CBS-KNAW Fungal Biodiversity Centre
An Institute of the Royal Netherlands Academy of Arts and Sciences
22042014 23042014
22-23 April 2014
Diversity and Barcoding of Medical Fungi:
Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract and
Programme book
.
CBS-KNAW Fungal Biodiversity Centre
An Institute of the Royal Netherlands Academy of Arts and Sciences
22042014 23042014
Diversity and Barcoding of Medical Fungi:
Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
22-23 April 2014
Introduction
Welcome to the ISHAM Workshop on Barcoding of Medical Fungi. 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])
or to Wieland Meyer ([email protected]).
Venue
CBS-KNAW Fungal Biodiversity Centre, Utrecht, Uppsalalaan 8, The Netherlands. The institute is easily reached
from Amsterdam Schiphol Airport within one hour 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.
Registration
Registration before 15 April is compulsory; for on-site registration and no-show €100,- will be charged. Abstract
submission deadline is 1 April 2014. The workshop including lunches and dinners is provided at no cost.
Sponsors
We are grateful to our sponsors ThermoFisher Inc., Astellas, Elsevier Publishing Co. 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 ISHAM Working group on Barcoding, contact
Wieland Meyer [email protected].
Welcome to Utrecht!
The organisers: Sybren de Hoog, Wieland Meyer, Michaela Lackner, Kathrin Tintelnot
1
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Programme
22 April 2014
08.00- 08.45 Registration, room open with coffee
08.45-09.00 Opening
Session 1 Chairs: Sharon Chen, Jens Frisvad
09.00-09.20
Dirk Stubbe, Marijke Hendrickx, Sam Roesems (BCCM/IHEM Culture Collection of Biomedical Fungi and
Yeasts, Service of Mycology and Aerobiology, Scientific Institute of Public Health, Brussels, Belgium):
Maldi-tof as an additional tool for strain characterisation in the BCCM/IHEM culture collection.
09.20-.9.40
Valérie Monnin, David Pincus (bioMérieux R & D Microbiology Department, France): Pseudallescheria
and Scedosporium species differentiation from agar cultures by MALDI-TOF Mass Spectrometry.
09.40-10.00
Andreas Zautner, Claudia Folba, Mareike Bernhard, Michael Weig, Uwe Groß and Oliver Bader (Institute
for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, D-37075 Göttigen)
Approaches to intra-species typing using MALDI-TOF MS
10.00-10.20
P.-M. Rath, J. Steinmann (Institute of Medical Microbiology, University Hospital, Essen, Germany):
Genotyping and susceptibility testing with inhalative agents.
10.20-10.50 Coffee break
Session 2 Chairs: Elena Piecková, Kathrin Tintelnot
10.50-11.10
Anne-Cécile Normand, Magali Gautier, Carole Cassagne, Coralie L’Ollivier, Stephane Ranque, Renaud
Piarroux (Parasitology & Mycology, Assistance Publique-Hôpitaux de Marseille, CHU Timone-Adultes,
Marseille, France): Routine identification of mold using MALDI TOF MS.
11.10-11.30
Sharon Chen (Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital,
ICPMR, Sydney, Australia): Antifungal Drugs: new approaches
11.30-11.50
Monica Ganan, Morten Sørli (Department of Microbiology, Rikshospitalet, Oslo University Hospital, Oslo,
Norway): New and alternative antifungals.
2
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Programme
11.50-12.10
Heinrich Anne1, Rehorska René2, Pfeifhofer Hartwig Wilfried2, Jamnig Julia2, Mueller Maria2, Buzina
Walter1 (1 Institute Of Hygiene, Microbiology And Environmental Medicine, Medical University Of Graz,
2
Institute Of Plant Sciences, University Of Graz, Austria) Susceptibility of Scedosporium species against
plant extracts from neophytes
12.10-12.30
Fabiola Fernández1, Josep Guarro2, Cornelia Lass–Flörl3, Michaela Lackner3 (1Unitat d´Anatomia Patològica,
Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus 2Unitat d´ Microbiologia,
Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus 3Division of Hygiene
and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria.) Azole-echinocandin
combination a potential therapeutic option against scedosporiosis - new in vitro and vivo study insights
and experiences from case studies
12.30-13.30 Lunch
Session 3 Chairs: Michaela Lackner, Dea Garcia-Hermoso
13.30-13.50
Laszlo Galgoczy, Monika Homa, Csaba Vagvolgyi, Tamas Papp (University of Szeged, Faculty of Science
and Informatics, Department of Microbiology, Hungary): Beyond the conventional antifungal agents:
Antifungal activity of non-antifungal drugs against Scedosporium and Pseudallescheria isolates.
13.50-14.10
Jens C. Frisvad & K.F. Nielsen (Department of Systems Biology DTU, Lyngby, Denmark): Can secondary
metabolites add significant taxonomic data in Scedosporium?
14.10-14.30
Dea Garcia-Hermoso1,2, Aude Sturny-Leclere1,2, Damien Hoinard1,2 , Alexandre Alanio1,2 & Françoise
Dromer1,2 (1Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses
Invasives et Antifongiques, Paris, France 2 CNRS URA3012, Paris, France): Evaluation of physiological
parameters of Scedosporium and Pseudallescheria species.
14.30-14.50
Ulrike Binder, Cornelia Lass-Flörl, Michaela Lackner (Medical University Innsbruck, Division of Hygiene and
Medical Microbiology): Galleria mellonella as an alternative model to study Scedosporium-infections.
14.50-15.20 Tea break
3
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Programme
Session 4 Chairs: Walter Buzina, Josep Cano
15.20-15.40
Ayse Kalkanci, Ali Adil Fouad, Merve Erdogan, Nevin Keskin (Gazi University School of Medicine Department
of Medical Microbiology, Ankara, Turkey): Mini model of Scedosporium infection.
15.40-16.00
Kjetil F. Christensen, Jørgen Bjørnholt, Reza Assalkhou (Department of Microbiology, Rikshospitalet, Oslo
University Hospital, Oslo, Norway): Animal models.
16.00-16.20
Stepanova A.A., Vasilyeva N.V. (I.I. Mechnikov North-Western State Medical University: Kashkin Research
Institute of Medical Mycology, Santiago-de-Cuba 1/28, 194291, Saint Petersburg, Russia): Ultrastructure of
lateral cell walls, septa and septal pore apparatus in the cells of vegetative mycelium of Pseudallescheria
species
16.20-16.40
Kathrin Tintelnot, Anne Bernhardt (Robert Koch Institut, Berlin, Germany): Identification of scedosporiosis
from FFPE tissue.
16.40-17.00
R.O.R. Calixto, J.M. Vieira, R. Rollin-Pinheiro, L.C.L. Lopes, G.L. Sassaki, L.M. de Souza, E. BarretoBergter (Departamento de Microbiologia Geral, Instituto de Microbiologia, UFRJ, Rio de Janeiro,
Brasil; Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmacia, UFRJ, Rio de Janeiro,
Brasil; Departamento de Bioquimica e Biologia Celular, UFPR, Paraná, Brasil): Structural analysis of
glucosylceramides (CMHs) from species of the Pseudallescheria / Scedosporium complex.
17.00-17.20
Ferry Hagen, Teun Boekhout (CBS-KNAW Fungal Biodiversity Centre, Yeast Research, Utrecht, The
Netherlands; Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina
Hospital, Nijmegen, The Netherlands) Towards a new taxonomy for the basidiomycetous yeasts and the
Cryptococcus neoformans and Cryptococcus gattii species complex in particular.
18.00 Departure Bus to Utrecht city centre
18.30 Dinner at Live Utrecht
4
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Programme
23 April 2014
08.30- 09.00 Room open with coffee
Session 5 Chairs: Johannes Rainer, Dirk Stubbe
09.00-09.20
Ilka McCormick Smith1, Ludwig Sedlacek2, Danny Jonigk3 , Volker Rickerts1 (1Robert Koch-Institut, Berlin,
Germany 2 Medizinische Hochschule Hannover, Hannover, Germany 3Medizinische Hochschule Hannover,
Hannover, Germany) Identification of Scedosporium and other fungi in formalin fixed, paraffin embedded
tissue from patients with cystic fibrosis by broadrange PCR and Fluorescence in situ hybridisation (FISH)
09.20-09.40
A. Bernhardt1, L. Sedlacek2, C. Schwarz3, K. Tintelnot1 ( 1 Mycotic and Parasitic Agents and Mycobacteria,
Robert Koch-Institut, Berlin, Germany, 2 Institut für Med. Mikrobiologie und Hygiene, Medizinische
Hochschule Hannover, Hannover, Germany, 3 Christiane Herzog-Zentrum/Mukoviszidose, Charité –
Universitätsmedizin, Berlin, Germany) Multilocus sequence typing of follow up Scedosporium isolates
from cystic fibrosis patients
09.40-10.00
Wieland Meyer (Molecular Mycology Research Laboratory, Sydney Medical School, Westmead Hospital,
Sydney, Australia): An update on the Scedosporium aurantiacum MLST analysis.
10.00-10.20
Lucia Kraková, Domenico Pangallo, Mária Majorošová, Elena Piecková (Institute of Molecular Biology,
Slovak Academy of Sciences, Bratislava, Medical Faculty, Slovak Medical University, Bratislava, Slovaki):
Discriminative potential of different PCR-based methods at Scedosporium strains
10.20-11.10 Coffee break
Session 6 Chairs: Ayse Kalkanci, Wieland Meyer
11.10-11.30
Min Chen, Shuwen Deng, Sybren de Hoog (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands;
Shanghai Key Laboratory of Molecular Medical Mycology, Department of Dermatology, Shanghai
Changzheng Hospital, Second Military Medical University, Shanghai, China): Loop-mediated isothermal
amplification as a diagnostic technique.
5
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Programme
11.30-11.50
Benjamin Stielow, Dea Garcia-Hermoso (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands
and National Reference Center of Invasive Mycosis and Antifungals, Institute Pasteur, Paris): DNA
barcoding to resolve Scedosporium apiospermum.
11.50-12.10
J. Rainer1 & J. Kaltseis2 (1Institute of Microbioology, Leopold-Franzens-University Innsbruck, Austria;
2
Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Austria): Can diesel fuel
tolerance and utilization by Scedosporium species be used as barcoding tool?
12.10-12.30
Walter Buzina, Anne Heinrich, Erich Leitner (Laboratory for Medical Mycology, Institute of Hygiene,
Medical University Graz, Austria): VOC spectrum of Scedosporium species.
