical virulence in the chicken embryo model to establish murine pulmonary infection models, based upon established models for Rhizopus and Aspergillus infection. Immu-nocompetent mice survived intranasal infection with up to 10*7 spores without developing clinical disease. However, L. corymbifera could be isolated in low numbers from the lungs of these mice 58 days after infec-tion. Immunosuppression with corticosteroids prior to infection led to 100% mortality in mice infected with 10*6 or 5 × 10*6 or spores. Disease in these animals ranged from peracute-acute to chronic. Acute disease was characterized by respiratory symptoms and extensive necrosis and hemorrhage in the lung. Histology revealed abundant mycelia, angioinvasion and thrombosis. Dissemination from the lung to other internal organs, especially the kidneys, was observed in all animals that became moribund ≥ 6 days post infection. Similar to the lung, abundant mycelia, angioinvasion and thrombosis characterized alterations in the various internal organs.

In summary, members of the genus Lichtheimia differ in their virulence potential as assessed in a chicken embryo model. Clinical infection of mice via the pulmonary route depends on immunosuppression and is char-acterized by pathological alterations typical for mucormycosis in humans. Thus, intranasal infection of mice is a feasible model to further study pathogenesis and virulence mechanisms, the role of other risk factors such as ketoacidosis, and treatment efficacy in Lichtheimia mucormycosis.

Oral


41


The genome of Lichtheimia corymbifera

V.U. Schwartze1,2, E. Shelest3, M. Marcet-Houben4, S. Winter5, V. Valiante2, K. Hoffmann1,2, M. Sammeth6, I.D. Jacobsen7, M. Nowrousian8, M. Marz5, T. Gabaldón4, S. Böcker5, A.A. Brakhage1,2, K. Voigt1,2

1 University of Jena, Institute of Microbiology, Department of Microbiology and Molecular Biology, Jena, Germany 2 Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Department of Molecular and Applied Microbiology, Jena, Germany 3 Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Systems Biology/Bioinformatics, Jena, Germany 4 Centre for Genomic Regulation (CRG), Barcelona, Spain 5 University of Jena, Institute of Informatics, Department of Bioinformatics, Jena, Germany 6 Centre Nacional d’Anàlisi Genòmica (CNAG), Functional Bioinformatics, Barcelona, Spain 7 Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Department of Microbial Pathogenicity Mechanisms,

Jena, Germany 8 Ruhr University Bochum, Department of General and Molecular Botany, Bochum, Germany

Mucoralean fungi are ubiquitous saprophytic fungi but are also able to cause life-threatening infections in animals and humans. Although mucormycoses are uncommon infections compared to aspergillosis or candidiasis the number of cases increased during the last decades. However, almost nothing is known about the pathogenicity mechanisms of mucoralean fungi. Genome sequences

are available for only two mucoralean pathogens (Mucor circinelloides and Rhizopus oryzae). Moreover, both species are closely related and represent more derived mucoralean fungi. In contrast, no information about the pathogenesis and genomes of ancient mucoralean pathogens are available. To shed light on molecular basis of the pathogenicity-associated traits of the basal Mucorales, the genome of the type strain of L. corymbifera was sequenced using a combination of 454 and Illumina sequencing. Our data show a high dissimilarity of the L. corymbifera genome compared to the other mucoralean genomes. In addition, massive gene expansions were found in several gene families including heterokaryon incompatibility protein, major facilitator superfamily, cytochrome P450 and several transcription factors.

Oral


42 43

Predicting potential virulence factors in the de novo sequenced genome of Conidiobolus coronatus

E. Shelest, V.U. Schwartze, K. Voigt

Hans Knoell Institute, Jena, Germany

Conidiobolus coronatus is an emerging human pathogen causing life-threatening infections. The treatment of this fungus is extremely problematic. Not much is known about the pathogenicity mechanisms of these organisms. In order to get the first insights into the molecular basis of the pathogenicity-associated traits of these pathogens, we sequenced and analysed the genome of C.

coronatus. Our first results reveal the sets of potentially virulence-associated factors, e.g.,secreted proteases and transcription factors (TFs). We show the gene expansion for some of these factors in the C. coronatus. genome. Additionally, I will particularly focus on transcription factors of the MADS box gene family, which demonstrate an interesting behaviour in basal lineages of fungi in general and in Zygomycetes in particular.