12.30-13.30 Lunch
Session 7 Chairs: Cornelia Lass-Flörl, Ferry Hagen
13.30-13.50
Tamas Papp, Gabor Nagy, Anita Farkas, Arpad Csernetics, Csaba Vagvolgyi (University of Szeged, Faculty
of Science and Informatics, Department of Microbiology, Szeged, Hungary): Transcription analysis of
certain genes of Scedosporium (Pseudallescheria) isolates.
13.50-14.10
Karolina Dukik, Benjamin Stielow, Sybren de Hoog, Mariceli Araujo, Yvonne Graeser (CBS-KNAW Fungal
Biodiversity Centre, Utrecht, The Netherlands, and Institute for Microbiology Charite, Humboldt University,
Berlin, Germany): Towards a multilocus phylogeny of dermatophytes.
14.10-14.30
José F. Cano-Lira, Alberto M. Stchigel, Dania García and Josep Guarro ( Mycology Unit, University Rovira i
Virgili and IISPV, C/ Sant LLorenç 21, 43201 Reus, Spain): Resolving the phylogeny of chrysosporia
14.30-14.50
Yu Zhang, Ferry Hagen, Benjamin Stielow, Sybren de Hoog (Tianjin Academy of Traditional Chinese Medicine
Affiliated Hospital, Tianjin, China; CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands;
Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen,
The Netherlands): Historical biogeography and evolutionary patterns in human- and animal-pathogenic
Sporothrix species
14.50-15.20 Tea break
6
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Programme
Session 8 Chairs: Tamas Papp, Ana Alastruey Izquierdo
15.20-15.40
László Kredics, András Szekeres, Lóránt Hatvani, László Manczinger, Csaba Vágvölgyi (Department of
Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary): Orphan fungal
infections caused by members of the genus Trichoderma.
15.40-16.00
Ana Alastruey Izquierdo (National Centre for Microbiology, ISCIII, Madrid, Spain): Antifungal susceptibility
profile of Scedosporium spp.
16.00-16.20
Wieland Meyer (Molecular Mycology Research Laboratory, Sydney Medical School, Westmead Hospital,
Sydney, Australia): Genome analysis of Scedosporium aurantiacum.
16.20-16.40
Anne D. van Diepeningen (CBS-KNAW Fungal Biodiversity centre, Utrecht, The Netherlands): DNA and
Protein based diagnostics of the Fusarium fujikuroi species complex.
17.30 Dinner at CBS
Accreditation
An application has been made to the EACCME for CME accreditation of this event.
7
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Maldi-tof as an additional tool for strain characterisation in the
BCCM/IHEM culture collection
Dirk Stubbe, Marijke Hendrickx, Sam Roesems
BCCM/IHEM Culture Collection of Biomedical Fungi and Yeasts, Service of Mycology and Aerobiology, Scientific Institute of Public Health, Brussels, Belgium
M
ALDI-TOF MS has been shown to be a rapid, reliable and straightforward tool that has recently
revolutionised the identification of micro-organisms. It is based on the comparison of species
specific protein spectra to a reference spectra database.It has already shown his use for the
identification of bacteria and yeasts, and also for filamentous fungi, more and more publications
underpin its value. The drawback for the use of MALDI-TOF MS for the identification of filamentous fungi is the
need for a standardised sample preparation method and a good and elaborate reference spectra database.
Together with the CHU Marseille, the BCCM/IHEM collection constructed a reference spectra database
using a standardised approach and using reference strains from the BCCM/IHEM collection. The database
consists of over 1000 strains, covering about 550 species and about 150 different genera. Meanwhile we
have challenged the database with clinical isolates coming from different hospitals in France and Belgium to
assess its performance. The results of these validation studies will be briefly presented. The 9 strains that we
obtained in the frame of this workshop were analysed with our MALDI-TOF MS approach and the results will
be presented here. Focus will be on the practical approach, the quality controls and validation we took into
account, the limitations of this technique and how it is implemented in our collection.
8
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Pseudallescheria and Scedosporium species differentiation from agar cultures
by MALDI-TOF Mass Spectrometry
Valérie Monnin1, Blandine Ducarre1 , David Pincus2and Victoria Girard1,
bioMérieux, R&D Microbiology, Route de Port Michaud, 38390 La Balme Les Grottes, France
2
bioMérieux Inc., R&D Microbiology, 595 Anglum Road, Hazelwood, MO
1
V
ITEK® MS is a MALDI-TOF (Matrix Assisted Laser Desorption Ionization – Time of Flight) mass
spectrometer for microbial identification. Sample preparation for moulds is more challenging than
for bacteria and yeasts. For moulds, a specific, rapid and safe inactivation / extraction method,
suitable for routine laboratory work, is used for spectrum acquisition. This protocol was set up on
the Master Class strain selection including Scedosporium aurantiacum (n=4), Pseudallescheria boydii (n=3),
P. angusta (n=1) and P. ellipsoidea (n=1) that were grown on three different solid media (Sabouraud dextrose
agar, Sabouraud dextrose agar with gentamicin and chloramphenicol, and potatoe dextrose agar) and tested
at two different incubation times (6 and 12days). Cluster analysis based on peaks similarity indicated a clear
discrimination between Scedosporium and Pseudallescheria, independently of the culture conditions. The
MALDI-TOF results showed a high percentage of similarity (> 65%), for spectra within the Pseudallescheria
complex, except for one strain of P. boydii (CBS 117410) which is out of the Pseudallescheria cluster.
9
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Approaches to intra-species typing using MALDI-TOF MS
Andreas Zautner, Claudia Folba, Mareike Bernhard, Michael Weig, Uwe Groß and Oliver Bader
I
Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, D-37075 Göttigen
ntra-species typing of fungi is used for the investigation of nosocomial outbreak situations and may be
of help to predict clinically relevant phenotypes. Common genetic typing methods such as MLST or RFLP,
however, require species-adapted protocols, are usually laborious and time consuming. We investigated
different MALDI-TOF MS methods for typing, including clustering of mass spectra or selected biomarker
masses.
A set of potentially outbreak-associated Aspergillus flavus as well as two sets of previously MLST-typed
Candida albicans and C. glabrata isolates were used to determine the degree of concordance between conventional typing and cluster analysis from mass spectra. Indeed, clustering of the mass spectra showed the
outbreak-associated A. flavus isolates as a distinct cluster, well separated from unrelated control isolates.
The MALDI-derived clusters were in congruence with PCR typing results and suggested a colonization of
the patients originating from the hospital environment. Similarly, clusters generated from C. albicans and C.
glabrata mass spectra showed a general overlap with MLST-typing.
Searching for biomarkers with intra-species typing capability is more difficult and necessitates optimization
of extraction methods.
Our studies indicate the usefulness of MALDI-TOF MS for phyloproteomic typing. Since the production of
spectra is an organism independent method, typing by MALDI-TOF MS may also prove useful for other species, given that sufficient independent control isolates are available.
10
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Genotyping and susceptibility testing with inhalative agents
P.-M. Rath, J. Steinmann
I
Institute of Medical Microbiology, University Hospital, Essen, Germany
n this study, we characterized the nine Scedosporium/Pseudallescheria strains by semiautomated
repetitive sequence-based PCR and tested their susceptibility against colistin and epigallocatechin-3gallate (EGCG) in broth microdilution assays. In a previous work we could show that colistin has antifungal
activity against Scedosporium spp. It is used as inhalative agent against multidrug resistant gramnegative
bacteria, especially in patients with cystic fibrosis. EGCG is the main component of green tea and showed
antibacterial and antifungal activity.
DNA extraction, amplification, and interpretation of band patterns were performed according to the instructions of the manufacturer (bioMérieux, Nürtingen, Germany). Isolates with similarity of band pattern
of > 97% were considered as ´indistinguishable´. The susceptibility against colistin and EGCG was tested by
using broth microdilution assays according to CLSI standards.
In the rep-PCR all strains could be discriminated from each other when taking a < 98% agreement in the
band patterns as a basis for dissimilarity (Figure). However, the strains of the same species did not cluster
together. Testing the strains by microdilution with colistin revealed MICs in the range of 32 to > 128 mg/l.
EGCG showed no inhibition in clinically relevant concentrations as the MICs were > 1024 mg/l.
In conclusion, the rep-PCR can be used for genotyping of isolates of a given species, but seems not to be
applicable for identification of analyzed species. Colistin exhibits in vitro antifungal activity in a speciesspecific manner, whereas the first data indicate no relevant effects of EGCG against Scedosporium/Pseudallescheria spp.
Figure 1: Band patterns of the strains in the rep-PCR
11
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Routine identification of mold using MALDI TOF MS
Anne-Cécile Normand, Magali Gautier, Carole Cassagne, Coralie L’Ollivier, Stephane Ranque, Renaud Piarroux
C
Parasitology & Mycology, Assistance Publique-Hôpitaux de Marseille, CHU Timone-Adultes, Marseille, France
linical diagnosis of mold infections currently involves complex species identification based on
morphological criteria, which is often prone to errors. Employing an extensive mold species reference
spectrum library, we assessed the extent to which MALDI-TOF mass spectrometry (MALDI-TOF MS)
enhanced the accuracy of species identification.
MALDI-TOF MS data were initially validated against morphology- and DNA sequencing-based results. We
then compared the mold identification results obtained before and after the implementation of MALDI-TOF
MS in routine analyses, by subculturing 247 filamentous samples from 2011, applying the new protocol on
them and comparing with the results obtained initially. Finally, fungal species diversity, species identification accuracy and corresponding clinical characteristics were evaluated for 956 specimens collected in 2012.
When comparing the results from the first 4 months of 2011 with the same period in 2013, the implementation of MALDI-TOF MS displayed a dramatic improvement in mold identification at the species level (100%
vs. 64.6%, p<10-4), a significant decrease in result turnaround time (8.7 vs. 10.6 days, p<10-4) and a marked
reduction in the misidentification rate of morphologically similar species. Re-assessment of a set of isolates
from 2011 using MALDI-TOF MS yielded the accurate species-level identification of 241 out of 247 (97.6%)
mold and an increase in species diversity from 16 to 42 species. Applying MALDI-TOF MS in 2012 with the
latest MS database enabled the identification of 98.5% of the clinical mold isolates (109 species).
MALDI-TOF MS-based mold species identification proved to be markedly more accurate and efficient than
the classical microscopy method. This novel identification approach may soon challenge traditional techniques in the clinical laboratory, as patient prognosis is largely contingent on rapid and accurate diagnosis
and appropriate treatment.
12
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Antifungal Drugs: new approaches
Sharon Chen
T
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, ICPMR, Sydney, Australia
he need for new antifungal agents or novel ways of delivering antifungals is urgent given the increasing
drug resistance of many pathogenic fungi. Few new drugs have fulfilled their potential since the
echinocandins in the early 2000s. A limited number of newer agents are on the horizon. Importantly,
novel approaches and combination therapies using existing and/or new drugs are being explored.
New agents under study include isavuconazole, the 2nd generation “Mycograb”, enfumafungin (an oral
echinocandin) and miltefosine. The only one to hit the market has been isavuconazole. With superior PK/PD
properties to exisiting azoles. It is likely to be useful alternative antifungal for treating candidiasis, has potential for first line therapy of aspergillosis and has activity against mucormycetes. A new oral formulation of
posaconazole and the long-awaited IV formulation are likely to fill the need to enable more efficacious drug
levels in seriously ill patients. Elegant studies show that posaconazole is contained in host cell membranes
and thereafter transferred to fungal cell membranes. This has implications for dosing strategies, options for
cellular loading and therapeutic drug monitoring. Combination antifungal therapies are increasing used –
with existing or novel agents functioning as synergents or chemosensitisers. Strategies employing agents
such as miltefosine and iron chelators have been studied with varying success in animal models of infection.
Other novel agents include amiodarone, and photodynamic agents.
In the absence of new antifungal agents, it is essential to explore strategies to maximize the therapeutic
potential of existing agents.
13
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Susceptibility of Scedosporium species against plant extracts from neophytes
Heinrich Anne, 2Rehorska René, 2Pfeifhofer Hartwig Wilfried, 2Jamnig Julia, 2Mueller Maria, 1Buzina Walter
1
Institute Of Hygiene, Microbiology And Environmental Medicine, Medical University Of Graz, 2Institute Of Plant Sciences, University Of Graz, Austria
1
B
ackground: Invasive plant species (neophytes) represent a major threat to the native flora due to their
growth characteristics and regenerative capabilities. Moreover, there is evidence that some of these
species produce potentially herbicidal and fungicidal/antimycotic compounds, which may influence
the growth of native plants and fungi, leading to competitive advantages of invasive plant species.
Objective of this study was to investigate compounds of neophytes and other plants for their antimycotic
activities against species of the Scedosporium/Pseudallescheria species complex.
Material and methods: The susceptibilities were tested with microdilution method. Resveratrol,
emodin, quercetin and physcion (secondary metabolites of Fallopia japonica, Japanese knotweed),
2-methoxy-1,4-naphthoquinone (of Impatiens glandulifera, Himalayan Balsam or Kiss-me-on-themountain), juglon and salicylic acid were chosen to study the fungicidal/antimycotic effects against
9 different strains of Scedosporium/Pseudallescheria species: P. angusta (CBS 254.72), P. boydii (CBS
117410), P. boydii (CBS 117432), P. boydii (CBS 120157), P. ellipsoidea (CBS 301.79), S. aurantiacum (CBS
116910), S. aurantiacum (CBS 136046), S. aurantiacum (CBS 136047), S. aurantiacum (CBS 136049).
The substances were dissolved in RPMI-solution and 5 serial dilutions (1:10) were made starting at 1 mg/
mL concentration for resveratrol and salicylic acid and at 0.1 mg/mL for the others due to solubility reasons.
RPMI-suspensions were made from the fungal strains corresponding to a McFarland standard of 0.5, containing approximately 5*106 conidia/mL. 100 µL suspension and 100 µL test solution were pipetted into a
microtiter plate. After incubation for 72 hours at 35°C the plates were controlled for turbidity showing fungal
growth.
Results: Strain P. boydii (CBS 117432) showed no growth in the growth control and was therefore excluded.
Resveratrol, quercetin, physcion and emodin were not able to inhibit fungal growth at all concentrations tested.
Salicylic acid had an MIC (minimal inhibitory concentration) of 1 mg/mL for all strains tested.
Juglon and 2-methoxy-1,4-naphthoquinone showed a different inhibition pattern depending on the
fungal strain: For all strains juglon had an MIC of 0.1 mg/mL except for the strains P. boydii (CBS
120157), P. ellipsoidea (CBS 301.79) and P. boydii (CBS 117410), which showed an MIC of 0.01 mg/mL.
2-methoxy-1,4-naphthoquinone showed an MIC of 0.1 mg/mL for all strains except for P. boydii (CBS 117410), P. boydii (CBS 120157), P. ellipsoidea (CBS 301.79) and S. aurantiacum (CBS 116910) which showed an MIC of 0.01 mg/mL.
For the antifungals voriconazole the strains showed an MIC of 0.047-0.25, for posaconazole the range differed from 0.75 (CBS 301.79) to >32 (CBS 136047).
Discussion: Despite some reports of candicidal effects of resveratrol, in our study the fungicidal effects of
the tested substances against Scedosporium/ Pseudallescheria were rather low. The differences within the
tested strains in susceptibility were not over one dilution step. The tested substances are neither able to differentiate between Scedosporium/Pseudallescheria species nor possible agents for treatment of infections
caused by these fungi.
14
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Azole-echinocandin combination a potential therapeutic option against
scedosporiosis - new in vitro and vivo study insights and experiences from case studies
Fabiola Fernández1, Josep Guarro2, Cornelia Lass–Flörl3, Michaela Lackner3*
Unitat d´Anatomia Patològica, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus 2Unitat d´ Microbiologia, Facultat de Medicina i
Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus 3Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria.
1
O
bjective Due to the therapy refractory nature of Scedosporium infections and the high number of
therapeutic failures, new treatment options are desperately needed to control this life-threatening
disease. The aim of this study was to investigate the efficacy of azole-echinocandin combination
therapy against systemic Scedosporium infections.
Methods The in vitro activity and in vivo efficacy of the combinations (a) micafungin (MFG)/posaconazole
(PSC) and (b) MFG/voriconazole (VRC) were evaluated against S. boydii and S. apiospermum; as these species are predominant in temperate climate zones. Both combinations were in vitro tested against S. boydii
(n = 17) and S. apiospermum (n = 26) using checkerboard method according to CLSI M38-A2 guidelines. As
characteristic measure for in vitro efficacy, the fractional inhibitory concentration index (FICI) was defined.
The in vivo efficacy against four isolates of the Scedosporium apiospermum complex were evaluated in a
well-established, immunocompromised, haematogenous murine model of systemic scedosporiosis. Measurement categories were (a) prolongation of survival and (b) reduction of tissue burden in kidneys and brain.
Results Most isolates, regardless of species-affiliation, showed for both azole-echinocandin combinations
in vitro ‘no interaction’ (FICI >0.5 - 4.0). In vitro ‘synergism’ and ‘antagonism’ was seldom found. The FICI
failed to predict the in vivo outcome of both azole-echinocandin therapies. The in vivo efficacy of the combination therapies were strain-dependent, rather than species-dependent, even though effects were more
pronounced for both S. boydii strains. All mice receiving azole-echinocandin combination therapy had a significantly prolong survival, when compared with the untreated control and MFG monotherapy group. The
strongest reduction in brain and kidney fungal load was found for mice infected with S. boydii strains and
receiving an azole-echinocandin combination therapy.
Conclusion Based on our in vivo study results, azole-echinocandin combination therapy is promising, in
particular for patients that fail to improve during PSC or VRC monotherapy administration. In vitro test results (FICI), failed to predict in vivo outcome for combination therapy.
15
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Beyond the conventional antifungal agents: Antifungal activity of non-antifungal
drugs against Scedosporium and Pseudallescheria isolates
László Galgóczy1, Mónika Homa1, Csaba Vágvölgyi1, Tamás Papp1
T
University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Hungary
1
he incidence of fatal systemic, cerebral and lung infections caused by members of the genera
Scedosporium and Pseudallescheria has been increasing continuously during recent years especially
among the immunocompromised, cystic-fibrosis and near-drowning patients. Application of an
appropriate antifungal drug for the therapy is not simple in these cases because of the frequent
occurrence of antimycotic resistant strains, the possibility of drug interactions between the therapeutic agents
and the severe side effects of the clinically available antifungals. Therefore, there is a substantial demand
for new, safely applicable antifungal strategies for the treatment of Scedosporium and Pseudallescheria
infections. The non-antifungal drugs which have been introduced in the drug-market and have a secondary
antifungal effect and/or can interact synergistically with conventional antifungals are promising candidates
to treat different mycotic infections. Previously, it was observed that mucolytic agents which are applied as
respiratory medicines (e.g. N-acetyl-cysteine) and antipsychotic drugs which can easily penetrate the bloodbrain barrier and can accumulate in the central nervous system (e.g. chlorpromazine, trifloupherazine) could
be appropriate compounds in the treatment of lung and cerebral fungal infections, respectively.
In the present work the in vitro antifungal activities of suramin (an antiparasitic/antitumor agent), six mucolytic compounds (D-, L-cysteine, L-cysteine-methyl-ester (LCME), N-acetyl-cysteine (NAC), N-isobutyryl-Dcysteine and N-isobutyryl-L-cysteine), five antipsychotic drugs (chlorpromazine (CPZ), trifluopherazine (TFP),
amantadine hydrochloride, R-(-)-deprenyl hydrochloride (R-DEP) and valproic acid) and their combinations
with four conventional antifungal agents (voriconazole, amphotericin B, terbinafine and caspofungin) were
investigated in broth microdilution test against nine fungal isolates belonging to the genera Scedosporium
and Pseudallescheria. When used alone, LCME, NAC, CPZ, TFP and R-DEP exerted remarkable antifungal effects. In combinations with antifungal agents, LCME and NAC acted synergistically with all investigated antifungal drugs. Similar interaction was observed when CPZ or TFP was combined with amphotericin B or terbinafine. In all other cases indifferent interactions were revealed. After in vivo studies, these combinations
could be bases of effective, less toxic therapies for treatment of different Scedosporium and Pseudallescheria
infections.
The research of L.G., M.H. and Cs.V. was supported by the European Union and the State of Hungary, cofinanced by the European Social Fund in the framework of TÁMOP 4.2.4.A/2-11-1-2012-0001 ‘National Excellence Program’.
16
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Can secondary metabolites add significant taxonomic data in Scedosporium?