Oral


43


Hyperthermia sensitizes Rhizopus oryzae to posaconazole and itraconazole action through apoptosis

Fazal Shirazi1, Michael A. Pontikos1, Thomas J. Walsh2, Nathaniel Albert1, Russell E. Lewis1, Dimitrios P. Kontoyiannis1

1 The Univ of Texas M.D. Anderson Cancer Center, Houston, TX, 2 Cornell Univ, New York, NY

Background. The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel combinations of antifungal agents. We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of the conventional fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) and that this is mediated through apoptosis.

Methods. Fungicidal activity and apoptosis were studied using standard microbiological techniques, hyphal metabolic and vital dye reduction assays at 25, 37 and 42°C in RPMI 1640. Apoptosis was characterized by phosphatidylserine externalization, DNA fragmentation, membrane integrity, chromatin condensation, reac-tive oxygen species (ROS) generation and metacaspase activity.

Results. MICs for PCZ and ICZ alone were significantly higher at 37°C (8-16 mg/mL) compared to 42°C (0.25-1 mg/mL). In addition, intracellular ROS increased notably when fungi exposed to antifungals at 42°C. At 42°C, 20-60% of cells were apoptotic at 0.25-0.5 mg/mL of PCZ or ICZ, whereas at 37°C higher concen-trations of PCZ and ICZ (4-16 mg/mL) were required to induce apoptosis. The addition of the ROS scaven-ger N-acetylcysteine increased fungal survival, prevented apoptosis through reduced ROS accumulation and metacaspase activation.

Conclusions. Hyperthermia induces apoptosis in combination with PCZ or ICZ in Rhizopus oryzae. Local thermal delivery could be a useful adjunct strategy for these Mucorales infections.

Poster


44 45

Mitochondrial respiratory pathways inhibition in Rhizopus oryzae potentiates activity of posaconazole and itraconazole via apoptosis

Fazal Shirazi1, Dimitrios P. Kontoyiannis1

1 The Univ of Texas M.D. Anderson Cancer Center, Houston, TX,USA

Background. The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. We hypothesized that targeting mitochondrial respiratory pathways potentiates triazole activity via

apoptosis.

Methods. Fungicidal activity and apoptosis were studied using standard microbiological techniques, hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by phosphati-dylserine externalization, DNA fragmentation, membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, metacaspase and superoxide dismutase activity and cytochrome c release.

Results. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes character-istics of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accu-mulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased metacaspase activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by addition of the ROS scavenger N-acetyl-cysteine.

Conclusions. Taken together, these findings suggest that use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for development of improved or novel antifungal agents.

Poster


45


Targeting the calcineurin pathway enhances ergosterol biosynthesis inhibitors activity against mucorales in vitro via apoptosis

Fazal Shirazi1, Dimitrios P. Kontoyiannis1

1 The Univ of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Background. Calcineurin is implicated in a myriad of human diseases as well as in homeostasis and virulence in all major human pathogenic fungi. The Mucorales Rhizopus oryzae, Cunninghamella bertholletiae and Mucor circinelloides are a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive mucormycosis

are often ineffective, and novel therapeutic approaches are urgently needed. We hypothesized that growth in the setting of the calcineurin inhibitor tacrolimus (TAC) results in fungicidal activity of the conventionally fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) and that this is mediated through apoptosis.

Methods. Fungicidal activity and apoptosis were studied using standard microbiological techniques, hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting Ca2+ levels, phosphatidylserine externalization, DNA fragmentation, membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, metacaspase activity, ATP and cytochrome c release assay.

Results. MICs for PCZ and ICZ alone were significantly higher (8-128 mg/ml) compared to PCZ/ICZ +TAC (0.25-4 mg/ml) against R. oryzae, C. bertholletiae and M. circinelloides. PCZ/ICZ in combination with TAC becomes fungicidal and this activity was mediated through increased apoptotic cell death R. oryzae (10-50%), C. bertholletiae (5-50%) and M. circinelloides (5-55%) germlings with morphological changes characterized by elevated Ca2+ levels, externalization of phosphatidylserine, generation of ROS, disturbance of mitochon-drial potential, nuclear condensation, and internucleosomal DNA fragmentation. Protein synthesis inhibitor cycloheximide, prevented TAC+PCZ induced apoptosis. Moreover, metacaspase activation was accompanied by TAC+PCZ/ICZ-induced cell death.