Jens C. Frisvad and Kristian F. Nielsen1
1 Department of Systems Biology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark, [email protected] & [email protected]
N
ine strains of Scedosporium and Pseudallescheria were inoculated on the media Czapek yeast
autolysate agar (CYA), Blakeslee malt extract agar (MEA), malt extract agar (Oxoid) (MEAOx), potato
dextrose agar (PDA), potato carrot agar (PCA), creatine sucrose agar (CREA), yeast extract sucrose
agar (YES) and Wickerhams antibiotic test medium (WATM) and examined for growth patterns after
7 days incubation at 25°C, and extracted for secondary metabolites and analyzed chemically after 15 days of
incubation. The extracts were analyzed by ultra high performance liquid chromatography (UHPLC)-qTOF (time
of flight) accurate mass and also by UHPLC-DAD (diode array detection). Secondary metabolitre production
was very dependent on the medium, and the best media for production of secondary metabolites were
MEA (Oxoid), PDA and YES. Pseurotin A and a metabolite related to altersetin were among the secondary
metabolites detected. Pseurotin A was produce by one strain of P. boydii (CBS 120157), one of P. angusta
(CBS 254.72) and one of P. ellipsoidea (CBS 301.79). Pseurotin A was earlier found in P. boydii IFM 4642
when grown on rice. Even though four isolates of S. aurantiaca were very similar macro-morphologically,
two produced few secondary metabolites (CBS 116910, CBS 136046), while two other produced different
metabolites, one of them in common with P. boydii and P. ellipsoidea. Even though chemotaxonomy show
some promise in these fungi, medium optimization and the use of more isolates is necessary to make
this kind of analysis efficient for identification. Some of the metabolites found or reported are common
with Aspergillus fumigatus: pseurotin A and gliotoxin, so this shows a functional relationship, rather than
a taxonomic relationship between these pathogenic fungi . A polyphasic approach to classification and
identification is recommendeds for these fungi. 17
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Evaluation of physiological parameters of Scedosporium and Pseudallescheria species
Dea Garcia-Hermoso1,2, Aude Sturny-Leclere1,2, Damien Hoinard1,2 , Alexandre Alanio1,2 & Françoise Dromer1,2
Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Paris, France 2 CNRS URA3012, Paris, France
1
W
e evaluated some physiological parameters for the different species of Scedosporium/
Pseudallescheria strains sent by the CBS. We initially applied the flow diagram of morphological
and molecular identification (purity check, slide culture, antifungal susceptibility testing,
sequencing, etc...) that we performed to every isolate arriving to the National Reference Center
of Invasive Mycoses and Antifungals (CNRMA). Different approaches to investigate the diversity of the strains
consisted on the examination of initial germination of the isolates and the monitoring of fungal growth over
time (using the recently acquired Bioscreen C Microbiology Reader (Labsystems Oy)) in presence or absence
of antifungal compounds. In addition, the interaction (phagocytosis, proliferation) between Scedosporium/
Pseudallescheria isolates and host cells was explored by adapting the in vitro model of yeast/macrophage
interaction, previously standardized in our laboratory (Alanio et al. mBio2011). Preliminary results will be
presented as well as the difficulties or pitfalls we encountered.
18
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Galleria mellonella as an alternative model to study Scedosporium-infections
Ulrike Binder, Cornelia Lass-Flörl and Michaela Lackner
I
Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Austria
1
nvasive fungal infections are increasing worldwide. A key tool to a better understanding of infections due
to opportunistic fungal pathogens, including Scedosporium 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 difference in virulence of the Scedopsorium-complex.
In preliminary assays we determined the suitable inoculum size and incubation temperature and evaluated
the model for its suitability to study Scedosporium infections. Similar to data obtained in other models, we
saw differences in virulence between strains of the same species. Furthermore, we aim to check for in vivo
fungal growth by histopathology, check for differences in haemocyte killing or differences in larval immune
response.
19
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Mini model of Scedosporium infection
Ayse Kalkanci1, Ali Adil Fouad1, Merve Erdogan1, Nevin Keskin2
1: Gazi University School of Medicine, Department of Medical Microbiology, Ankara, Turkey; 2: Hacettepe University, Faculty of Science, Department of Biology,
Ankara, Turkey
N
onmammalian model systems of infection such as Galleria mellonella have significant logistical and
ethical advantages over mammalian models since they are exact mini models. Larvae of wax month,
G. mellonella, is inexpensive to purchase and easy to handling even for inexperienced research
personel. G. mellonella were inoculated using Scedosporium aurantiacum and Pseudallescheria
boydii CBS reference strains. Twelve randomly selected larvae were inoculated with each isolate. The
larvae were inoculated by injecting 1 x 106 CFU per 10 microliter aliquot using a 10 μL Hamilton animal syringe.
Caterpillars were incubated at 30°C after infection and observed for 24 and 48 hours. Extent and rapidity of
larvae mortality were both analyzed by using the rates of survived and died larvae numbers. A saline injected
control group was also included into study. The outcome for the mini model was the rate of mortality of the G.
mellonella. In the G. mellonella model at 24 hours, S. aurantiacum isolates killed 25% of the larvae compared
with 16% mortality with larvae injected with P. boydii isolates. Survived, however seriously sick (nonmotile,
brown colored) larvae were sacrified at 48 hours of the study. Larvae infections were checked by the colony
counts on blood agar plates. Mortality rates at 24 hours were identified as the key determinant of virulence
in the wax moth larvae. G. mellonella infection model is suitable for assessing aspects of Scedosporium
virulence function.
20
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Ultrastructure of lateral cell walls, septa and septal pore apparatus in
the cells of vegetative mycelium of Pseudallescheria species
Stepanova A.A., Vasilyeva N.V.
I.I. Mechnikov North-Western State Medical University: Kashkin Research Institute of Medical Mycology, Santiago-de-Cuba 1/28, 194291, Saint Petersburg, Russia
T
he aim of the present work was a comparative ultrastructural analysis of the lateral cell walls, septa
and septal pore apparatus (SPA) of different Pseudallescheria genus species (P. ellipsoidea, P. angusta
and P. boydii), and determine the variability of this marker among different strains (CBS 117410,
117432, 120157) of a single species (P. boydii).
Materials and methods: Strains of P. ellipsoidea (CBS 301.79), P. angusta (CBS 254.72) and P. boydii (CBS
117410, 117432, 120157) were cultured in vitro on potato dextrose agar (20 days incubation at 28ºC). For
transmission electron microscopy the pieces of medium with different part of fungal colonies after 7 and 20
days of cultivation were fixed for 3 h in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide during 10
h. Samples were dehydrated through series of ethanol and acetone and embedded in epon-araldite epoxy
resin. Ultrathin sections were examined under a transmission electron microscope JEM-100 X II.
Result and discussion: The thickness of the lateral cell walls of aerial and submerged vegetative mycelium
was similar for analyzed Pseudallescheria species (Table). The cell walls thickness was identical 0.05 µm for
hyphal cells of P. ellipsoidea and P. angusta. For analyzed cells of mycelium this parameter varied from 0.04
to 0.06 µm. The cells of submerged mycelium differ from aerial by the presence the outer thick (0.30‒0. 33
µm) extracellular matrix. This layer was covered by tightly and random localized, dark microfibrils forming a
layer which was 6 time thicker than lateral cell walls. Hyphal cells of investigated species had thin (0.04‒0.06
µm), straight, light septa with central pore. The pore diameter varied from 0.11 to 0.16 µm. The SPA consisted of Woronin bodies (WB) acting as septal pore plugs, in all investigated species and large with irregular
satellites (P. boydii, strains CBS 117410, 117432). The WB is a membrane-bound component of SPA, and
the dense irregularly crystalline core possibly is proteinaceous in nature. The shape of WB was spherical in
P. boydii and ellipsoidal in P. ellipsoidea and P. angusta. The median diameter of WB was similar (0.22 µm)
in the hyphal cells of P. ellipsoidea and P. angusta, but varied from 0.17 to 0.22 µm in the same of different
strains of P. boydii. The diameter of WB was larger than the septal pore. It was interesting that in addition to
WB we revealed in the hyphal cells of all investigated Pseudallescheria species the presence special spherical median electron density crystalline-satellites without boundary membrane, of a diameter smaller than
that of WB (Table). Maximally two crystalline satellites on medial hyphal cells were found in P. boydii, while
in P. ellipsoidea and P. angusta the maximum was 3 and 4, respectively. The SPA of hyphal cells of two strains
(CBS 117410, 11743) P. boydii strains with respect to another 2 species posses with large irregular crystalline inclusions which, as a rule, localized near and inside septal pore. Repetitions, please describe crystalline
only once. Plugs were very frequent components of SPA of mature hyphal cells. They were large, dark, homogeneous, variable in form on median sections (spherical, irregular or rectangular), symmetrical or asymmetrical, completely or partially filling the septal pore. Very rare the thin dark hemispherical diaphragm was
revealed in septal pore of P. boydii (CBS 117432).
21
Species
Strains number (CBS)
Thickness of lateral cell walls of the aerial
and submerged hyphae, µm
Thickness of extra-cellular matrix of
submerged hyphae, µm
Septum thick-ness,
µm
Diame-ter of the septal pore,
µm
Woronin bodies diameter,
µm
The number of the Woronin bodies on
median section of septae
Diameter of crystallic satelitts
in septal pore apparatus,
µm
The num-ber of crystallic satellits
on median section of septae
Presence/ absence of shapeless crystallic
components in septal pore apparatus
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
P. ellipsoidea
301.79
0.05
0.31
0.06
0.13
0.22x0.19
2-Apr
0.15
1-May
-
P. angusta
254.72
0.05
0.32
0.05
0.11
0.22x0.19
2-Apr
0.13
1-Apr
-
P. boydii
117410
0.04
0.30
0.04
0.12
0.17
2-Mar
0.13
1-Feb
+
P. boydii
117432
0.06
0.33
0.04
0.16
0.20
2-Mar
0.15
1-Feb
+
P. boydii
120157
0.06
0.30
0.05
0.15
0.22
2-Mar
0.16
1-Feb
-
22
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Identification of scedosporiosis and other fungal infections from FFPE tissue.
Kathrin Tintelnot, Anne Bernhardt
T
Robert Koch Institut, Berlin, Germany
he detection of hyphae in tissue unmistakably suggests a deep mycosis, but micromorphology
alone rarely allows a specific diagnosis. Thus confusion is possible between Aspergillus, Fusarium,
Scedosporium species and several other fungal agents. If broad, nearly unseptated hyphae are
detected histologically, a mucormycosis can be suspected. Because therapeutic strategies may differ
depending on the specific fungal agent, so in scedosporiosis, a suspected diagnosis should be supplemented
by other, e.g. molecular methods.
Since 2010 203 formalin fixed paraffin embedded (FFPE) tissue blocks (78 x veterinary samples, 125 x of
human origin) have been studied to identify the infectious fungal agent. The examined tissues included 52
cases of cats with proven subcutaneous mycosis, 24 cases of a retrospective study of cerebral fungal infections and routinely sent FFPE samples to confirm a fungal infection.