Conclusions. Taken together, these findings suggest that use of PCZ or ICZ in combination with TAC makes R. oryzae, C. bertholletiae and M. circinelloides exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for development of improved or novel antifungal agents.

Poster


46 47

Interactions of Antifungal Agents and Host Defense against Zygomycetes

Maria Simitsopoulou

Aristotle University of Thessaloniki, Thessaloniki, Greece

Fungal infections caused by zygomycetes have been increasing among various groups of immunocompromised patients including those with organ or stem cell transplantations, haematological malignancies or diabetes mellitus. Zygomycetes show a resistant phenotype to most existing azoles and echinocandins with high MIC values and generally decreased susceptibility as compared to AmB

formulations. AmB monotherapy remains the gold standard in the treatment of zygomycosis, despite its potential toxicity. Drug efficacy is often compromised by the lack of selective fungicidal activity to fungi but also by the evolution of drug resistance, which could arise after prolonged exposure of fungal organisms to agents with fungistatic effects. In the absence of new drugs in the market, there is a growing need for implementing new antifungal strategies to enhance antifungal drug efficacy against zygomycetes. In vitro and in vivo evidence shows that combinational schemes including drug-to-drug or drug-to-host interactions augment antifungal potency, decrease emergence of resistance and reduce drug toxicity. It has been shown that, while echinocandins have minimal activity against Rhizopus oryzae, when used in combination with amphotericin B lipid formulations show synergistic activity in the treatment of mucormycosis in diabetic ketoacidotic mice. Beta-glucan unmasking by echinocandins lead to an enhancement of human neutrophils (PMNs) against R. oryzae hyphae, while AmB lipid complex exhibits synergistic or additive activity with PMNs against hyphae of Lichtheimia corymbifera, R. oryzae and Rhizopus microsporus. It is noteworthy that, although several studies exist on the immunomodulating properties that AmB formulations, voriconazole, caspofungin or micafungin exert on immune cells challenged with Aspergillus fumigatus, the second most common invasive mold among immunocompromised patients, comparative data are still lacking for zygomycete species. In recent years, except for combination strategies that involve conventional fungal cell wall or cell membrane inhibitors, several studies have investigated novel combinational applications that have an effect on signal transduction pathways blocking fungal stress responses or on protein prenylation affecting intracellular posttranslational modifications and cell apoptosis processes. Combinations of calcineurin inhibitors or statins with conventional antifungal agents have shown synergistic interactions. However, both compounds should be used with caution as they induce pleiotropic effects in the cellular metabolism of both the fungus and the host. Further studies are needed to explore the mechanisms of their antifungal activity against zygomycetes and gain insights as to how these compounds modulate pivotal host defenses for fungal detection and elimination.

Oral


47


Unusual agents of Mucormycosis

Alberto M. Stchigel

Mycology Unit, Medical School, Universitat Rovira i Virgili, Reus, Spain

Molecular biology techniques have allowed discovering in Mucorales numerous cryptic species of clinical interest. Clear examples can be found in Apophysomyces, Saksenaea and Mucor. The availability of reference sequences and phenotypic tests has been very useful in the diagnosis of recent clinical cases and outbreaks in which some of these species were involved. One of

the emerging mucoralean genera, Apophysomyces, has been reported in a series of cases of necrotizing fasciitis in India, in which was identified for the first time A. elegans, the type species of the genus, as agent of human infection. A new species of this genus has also been identified in a case of human infection in Mexico. The genus Syncephalastrum up to now has two accepted species, S. racemosum and S. monosporum (including three varieties). However, recent molecular studies have demonstrated that S. monosporum clade also includes some strains morphologically identified as S. racemosum. Two additional clades have been also detected when more nuclear genes were sequenced and analyzed.

Oral


48 49

CYTOLOGY OF LICHTHEIMIA SPP. IN VIVO AND IN VITRO

Stepanova А.А., Khostelidi S.N.2, Araviyskiy R.A1, Zuzgin I.S.3, Ruzjinskaiya O.S.3, S.M.3, Kri-volapov Y.V.3, Sinitskaya LA.1, Klimko N.N.2

¹Kashkin Research Institute of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint Petersburg, Russia 2Department of Clinical Mycology, Allergy and Immunology, North-Western State Medical University named after I.I. Mechnikov,

St. Petersburg, Russia ³Leningrad Region Clinical Hospital, St. Petersburg, Russia

The comparative ultrastructural analysis of Lichtheimia spp. from the lesion of patient with soft tissue mucormycosis and after pathogen isolation in culture (Saburo agar, 3 days after sowing, 28º) with the using of light microscopy, TEM and SEM was made. It was showed that the hyphal profiles (HP) in vivo and in vitro were similar by number and structure of nuclei, mitochondries, by type of

accumulated storage substances (lipids inclusions and polyphosphate granules) and level of development of components of endomembrane system, but strongly differed on a structure of lateral cell walls (thickness, shape, electron density, the number of layers and its structure).

Four main types of a structure of cellular walls of mature hypha of vegetative mycelium were revealed: 1) uniform thick (1,1 μm), two-layered: with thick, dark, dense internal (0,8 μm) and external thin (0,3 μm), moderate electron density and thin-granular layer; 2) thick three-layered with an average thin uniform (0,1 μm) dark homogeneous, internal, with variable thickness (0,5-1,0 μm) dark homogeneous internal and (0,4-0,6 μm) moderate electron density are thin-granular external layer; 3) thin (0,1 μm) dark homogeneous lat-eral walls, with integral adjacent well developed variable in thickness (0,5-2,5 μm) external layer consisting from densely located contrast microfibrils; 4) irregularly thick (1,5-3,0 μm), one-layered, dark, homogeneous with various, often intricate (on cross sections) forms. Most often were visible HP with cell walls of the fours type (75% from total number), and the most rare (1%) – the third type. HP with cell walls of the second type composed 5%, whereas the first – 19%. The variety of cell walls structures of HP in vivo in comparison with HP in vitro, may be connected with Lichtheimia protective mechanism in reply to influence of patient immune system and antifungal therapy.

Oral


49


The transcriptional response to light in Phycomyces blakesleeanus

J.M. Villalobos-Escobedo1, V.G. Tagua2, E.M. Luque2, A. Herrera-Estrella1, L.M. Corrochano2

1Laboratorio Nacional de Genómica para la Biodiversidad. CINVESTAV, Irapuato. Mexico. 2Department of Genetics, University of Seville,

Spain

Phycomyces blakesleeanus is a model microorganism for research on sensory biology. The giant fruiting bodies, sporangiophores, of Phycomyces change their speed and direction of growth in response to a variety of environmental signals, including light, gravity, wind, touch, and the presence of nearby objects. Additionally, light regulates sporangiophore development and induces the synthesis of the

pigment beta-carotene. The genome of Phycomyces blakesleeanus has been sequenced to 7.49X depth and assembled into 475 scaffolds totaling 56Mbp, and 47847 ESTs have been assembled from cDNAs of light and dark cultures. A total of 14792 protein-coding genes have been predicted. The characterization of the cDNA libraries obtained from mRNA isolated from mycelia kept in the dark or exposed to light for 30 min allowed the identification of a number of light-regulated genes. We identified 113 genes as putatively regulated by light from a total of 5,713 cDNA clusters, and light-dependent mRNA accumulation was confirmed for the majority of the genes examined by quantitative RT-PCR (23 out of 27 genes). If our cDNA set is a representative sample from the transcriptome, then about 2% of the Phycomyces genes are regulated by light. Several light-regulated genes that have been characterized previously were not uncovered by our analysis, which suggest that the number of light-regulated genes in Phycomyces is higher than 2%.