Methods: Three to five sections of 5 m were cut from FFPE tissue blocks for. DNA extraction and purification
were performed with the Maxwell® 16 Clinical Instrument. After amplification of the ITS2 region of rDNA and
sequencing the fungal pathogens were identified by data bank analysis (NCBI-BLAST®) and by a microarray, if
possible. If an imported systemic mycosis should be diagnosed, a nested PCR within the 18SrDNA region was
performed. Parts of the human β-globulin or the Canis familiaris interphotoreceptor retinoid-binding protein
(IRBP)-gene were amplified to document a successful DNA preparation.
Two Haematoxylin & Eosin (HE) and silver (Grocott) stained tissue sections (alpha and omega) were reexamined for histomorphological characteristics, especially the presence and form of yeast cells or hyphae
and the presence of melanin within the fungal walls.
Results: In total 132 / 203 samples (64%) it was possible to identify the infectious agent from FFPE tissue.
In most cases the molecular result correlated with the histological pattern. The detection rate of fungal DNA
was strongly dependent on the quality of the FFPE tissue; if a tissue was exposed to formalin for several days
or even weeks, like brain tissue after autopsy, the chance to identify the fungus by PCR was nearly zero. In
human samples the frequent identified pathogens were Candida species > Histoplasma capsulatum > Aspergillus species > Mucorales, in two patients a scedosporiosis was proven. In cats, especially in subcutaneous
infections, Alternaria was the predominant identified pathogen.
23
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Structural analysis of glucosylceramides (CMHs) from species of
the Pseudallescheria/Scedosporium complex
Calixto R.O.R.1 , Rollin-Pinheiro, R.1, da Silva M.I.D 1., Lopes, L.C.L.2, Vieira, J.M.1 , Sassaki, G.L.3, and Barreto-Bergter, E.1
1
Departamento de Microbiologia Geral, Instituto de Microbiologia, UFRJ, Rio de Janeiro, Brasil; 2 Departamento de Análises Clínicas e Toxicológicas , Faculdade
de Farmacia, UFRJ, Rio de Janeiro, Brasil; 3 Departamento de Bioquimica e Biologia Celular, UFPR, Paraná, Brasil
G
lucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal
cells. In this work, glucosylceramides (GlcCer) were extracted from three strains of Scedosporium
(Pseudallescheria) boydii and one strain of Pseudallescheria ellipsoidea and purified by several
chromatographic steps. By using high-performance thin layer chromatography (HPTLC) we
found a similarity between CMHs obtained from all the strains analyzed. A detailed structural analysis of
the P.ellipsoidea CMH was performed by electrospray ionization mass spectrometry (ESI-MS) and 1- and
2-D heteronuclear NMR experiments (1H-13 C HSQC) and the glucosylceramide was identified as N-2´hydroxyoctadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine. This CMH species produced by
mycelial forms of P.ellipsoidea, displayed the same structure previously demonstrated by our group for
P.boydii , Cryptococcus neoformans, P.minustipora, Fusarium solani and Colletotrichum gloesporioides [1]
A monoclonal antibody (Mab) against this molecule was used for immunofluorescence experiments. Our
preliminary results revealed that CMH is present on the surface of P.ellipsoidea and the distribution of GlcCer
in this strain is similar from those already described in S.apiospermum , S.aurantiacum and P.minutispora [2].
The surface distribution of GlcCer in P.ellipsoidea is suggestive of an involvement of this molecule in fungal
growth. Previous results from our group showed that GlcCer seem to be cell wall components targeted by
antifungal antibodies that directly inhibit fungal development and also enhance macrophage function [3].
Since this molecule is produced and conserved among fungal species, the use of monoclonal antibodies to
GlcCer alone or associated with antifungal drugs in antifungal immunotherapy should improve the outcome
of fungal infection diseases.
1. Barreto-Bergter et al., 2011. Front. Microbiol., 2:239
2. Vieira et al., 2013. Abstracts 4th International Workshop on Pseudallescheria/Scedosporium Infections,
pp 14-15
3. Nimrichter et al., 2005.Infect.Immun., 73: 7860
Supported by: CNPq, CAPES, FAPERJ, CEMBIO, UFRJ
24
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Towards a new taxonomy for the basidiomycetous yeasts and the
Cryptococcus neoformans and Cryptococcus gattii species complex in particular
Ferry Hagen1,2*, Kantarawee Khayhan1,3, Bart Theelen1, Anna Kolecka1, Itzhack Polacheck4, Rama Falk4,5, Sittiporn Parnmen6, H.
Thorsten Lumbsch6, Xin-Zhan Liu7, Qi-Ming Wang7, Salvador Capella1,8, Marizeth Groenewald1, Benjamin Stielow1, Saad TajAldeen9, Toni Gabaldon8, Feng-Yan Bai1,7 and Teun Boekhout1,7,10
1
CBS-KNAW Fungal Biodiversity Centre, Yeast Research, Utrecht, The Netherlands; 2 Department of Medical Microbiology and Infectious Diseases, CanisiusWilhelmina Hospital, Nijmegen, The Netherlands; 3 Department of Microbiology and Parasitology, Faculty of Medical Sciences, University of Phayao, Phayao,
Thailand; 4 Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, Ein Kerem, Jerusalem, Israel; 5 Department
of Fisheries and Aquaculture, Ministry of Agriculture and Rural Development, Nir-David, Israel; 6 Science & Education, The Field Museum, Chicago, IL, U.S.A.;
7
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China;8 Centro Regulacion Genomica, Barcelona, Spain; 9
Hamad Medical Corporation, Doha, Qatar ;10 Department of Dermatology, Shanghai Key Laboratory of Molecular Medical Mycology, Institute of Dermatology and
Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
M
any of the currently used genera of yeasts are highly polyphyletic and, consequently, the
taxonomy of yeasts needs to be completely revised. The following developments contribute
to this new taxonomy: 1. Improved phylogenetic signal in multigene-based phylogenies; 2.
The impact of phylogenomics; 3. The introduction of the one species-one name concept; and
3. Ongoing species discovery that add species to new or sparingly sampled lineages. Attempts in order to
make the classification of the yeasts more natural, i.e. in line with phylogenetic principles, are ongoing by
comparative multigene sequencing and whole genome sequencing of currently described yeast species. We
will show these developments by presenting data from tremellaceous yeasts.
In addition considerable genetic heterogeneity has been demonstrated to occur in the C. neoformans/C.
gattii species complex by a plethora of molecular methods. Phylogenetic analysis of 114 globally collected
isolates of the species complex was done using 11 nuclear loci CAP59, GPD1, IGS1, ITS, LAC1, PLB1, RPB1,
RPB2, SOD1, TEF1 and URA5. These molecular data were used to perform gene tree analyses in a maximum
likelihood (ML) and Bayesian (B/MCMC) framework and coalescent-based species trees. We employed a
combination of methods to address the species delimitation, using gene tree estimation from single-locus and concatenated data sets, and species tree estimations, including a genealogical species recognition
method in which presence of clades in the majority of single-locus genealogies is taken as evidence that
these represent distinct lineages. We also used the coalescent-based general mixed Yule coalescent (GMYC)
method, which aims at locating the nodes that define the transitions between intraspecific (tokogenetic)
and interspecific relationships using branch lengths. Identification of the new species using a test set of 425
isolates was tested by MALDI-TOF MS.
Phylogenetic analysis of 11 loci and various genotyping studies revealed significant genetic diversity with
the pathogenic C. neoformans – C. gattii basidiomycetous yeast species complex. Genealogical concordance,
cohesion-based, and species tree approaches all supported the presence of distinct lineages within the complex. The type strain of C. neoformans CBS 132 represents a serotype AD hybrid and needs to be replaced.
The ‘newly recognized’ species differ in pathogenicity, prevalence for patient groups, as well as biochemical
and physiological aspects, such as susceptibility to flucytosine and fluconazole antifungals. MALDI-TOF MS
proved to be a reliable and easy-to-use identification tool as no major errors were observed.
25
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Tissue from patients with cystic fibrosis by broadrange PCR and Fluorescence in
situ hybridisation (FISH)
Ilka McCormick Smith1, Ludwig Sedlacek2, Danny Jonigk3 , Volker Rickerts1
1
Robert Koch-Institut, Berlin, Germany 2 Medizinische Hochschule Hannover, Hannover, Germany 3Medizinische Hochschule Hannover, Hannover, Germany
B
ackground: Microbial communities including various fungi inhabit the respiratory tract of
patients with cystic fibrosis (CF). Scedosporium spp. can be cultivated from patients with CF.
They have been described as agents of invasive fungal infections in these patients, although
their clinical relevance is difficult to assess in patients colonized with mixed fungal species.
However, they are generally thought to be associated with a decline in respiratory function. We aimed
to develop specific FISH probes targeting the ribosomal RNA of Scedosporium in order to identify
and localize these fungi in formalin-fixed, paraffin-embedded (FFPE) tissue samples from CF patients.
Methods: Sequences of the 18S-, and 28S rRNA-genes of clinically important Scedosporium were generated by overlapping PCR amplicons. We selected sequences for areas which are conserved among Scedosporium that significantly differ from other fungi. Potentially useful probes were evaluated in silico for
specificity and physicochemical properties. Selected probes were synthesized and evaluated using germlings of target and non-target fungi in order to establish probe sensitivity and specificity in comparison
with the resulting fluorescence signal of a positive control (EUK516) and a nonsense control (nonEUB).
Selected probes were further evaluated using FFPE tissue samples of explanted lungs from patients undergoing lung transplantation with and without culture confirmed colonisation with Scedosporium.
The presence of fungal DNA in the samples was confirmed by broadrange PCR assays and sequencing.
Results: Sequences of the 18S and 28S rRNA genes were generated for 8 Scedosporium strains. Loci able
to discriminate between Scedosporium and other pathogenic fungi were identified in both genes. Our experiments documented the specific hybridization with target fungi yielding a fluorescence signal comparable to the positive control probe. In some probes, hybridization was also detected with Fusarium spp.
but not with typical respiratory pathogens such as Aspergillus. Broadrange PCR of tissue samples amplified Scedosporium DNA from one sample from a patient with proven invasive fungal infection but not
from two patients colonized with Scedosporium and three patients without documented colonisation. Hybridization experiments using our probes and additional tissue samples are underway to evaluate if the
newly designed FISH probes allow for the reliable identification and localization of Scedosporium in tissue.
Conclusions: Fluorescently labeled probes targeting the ribosomal RNA Gene of Scedosporium can be designed to differentiate these molds from other fungi. Evaluation of the probes with additional tissue samples
are underway in order to verify their potential to differentiate Scedosporium from other fungi and localize
them within human tissue in a diagnostic setting.