A more detailed characterization of the transcriptional response to light has been performed by high-throughput sequencing of cDNAs (RNAseq) obtained from the wild type strain and a strain carrying muta-tions in the major photoreceptor complex from Phycomyces (the Mad complex) prepared from RNA isolated from two developmental stages, mycelium and sporangiophore (fruiting body). A total of 554 differentially expressed genes (312 induced and 242 repressed) were detected. In the mycelium of the wild-type strain the, overexpressed genes are, mainly, involved in translation, biosynthetic process, and responses to stimuli, while in the mutant mycelium only 60 were induced, and 54 repressed, showing a clear decrease in the tran-scriptional response to light. Interestingly, in the sporangiophore of the wild-type 874 genes were induced, 774 of which are repressed in the mutant strain-sporangiophore, The functional categories of translation, cellular respiration and carbohydrate metabolism are enriched in this group of genes, indicating the partici-pation of the Mad complex in the activation of such processes in the sporangiophore. Our results indicate that the two developmental stages of Phycomyces show different transcriptional responses to light. The results of the RNAseq analysis show that up to 9% of the predicted genes in the wild-type genome are differentially expressed upon exposure to light.

Oral


50 51

Phylogeny of the Zygomycetes

K. Voigt1, G. Walther1, G.S. de Hoog2

1Leibniz Institute for Natural Product Research ad Infection Biology - Hans Knoell Institut, and University of Jena Jena, Germany 2CBS-KNAW Fungal Biodiversity Center, Utrecht, The Netherlands

Zygomycetes, formerly described as class within the fungal kingdom, are polyphyletic, and therefore, split into five distinct subphyla, which are the Entomophthoromycotina, Mucoromycotina, Mortierellomycotina, Kickxellomycotina and Zoopagomycotina. The former two subphyla contain species, which are human pathogenic causing infections with diverse predisposition and etiologies.

They encompass ubiquitously distributed insect-killing, saprotrophic soil- or dead plant material-inhabiting fungi of the orders Entomophthorales and Mucorales, respectively. Human pathogenic species inhabit different growth temperature optima ranging from 33 °C to 42 °C, just members of these two orders are capable of causing diseases, nmaely entomophthoromycosis and mucormycosis, in immunocompromised and immuncompetent humans, respectively. The evolution and phylogenetic relationships of the fungi formerly summarized as Zygomycetes are described with special emphasis on clinically relevant groups. Moreover, phylogenomic aspects are discussed with respect to future output of the 1000 Fungal Genome Project launched in 2011 (http://1000.fungalgenomes.org/home/).

Oral


51


Advances in Mucormycosis: Integrating Epidemiology, Animal Model Systems and Diagnostic Tools from Bench to Bedside

Thomas J. Walsh

Schueler Foundation Scholar in Mucormycosis Chief, Medical Mycology Research Laboratory Transplantation-Oncology Infectious Diseases Program Departments of Medicine, Pediatrics, and Microbiology & Immunology Weill Cornell Medical Center and Cornell University New

York, NY USA

Mucormycosis is an emerging cause of increased infection-related morbidity and mortality in immunocompromised patients. Characterized by a rapidly progressive disease of the paranasal sinuses, lower respiratory tract, central nervous system, gastrointestinal tract, skin and soft tissues, as well multiple deep tissues, mucormycosis is difficult to diagnosis in its early stages.