26
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Multilocus sequence typing of follow up Scedosporium isolates from
cystic fibrosis patients
A. Bernhardt1, L. Sedlacek2, C. Schwarz3, K. Tintelnot1
1
Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institut, Berlin, Germany, 2 Institut für Med. Mikrobiologie und Hygiene, Medizinische Hochschule
Hannover, Hannover, Germany, 3 Christiane Herzog-Zentrum/Mukoviszidose, Charité – Universitätsmedizin, Berlin, Germany
S
cedosporium species are the second most common lung-colonizing fungi in cystic fibrosis (CF) patients.
For epidemiological reasons it is important to trace sources of infection and routes of transmission
and to determine whether these fungi are transient or permanent colonizers of the respiratory tract.
Molecular typing methods like multilocus sequence typing (MLST) help provide this data. Clinical
isolates of S. apiospermum and P. boydii isolates from CF patients in different regions of Germany were
studied using MLST. Five gene loci, ACT, CAL, RPB2, BT2 and SOD2, were analysed. The S. apiospermum
isolates from 53 patients were assigned to 44 sequence types (ST), and the P. boydii isolates from 32 patients
to 12 ST. The results revealed that patients can be colonized by individual strains for years. The MLST scheme
developed for S. apiospermum and P. boydii is a highly effective tool for epidemiologic studies worldwide.
27
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
An update on the Scedosporium aurantiacum MLST analysis
Meyer W1, Schwabenbauer K1, Harun A1,2 & Firacative C1
1
Molecular Mycology Research Laboratory, CIDM, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School-Westmead Hospital,
University of Sydney, Westmead Millennium Institute, Westmead, NSW, Australia; 2School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Kelantan,
Malaysia.
S
cedosporium spp. are amongst the most important fungal infections for patients with cystic fibrosis
(CF). Invasive Scedosporium infections are also problematic after near-drowning accidents and in
transplant patients. Scedosporium aurantiacum is an emerging human pathogen causing a wide
range of invasive infections. To identify possible environmental sources of infections and to determine
the spread of this emerging pathogen a better understanding of its epidemiology is required. A total of 188
clinical, environmental and veterinary S. aurantiacum isolates from Australia, Europe, Asia and the America
were characterised by multilocus sequence typing (MLST). The scheme is based on the combined sequence
analysis of six genes: actin (ACT), calmodulin (CAL), elogation factor-1a (EF1a), RNA polymerase subunit II
(RPB2), manganese superoxide dismutase (SOD2), and b-tubulin (TUB) at: mlst.mycologylab.org. Among this
geographically diverse collection 21 ACT, 8 CAL, 23 EF1a, 13 RPB2, 18 SOD2 and 12 TUB alleles resulting in
159 unique sequence types were identified. Allele and sequence types can be determined online at http://
mlst.mycologylab.org. Phylogenetic analysis revealed separate clustering of the Australian and European
samples. The high diversity among the Australian strains suggests that S. aurantiacum may have originated
within Australia and was subsequently dispersed to other regions. A fact revealed by the close phylogenetic
relationships between some of the Australian sequence types and those found in other parts of the world.
28
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Discriminative potential of different PCR-based methods at Scedosporium strains
Lucia Kraková*, Domenico Pangallo*, Mária Majorošová, Elena Piecková
T
*Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Medical Faculty, Slovak Medical University, Bratislava, Slovakia
hree different PCR-based methods were evaluated in order to discriminate 9 strains of Scedosporium.
The ITS PCR (f-ITS) by primers ITS3 (GCA TCG ATG AAG AAC GCA GC) and ITS4-FAM (6-carboxyfluorescein;
6-FAM e TCC TCC GCT TAT TGA TAT GC) were combined with capillary electrophoresis to analyze the
amplified PCR products.
Denaturing gradient gel electrophoresis (PCR-DGGE) was used for analysis of the PCR products of the
strains obtained by the primers ITS1f-GC (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG
GTC CGT AGG TGA ACC TGC GG-3′) and ITS2 (5′-GCT GCG TTC TTC ATC GAT GC-3′). In addition, another PCR
assay (f-CBH) oriented to cellobiohydrolase gene (primers CbH-fw-FAM – TCG AYG CSA ACT GGC GCT GG;
CbH-rv – TTG GCY TCC CAG AYA TCC ATC TC) was also performed and the PCR products were separated by
the same capillary electrophoresis.
The f-ITS PCR was able to cluster the strains in different groups on the basis of f-ITS profile, the first group
was composed by Pseudallecheria ellipsoidea and two P. boydii strains (CBS 117432 and CBS 120157). Another group was formed by all of the four Scedosporium aurantiacum strains. While the strains P. angusta
and P. boydii CBS 117410 showed different f-ITS profiles.
By PCR-DGGE the strains were grouped too but in a different order: i) P. angusta and P. ellipsoidea, ii) P.
boydii strains CBS 117432 and CBS 120157; iii) S. aurantiacum strains CBS 136046 and CBS 136047, and iv)
the other two S. aurantiacum strains CBS 136049 and CBS 116910. The strain P. boydii CBS 117410 displayed
a specific DGGE profile.
The polymorphism of the f-CBH showed a great heterogeneity among the analyzed strains and it was difficult to cluster them, except of S. aurantiacum CBS 136046 and CBS 116910 with almost the identical profiles.
PCR epidemiological methods of RAMP, ERIC, REP, and PCR-BOX are tested on the strains recently.
29
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Loop-mediated isothermal amplification as a diagnostic technique
Min Chen, Shuwen Deng, Sybren de Hoog
CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; Shanghai Key Laboratory of Molecular Medical Mycology, Department of Dermatology,
Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China): Loop-mediated isothermal amplification as a diagnostic technique.
L
oop-Mediated Isothermal DNA amplification (LAMP) is an isothermal detection method which was
invented a decade ago, and has since widely been applied in laboratory settings to medical pathogens.
The advantages of LAMP, such as rapidity, specificity and sensitivity make it a potential diagnostic
platform that could meet requirements of diagnostic mycology. The method has been commercialized
with several kinds of kits for clinical pathogens such as tuberculosis and the hepatitis B virus. Two major
formats of LAMP are therefore possible: one using the commercial LAMP DNA Amplification Kit, and
another using raw reagents. We successively introduced a LAMP assays for identification of all members
of the Cryptococcus neoformans / C. gattii species complex by the commercial kit. For Cladophialophora
carrionii we used raw reagents. In our study, results of specificity and sensitivity from both the commercial
kit and raw reagents are good, but less unproblematic as generally supposed. Using raw reagents careful
optimization is necessary for every individual application; large differences exist between published reports.
It is recommended to detect LAMP products using a Loopamp real-time turbidity meter or adding calcein
before amplification in order to prevent aerosol contamination which easily leads to false-positives results.
In our opinion, LAMP is a useful diagnostic platform only under strict operational conditions and a carefully
optimized reaction system.
30
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
DNA barcoding to resolve Scedosporium apiospermum
Dea Garcia Hermoso1 and J. Benjamin Stielow2
National Reference Center of Invasive Mycosis and Antifungals, Molecular Mycology Unit, Institute Pasteur, Paris, France, 2CBS-KNAW Fungal Biodiversity Centre,
Utrecht, The Netherlands
1
T
he Scedosporium apiospermum / boydii complex represents the clinically most relevant Scedosporium
taxa, and both species are assumed to represent two distinct evolutionary lineages. Within S.
apiospermum significant genetic variability has been detected (de Hoog et al. unpublished data), and
thus it has been argued whether to accept more than one distinct taxon or the opposite. Produced
as a sub dataset within the framework of the Embarc project (Joint research of the European culture
collections), ‘Set of new genes to use in addition or replacement of ribosomal sequences’, 11 distinct gene
sections were sequenced and analyzed for 30 strains of the Scedosporium apiospermum / boydii complex
isolated at Institute Pasteur, France. We employed an analysis strategy using the state of the art R package
‘Spider’ based on ‘Ape’ to investigate genetic diversity within the S. apiospermum / boydii complex. Our
results confirm S. apiospermum as a single taxon.
31
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Can diesel fuel tolerance and utilization by Scedosporium species be used as
barcoding tool?
J. Rainer1 & J. Kaltseis2
Institute of Microbioology, Leopold-Franzens-University Innsbruck, Austria; 2Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Austria
1
S
trains of the genus Scedosporium are known to inhabit environments strongly influenced by humans.
Artificially enrichment with nutrients like nitrogen compounds, elevated temperature and increased
concentration of hydrocarbons are the main characteristics of these soils. Hydrocarbon entry in many
cases is due to industrial sources, pest control agents and modern mobility. Soil adjacent to streets
and petrol-stations had been described to be more or less infested with hydrocarbons.
Several authors described the ability of microbial communities to degrade aliphatic and aromatic hydrocarbons, but without referring to special strains or species. Scedosporium spp. had been proven to metabolize
some forms of alkanes, phenoles and dioxins. A hydrocarbon containing medium was shown to have diagnostic value for clades in S. dehoogii (ref. to S. deficiens). This concept was extended to the standard set of
strains spread previous to the workshop. At first the diesel tolerance of the set strains was tested in complex
media. Secondly the utilization of diesel as sole carbon source was tested in different concentrations on solid
as well as in liquid media.
In this presentation we outline the correlation of diesel tolerance and utilization to certain strains or taxonomic entities together with the concept of barcoding. Possible accordance with phylogenetic analyses and
ecological conditions will be discussed.
32
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
VOC spectrum of Scedosporium species
HEINRICH Anne, 2LEITNER Erich, 1KÖLLI Bettina, 1BUZINA Walter
1
1Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 2 Institute of Analytical Chemistry and Food Chemistry at the Graz
University of Technology, Austria
B
ackground: All living organisms produce secondary metabolites. Some of them are volatile; in the
case of microorganisms they are called microbial volatile organic compounds (MVOC). Recently
it was shown that bacteria and some fungi (i.e. Aspergillus fumigatus, Candida albicans) produce
characteristic spectra of MVOC, which can be detected by means of gas chromatography and mass
spectroscopy (GC-MS). The aim of our study was to find suitable media and to examine species of the
Scedosporium/Pseudallescheria species complex for characteristic GC-MS profiles.
Material and methods: Nine different strains of Scedosporium/Pseudallescheria species: P. angusta (CBS
254.72), P. boydii (CBS 117410), P. boydii (CBS 117432), P. boydii (CBS 120157), P. ellipsoidea (CBS 301.79), S.
aurantiacum (CBS 116910), S. aurantiacum (CBS 136046), S. aurantiacum (CBS 136047), S. aurantiacum (CBS
136049) were cultured in 20-mL headspace vials filled with 5 mL of Sabouraud agar (SAB) at a slant. After
inoculation of the microorganisms the vials were closed with a screw seal and incubated at 37°C for 3-5 days.
Enrichment of the volatile fraction was done by using Solid Phase Microextraction (SPME) directly in the
head space of the isolated cultures. For identification of VOC mainly gas chromatography coupled to mass
spectrometry (GC-MS, single quadrupole, triple quadrupole and GCxGC) was be used. For differentiation and
classification chemometric methods based on principal component analysis (PCA) was applied.
Results: Of the five tested media (SAB, malt extract, CandidaID (BioMérieux), DG-18 and SceSel+) SAB
and SceSel+ showed the highest difference in GC-MS spectra between the medium and the grown fungi.
Specific MVOCs were produced by all four species which grouped the different species in distinct clades by
principal component analysis. These characteristic MVOC comprised 3-octanone, farnesene, 2-pinene, acetoin and others.
Discussion: The analysis of MVOC produced by Scedosporium/Pseudallescheria species may be a promising tool for the detection and differentiation of or between these species.
33
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Transcription analysis of certain genes of Scedosporium (Pseudallescheria) isolates
Tamás Papp, Gábor Nagy, Anita Farkas, Árpád Csernetics, Csaba Vágvölgyi
P
University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Szeged, Hungary
seudallescheria boydii (anamorph Scedosporium apiospermum) is a significant opportunistic pathogen
with a worldwide distribution that can cause various and frequently fatal infections in humans. It is
notorious for its high antifungal-resistance and difficult diagnosis. Analysis of the molecular and the
genetic background of pathogenicity can help to identify new targets for antifungal therapy and
to understand the emerging and spreading of the infection. Unfortunately, possible virulence factors of
this fungal pathogen are poorly characterized and the genetic background of its pathogenicity is almost
completely unknown. At the same time, Pseudallescheria/Scedosporium species have several interesting
ecological and physiological characteristics, such as environmental distribuiton, heat tolerance varying
among the different species and strains, salt tolerance and their ability to growth under poorly aerated or
anaerobic conditions (1).
HMG-CoA reductase is a key enzyme of the mevalonate pathway and its activity highly affects the ergosterol biosynthesis and the formation of the prenyl groups of different signal and regulator proteins. The
enzyme therefore affects many biological processes including morphogenesis, membrane fluidity, synthesis
of different metabolites or adaptation to environmental changes (e.g. changes in the oxygen concentration
or salinity of the medium).
In this study, a fragment of the HMG-CoA reductase gene was amplified using degenerated primers from
the genomic DNA of one P. angusta and P. ellipsoidea, three P. boydii and four S. aurantiacum strains. Sequences of the resulting fragments were determined and used to design primers for quantitative real-time
PCR analysis. Using these primers, relative transcript levels of the HMG-CoA reductase gene was analysed
after cultivating the fungi under various conditions, such as at different temperatures, salt concentrations
and oxygen tensions. To normalise the data, the actin gene, which was also amplified from each strains, was
used. Differences among the strains and the effect of the cultivation conditions on the transcript levels will
be discussed.
This research was supported by the grant OTKA NN106394. GN, ÁC and CV were supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP
4.2.4.A/2-11-1-2012-0001 ‹National Excellence Program›.
1. Guarro et al. 2006. Medical Mycology 44, 295-327.
34
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Towards a multilocus phylogeny of dermatophytes
Karolina Dukik1, Benjamin Stielow1, Sybren de Hoog1, Mariceli Araujo2, Yvonne Graeser3
CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, 2Department of Pathology, Federal University of Espírito Santo, Vitória, Brazil, 3Institute for
Microbiology Charité Berlin, Humboldt University, Berlin, Germany
1
T
he dermatophytes form a separate monophyletic group of closely related fungal taxa in the
order Onygenales. Their taxonomy has been refined multiple times over the last decades, often
being conflicting, when different methodologies were applied to resolve their morphological and
phylogenetic relationships. For example, methods based on morphology and/or reproductive
behavior, when compared with genetic data, are partly in conflict with conventional and clinical data.
Morphologically clearly defined species may be indistinguishable in molecular phylogenetic analyses, and vice
versa. Phylogenetic inference based on different gene sections often results in incongruent tree topologies.
Uncertainty about species limits on the basis of single gene phylogenies can be resolved by simultaneous
analysis of several gene genealogies. Most molecular studies investigating the dermatophytes were based
on a single or in some cases on up to four gene sections. We here present multilocus sequencing data of
9 genes (11 sections) and a comprehensive analysis was performed using the R package ‘Spider’ based on
‘Ape’, with the aim to determine the optimal biomarker for detection and species delimitation in the fungi
causing dermatomycosis.
35
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Resolving the phylogeny of chrysosporia
José F. Cano-Lira, Alberto M. Stchigel, Dania García and Josep Guarro
C
Mycology Unit, University Rovira i Virgili and IISPV, C/ Sant LLorenç 21, 43201 Reus, Spain
hrysosporium Corda is an anamorphic genus that comprises a broad number of ubiquitous, mostly
soil-borne keratinophilic species (ca. 70) which are responsible of occasional opportunistic infections
in humans. The reproductive structures of these fungi are very poorly differentiated. They produce
unicellular, sessile, solitary and hyaline conidia that usually born laterally or terminally from the
vegetative hyphae. Currently, Chryssosporium is considered a polyphyletic genus, with representatives
in phylogenetically distant orders, i.e.Onygenales, Sordariales,.. The molecular boundaries among
Chrysosporium and related genera such as Geomyces, Malbranchea, Trichophyton, etc have not been
established and their taxonomy needs further studies. Vidal et al (2000), on the basis of ITSs sequences,
concluded that most of the species of Chysosporium are distributed in nine well supported clades, grouped
according the morphology of their asexual and sexual forms. However, the taxonomic position of several taxa
(C. merdarium, Nannizziopsis vriesii or C. pilosum) remained unsolved. We have revised and reanalyzed the
most recent data on Chrysosporium and relatives (Gargas et al., 2009; Pettersson et al., 2011; Minnis and
Lindner, 2013; Pitt et al., 2013; Sigler et al., 2013 and Stchigel et al., 2013) in order to clarify and propose a
modern circumscription of these fungi.
REFERENCES
Gargas, A.; Trest, M.T.; Christensen, M; Volk, T.J. and Blehert, D.S. (2009). Geomyces destructans sp nov associated with bat white-nose
syndrome. MYCOTAXON, 108: 147-154.
Minnis, A.M. and Lindner, D.L. (2013). Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus
destructans, comb. nov., in bat hibernacula of eastern North America. FUNGAL BIOLOGY, 117: 638-649.
Pettersson, O.V.; Leong, S.L.L.; Lantz, H.; Rice, T.; Dijksterhuis, J.; Houbraken, J.; Samson, R.A.; Schnurer, J. (2011). Phylogeny and
intraspecific variation of the extreme xerophile, Xeromyces bisporus. FUNGAL BIOLOGY, 115: 1100-1111.
Pitt, J. I.; Lantz, H. Petterson, O.V. and Leong S. L. (2013). Xerochrysium gen. nov. and Bettsia, genera encompassing xerophilic species of
Chrysosporium. IMA FUNGUS, 4: 229-241.
Sigler, L.; Hambleton, S.; Pare, J. A. (2013). Molecular Characterization of Reptile Pathogens Currently Known as Members of the
Chrysosporium Anamorph of Nannizziopsis vriesii Complex and Relationship with Some Human-Associated Isolates.JOURNAL OF CLINICAL
MICROBIOLOGY, 51: 3338-3357
Stchigel, A.M.; Sutton, D.A.; Cano-Lira, J.F.; Cabañes, F.J.; Abarca, L.; Tintelnot, K.; Wickes, B.L.; Garcia, D. and Guarro, J. (2013).
Phylogeny of chrysosporia infecting reptiles: proposal of the new family Nannizziopsiaceae and five new species. PERSOONIA, 31: 86-100.
Vidal, P.; Vinuesa, M.A.; Sánchez-Puelles, J.M. and Guarro, J. (2000). Phylogeny of the anamorphic genus Chrysosporium and related taxa
based on rDNA internal transcribed spacer sequences. PP. 22-29. In: In: Kushwaha RKS, Guarro J (Eds.). Biology of Dermatophytes and other
Keratinophilic Fungi. REVISTA IBEROAMERICANA DE MICOLOGÍA, Bilbao.
36
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Historical biogeography and evolutionary patterns in human- and
animal-pathogenic Sporothrix species
Yu Zhang1,2, Ferry Hagen3, Benjamin Stielow2, Anderson Rodrigues4, G. Sybren de Hoog2
Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital, Tianjin, China; 2 CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; 3
Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands; 4 Department of Microbiology,
Immunology and Parasitology, Cellular Biology Division, Federal University of São Paulo, São Paulo, Brazil
1
V
ertebrate pathology has emerged repeatedly in the order Ophiostomatales. The rare ancestral species
Sporothrix mexicana is known to have a low degree of virulence, whereas the main pathogenic
species cluster in the derived S. schenckii clade. Phylogeny and epidemiology of Sporothrix was
studied for 135 strains (clinical n=99; environmental n=36) using ITS and partial CAL, TEF1 and
TEF3 genes, and compared with results of cluster analysis with amplified fragment length polymorphism
(AFLP) data. Molecular data of endemic species were compared to historical data, which enabled meaningful
interpretation of thousands of cases published in the literature.
The Sporothrix schenckii clade comprised nine subclusters, most of which were separate from each other
in all partitions, suggesting low degrees of interbreeding. Subgroups of S. brasiliensis, however showed conflicting topologies. Two small groups were intermediate between S. schenckii and S. brasiliensis. Sporothrix
brasiliensis subclusters show limited geographic distribution, suggesting slow vectors of dispersal. In contrast, S. globosa exhibited consistent global distribution of identical AFLP types, suggesting another type of
dispersal. The two species had different pathologies and dispersal mechanisms, i.e. the former was involved
in an expanding zoonosis and transmitted by cats, whereas S. globosa infections originated from plant material. Sporothrix schenckii, the most variable species within the clade, also had a plant origin, with ecological
similarities to S. globosa. In both species the type of plant material was relatively variable, but nevertheless
Sporothrix infections consistently in the form of outbreaks, which sometimes are very large. A hypothesis
was therefore put forward that highly specific conditions in the plant material are required to promote the
growth of Sporothrix, leading to outbreaks when these conditions are met. Fermented, self-heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates infections
in mammals. In the clonal offshoot of S. schenckii, S. brasiliensis a habitat shift to prevalently cat-transmission is observed.