Recent single-institution and multi-institutional studies illustrate the increasing incidence, heterogeneous organisms, and expanding host populations. Despite the availability of lipid formulations of amphotericin B, mortality remains high and morbidity severe. While early diagnosis with prompt therapeutic intervention is a key determinant of survival and reduced morbidity, detection of the agents of mucormycosis remains elusive. Recent advances in development of molecular diagnostic, metabolic, proteomic, and immunochemistry platforms may provide new systems by which to rapidly diagnosis invasive mucormycosis at the earliest possible stage before irrevocable organ damage develops. Animal models of mucormycosis are critical to developing these diagnostic systems. Animal models can provide an abundance of standardized bronchoalveolar lavage fluid, lung tissue, and blood in specific host-based backgrounds for diabetes mellitus, neutropenia, and solid organ transplantation. Animal models may be rigorously control and adjust for size of inoculum, species of Mucorales, and type, duration, and dosage of antifungal therapy. Model-derived tissues and fluids can be then analyzed robustly to assess the effect of residual fungal burden, species, and antifungal therapy that may determine the analytical sensitivity and specificity of a given assay. As fluids and tissue from patients are difficult to acquire and archive, development of new assays may be greatly accelerated by initial studies conducted in specimens from clinically applicable laboratory animal models of mucormycosis. Following the characterization, optimization, and characterization of a diagnostic biomarker in laboratory animal models, the assay can be studied in human tissue, blood and fluids. Such human samples are largely restricted to a small number of samples within single institutions, thus limiting the timely development of rapid diagnostic assays for mucormycosis. The current protocol being developed through the ISHAM Zygomycosis Working Group (ZWG) will be the next generation study (ZWG2) that builds upon the earlier surveillance project. The ZWG2 protocol will build an international human specimen archive that will serve as a key resource in development of advanced molecular, metabolic, proteomic, and immunochemical diagnostic systems. The international archive also will preserve the isolates of patients enrolled into the study. The next objective of ZWG2 will be to develop a comprehensive database of the epidemiological, demographic, clinical, microbiological, and outcome variables of patients whose specimens are archived. From that database, a predictive risk model will be developed as a tool for early bedside identification of patients at increased risk for mucormycosis.

Oral


52 53

Main ecological trends in the Mucorales

Grit Walther1,2

1Institute of Microbiology, Department of Microbiology and Molecular Biology, University of Jena, Jena, Germany;2Leibniz-Institute for Natural Product Research and Infection Biology – Hans-Knöll-Institute, Jena Microbial Resource Collection, Jena,

Germany

Mucorales are considered to grow ubiquitous on wet organic material and to produce a high amount of asexual spores that are efficiently dispersed by air resulting in worldwide distribution. Based on data of the literature and of own preliminary studies I will show that this picture is too simplistic and that there are several understudied aspects in the ecology of Mucorales: genera such as Mucor

and Lichtheimia produce wet, less hydrophobic sporangiospores. In most indoor air samplings the Mucorales are strongly underrepresented. In addition, recent molecular studies of single mucoralean species revealed geographic patterns indicating a less efficient air-dispersal. Another neglected aspect is the dimorphism. In a current study on yeast formation in the Mucorales I found a high proportion of yeast-forming species in the Mucoraceae and the Backusellaceae. Triggered by liquid medium with a glucose content of at least 3 % the sporangiospores of these species germinate with yeast cells. The function of the yeast state in nature is unknown. Detailed and species based ecological studies are urgently needed also to understand where and when patients are exposed to these fungi and to provide best possible prophylaxis.

Oral


53


Mucormycosis acquired from herbal tea

B. Willinger, F. Wimazal , W.R. Sperr, A. Böhm, W.D. Baumgartner, C. Herold, L. Müllauer, U. Jäger, P. Valent, P. Knöbl

Division of Clinical Microbiology, Department of Laboratory Medicine and Division of Hematology, Department of Medicine, Vienna, Austria

A 46 year old male patient was diagnosed with acute myeloic leukemia (AML) and was admitted for induction therapy to be followed by stem cell transplantation. Induction therapy was started, but 11 days later the patient developed fever which was unresponsive to piperacillin/tazobactam, meropenem and vancomycin. However, serum reactive protein (CRP) continued to increase (11-20

mg/dL), but chest X-ray and blood culture were negative. As a few days later a swelling of the right periorbital area was observed clindamycin and caspofungin were added. A few days later proptosis was noticed and computed tomography (CT) of the paranasal sinuses and the orbita was performed showing a pansinusitis with accentuation at the right upper jaw. An endonasal drainage of the right upper jaw and the right ethmoid was performed and respective specimens were collected. Histology showed broad, sparsely septated and irregularly branched hyphae. Culture showed a rapid growing pale brownish-grey colony. Microscopically, simple rhizoids and brownish sporangiophores were detected. Sporangia were greyish-black and spherical. Columellae were subglobose to globose to conical. Also, sequence analysis was performed and identified the detected fungus as as Rhizopus microsporus var. rhizopodiformis.

At first the origin of the infection was unclear. There were no other patients with similiar infections. How-ever, after inquiry the patient informed the clinicians that he regularly drank a special herbal tea prepared from teapowder as used in traditional Chinese medicine. As a consequence we investigated the teapowder performing several cultures at different temperatures. All of these cultures showed the growth of Rhizopus microsporus var. rhizopodiformis and Syncephalastrum sp. thus confirming our speculation that the teapow-der was the most likely cause for the infection.

After the detection of Rhizopus microsporus from caspofungin was stopped and antifungal therapy with liposomal amphotericin B (AmB) at a dose of 5 mg/kg/day was started. After three weeks AmB was changed to posaconazole and continued for nine months. The patient responded very well, cultures became negative and Rhizopus couldn’t be detected when the antifungal therapy was stopped. However, due to mucormycosis the planned therapy of AML had to be stopped. In the meantime myelosarcoma developed and the patient eventually died from his underlying disease. On autopsy, no mucormycosis was present.

Oral


54 55

Pathogenesis mechanisms during invasive zygomycosis

Katherine Wong2, Robin C. May1, Deborah Mortiboy1 and Kerstin Voelz1,2

1 National Institute for Health Research Surgical Reconstruction and Microbiology Research Centre, Queen Elizabeth Hospital, Birmingham, UK, 2 Institute of Infection and Microbiology, School of Biosciences, University of Birmingham, Birmingham, UK

Life-threatening infections with zygomyceteous fungi occur mainly in patients with risk factors such as immunosuppression, iron overload, acidosis and anatomic barrier breakdown. The latter is particularly a problem in military personnel presenting with explosive blast wounds. A recent study showed that 31 of 37 soldiers (84%) with injuries secondary to explosive blasts developed an invasive fungal infection,

47.2% of which were caused by zygomycete species (1).

During the developmental cycle zygomycetes produce sexual and asexual spores to overcome disadvanta-geous conditions. Once conditions improve, the spores germinate in response to environmental cues such as temperature, humidity and day length. These spores are also the infectious agent, with disease being initi-ated upon spore germination. Despite the high incidence rates in susceptible populations, little research has been undertaken into the regulation of germination, and associated pathogenesis, of zygomycetes.

We obtained 13 clinical zygomycete isolates from patients with invasive and non-invasive zygomycosis, as well as three reference strains. These were used to investigate the relevance of spore size to virulence and to identify factors that influence the rate of spore germination during disease initiation.

We demonstrate that the onset of spore germination is synchronized between individual spores after ~ 300 minutes of incubation at 37°C and 5% CO2 in vitro. Spore size did not significantly differ between invasive and non-invasive isolates. However, under hypoxic conditions, invasive isolates showed a decreased germina-tion time whereas germination time increased in non-invasive isolates. Spore germination in all isolates was dramatically delayed after encounter of the intracellular macrophage environment. Intracellular spores ap-peared to be inactive and germination only occurred after release from the phagocyte. In addition, we also observed events of spore vomocytosis from and lateral transfer between macrophages.

In summary, these results demonstrate that host-pathogen interactions play a major role in initiation of zygomycosis and highlights the need for a deeper understanding of this relationship.

Warkentien et al. (2012) Clin. Infec. Dis. 55:1441-9

Oral


55


Drug combinations against Mucor irregularis: in vitro susceptibility test and successfully cured three patients

Jin Yu1 , Ruoyu Li1

1Department of Dermatology and Venereology, Peking University First Hospital; Research Center for Medical Mycology, Peking University, Beijing, China

Combinations of amphotericin B/terbinafine, amphotericin B/caspofungin, posaconazole/terbinafine, posaconazole/caspofungin, itraconazole/terbinafine, and itraconazole/caspofungin were studied as potential treatments against 18 isolates of Mucor irregularis in vitro. The synergism observed in 99.4% versus 38.9%, 66.7% versus 33.3%, or 94.4% versus 44.4% isolates noted for caspofungin versus

terbinafine with amphotericin B, posaconazole, or itraconazole, respectively. No antagonism was observed. Moreover, we present primary cutaneous mucormycosis caused by M. irregularis in three immunocompetent patients. By amphotericin B, itraconazole and terbinafine alone or combined therapy, they all were cured successfully.

Oral

Date post:	22-Jan-2022
Category:	Documents
Upload:	others
View:	3 times
Download:	0 times

Programe Abstracts - Zygomycota

Documents