37
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Orphan fungal infections caused by members of the genus Trichoderma
László Kredics, András Szekeres, Lóránt Hatvani, László Manczinger, Csaba Vágvölgyi
T
Department of Microbiology, Faculty of Science and Informatics, University of Szeged,
richoderma species are wide-spread saprophytic filamentous fungi from the Hypocreaceae family
of Ascomycota. Members of this genus take part in plant residue decomposition in the soil. Certain
species are excellent producers of cellulolytic enzymes and therefore applicable for the degradation
of cellulose in the biotechnological industry. Other Trichoderma species are potential agents of
biological control with the ability of antagonizing different plant pathogenic fungi. Some representatives of
the genus are also known to be harmful as the causal agents of the green mold disease, a disorder resulting
in substantial losses in mushroom cultivation. Moreover, a few Trichoderma species are emerging as causal
agents of opportunistic infections in humans.
In certain risk populations, Trichoderma strains are able to cause diseases varying from localized infections
to fatal disseminated diseases. There is a clearly emerging pattern of Trichoderma infections: most of the
cases are reported from patients undergoing peritoneal dialysis, from transplant recipients as well as from
patients with hematologic malignancies. Further risk factors include HIV infection and the receiving of chemotherapy, corticosteroids and antibiotics.
Trichoderma longibrachiatum is the most frequently occurring etiologic agent within the genus. Previous
reports about the occurrence of other species from the genus in clinical infections should be handled with
care, as the molecular reidentification of the available clinical isolates revealed that many of them were
incorrectly identified at the species level based on their morphological characteristics. The identification of
Trichoderma species is problematic if only morphological characters are considered; therefore, the identity
of clinical isolates is suggested to be confirmed by barcoding based on the sequence analysis of the ITS region and a segment of the tef1 gene.
Possible sources of infection include water-related sites, air, foods and catheters. Antifungal susceptibility
data are available in the literature, providing useful information for the planning of the therapy in cases of
suspected or confirmed Trichoderma infections. Ecophysiological and enzymological investigations of clinical
Trichoderma isolates revealed that there are several potential virulence factors of Trichoderma as a human
pathogen, like growth at elevated temperatures and neutral pH, the ability to utilize amino acids as carbon
and nitrogen sources, as well as the production of extracellular proteases, and bioactive secondary metabolites including peptaibol compounds with toxicity to mammalian cells.
László Kredics is supported by grant OTKA K-105972 from the Hungarian Scientific Research Fund.
38
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Antifungal susceptibility profile of Scedosporium spp.
Ana Alastruey-Izquierdo
S
Mycology Reference Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain
cedosporium the second or third most frequent genera of moulds isolated in human infections1. Several
cryptic species have been described in the last decade being some of them (such as S. aurantiacum, S.
boydii or S. dehoogii) also isolated in clinical samples. In addition Scedosporium species are known to
be emerging resistant pathogens with a poor response to most antifungals. The susceptibility profile
of nine CBS reference strains was analyzed and their susceptibility profile by EUCAST reference procedure2
determined. All strains showed high MICs to amphotericin B, itraconazole, terbinafine and echinocandins.
Voriconazole was the only drug showing activity against all strains while posaconazole showed a discrete
activity against some strains of P. boydii. In conclusion, Scedosporium species are multiresistant fungi.
Voriconazole is the best drug to treat these infections.
1. Alastruey-Izquierdo A, Mellado E, Peláez T, Pemán J, Zapico S, Alvarez M, Rodríguez-Tudela JL, CuencaEstrella M; FILPOP Study Group. Population-based survey of filamentous fungi and antifungal resistance
in Spain (FILPOP Study). Antimicrob Agents Chemother. 2013 Jul;57(7):3380-7.
2. Subcommittee on Antifungal Susceptibility Testing of the ESCMID European Committee for Antimicrobial
Susceptibility Testing 2008. EUCAST technical note on the method for the determination of broth dilution
minimum inhibitory concentrations of antifungal agents for conidia-forming moulds. Clin. Microbiol.
Infect. 14:982–984.
39
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
Genome analysis of Scedosporium aurantiacum
Meyer W1, Pérez-Bercoff Å1, Ramsperger M2, Nevalainen H3 & Huttley GA1
1
Molecular Mycology Research Laboratory, CIDM, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School-Westmead Hospital,
University of Sydney, Westmead Millennium Institute, Westmead, NSW, Australia; 2 John Curtin School of Medical Research, Australian National University,
Canberra ACT, Australia; 3 Department of Chemistry and Biomolecular Sciences, Macquarie University Sydney, NSW, Australia
A
bstract. Scedosporium aurantiacum is an emerging pathogen of humans and animals. It is commonly
present in urban environments. Animal studies, using the Galleria mellonella model, have revealed
virulence differences among strains. To establish a molecular basis for future studies to understand
the origin of those differences, we have sequenced and assembled for the first time the genome
of S. auarantiacum strain WM 09.24 using different genome assemblers. To project the genome annotation
and develop gene models we performed whole-genome alignment with the next closest species for which
an annotated genome was available, Trichoderma virens. However, this species is still so distant that only
approximately 30 % of the two genomes could be aligned through whole-genome alignment. Therefore,
neither direct-gene projection nor BLAST searches could be applied in order to annotate the S. aurantiacum
genome. To overcome this problem we performed RNA-sequencing of strain WM 09.24 under different
growth conditions (to maximise the number of transcribed genes), and used the JAMg pipeline to annotate
the genome. The annotated genome of strain WM 09.24 was used to identify functional genes in will is also
being used to annotate three additional S. aurantiacum strains with varying virulence to enable a wholegenome comparison to identify genes or genetic signatures that are associated with virulence.
40
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Abstract
DNA and Protein based diagnostics of the Fusarium fujikuroi species complex
Anne D. van Diepeningen1,*, Abdullah M.S. Al-Hatmi1, Balazs Brankovics1, G. Sybren de Hoog1, Anne-Cécile Normand2, Renaud
Piarroux2, Theo A.J. van der Lee3, and Cees Waalwijk3
) CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.2) Laboratoire de parasitologie, Hôpital de la Timone, Marseille, France. 3) PRI Plant Research
International, Plant Sciences Group, Wageningen University, Wageningen, The Netherlands. *) [email protected].
1
T
he large genus Fusarium is well-known for its many plant pathogens, mycotoxin producers, and
human and animal pathogens. The genus is divided into many species complexes that contain multiple
species that are morphologically often nearly indistinguishable and rely on multiple locus sequence
typing (MLST) for recognition. Human infections are caused by at least seven species complexes and
range from onychomycosis and keratitis in mainly healthy individuals to deep and disseminated infections in
the immunocompromised patients.
The Fusarium fujikuroi species complex (FFSC; formerly also known as Gibberella fujikuroi species complex)
contains at least 11 species that have been found in human infections, causing the whole range of infections.
Looking at population studies on fusariosis the FFSC is especially involved in the deep and disseminated infections causing approximately 1/3 of these infections next to members of the Fusarium solani and Fusarium
oxysporum species complexes.
We collected more than 100 clinical and environmental samples, including type strains, of all the known
human pathogenic species and some non-pathogenic species within the FFSC. Based on 15 loci we made an
MLST analysis for all these strains, grouping them in well-supported clades and confirming their identifications. All these strains can now be used to develop (databases for) DNA and protein based diagnostic tools.
MALDI-TOF analysis (Matrix-assisted laser desorption/ionization-Time of Flight) is a soft ionization technique used in mass spectrometry, which allows the analysis of biomolecules and proteins. Well-characterised
strains were used to build a database with reference spectra, which can now be used for the identification
of etiological agents. Luminex-analysis depends on probes based on species-specific DNA sequences. Combining spectrometry with specifically labelled probes this technique allows screening for multiple species in
one reaction, as well as targeting multiple loci simultaneously. The technique has been used successfully for
studies on mycotoxigenic plant pathogens of the Fusarium graminearum species complex. Based on all collected MLST data we will expand the probe set to include FFSC members.
41
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Poster
Translational medical mycology guides the clinic and laboratory practice
on fungal infectious diseases in China
Yuping Ran
T
Department of Dermatology, West China Hospital, Sichuan University, Chengdu, China
he fungal infected patients with skin lesions may consult to dermatologists, that is a challenge to
diagnose and treatment accurately. Dermatologists take samples from lesion to check the fungal
elements under the microscopy by KOH preparation, then to treat the patient. This model has
advanced as from bedside to bench, and, from bench to bedside (B to B to B), defined as Translational
Medical Mycology. Dermatologists have an advantageous position in finding, isolate, identification the
pathogenic fungi and treatment by familiar the usage of antifungal drugs. Samples should be cultured on
different media with or without chloramphenicol and cycloheximide and incubated at room temperature
and 37°C. No-culture techniques such as PCR based molecular identification, TEM, SEM, bio-chemistry
tests, and histopathology are also necessary to confirm identification of the species, especially when the
routine culture is negative. We start treatment upon obtaining proof of fungal infection, i.e., KOH positive.
Itraconazole, fluconazole, terbinafine, and amphotericin B can be used alone or in combination based on
the fungal species and the location. Practice on fungal infection including screen the patient, merge all of
the laboratory technique and methods from the microbiologists, pathologists, molecular researchers and
other specialists, to find the pathogen and to treatment as earlier as possible, to determine the optimum
antifungal drugs and the duration of therapy, based on the drug sensitive test and the time-course culture
monitor, from each individual patient.
42
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Poster
Pulmonary scedosporiosis after hydatid cyst surgery
Mustafa Altay ATALAY1, Ayşe Nedret KOÇ1, Mehmet BİLGİN2, Hafize SAV3,
1
Department of Medica Microbiology, Faculty of Medicine, Erciyes University, Kayseri, Turkey, 2 Department of Thoracic Surgery, Faculty of Medicine Erciyes
University, Kayseri, Turkey, 3 Department of Medical Microbiology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
I
ntroduction: Turkey is in one of the endemic regions for echinococcosis. The most common localization is
the liver and the lungs are the second most common site of involvement. Scedosporium apiospermum is
a saprophytic fungus which is the cause of mycetoma, a subcutaneous infection, characterized by granule
formation. It may also cause severe local or diffuse infections especially in immunosuppressive patients.
Scedosporium apiospermum-induced arthritis, endocarditis, keratitis, scleritis, endophthalmitis, meningitis,
osteomyelitis, otomycosis, onychomycosis, chronic prostatitis, peritonitis, esophagitis, renal infection, and
pulmonary and hepatosplenic abscess have been also reported in the literature.
On the other hand the coexistence of a saprophytic fungus and hydatid cyst is extremely rare. Herein is
reported a case of a patient presenting a solitary pulmonary abscess due to S. apiospermum after hydatid
cyst surgery.
Case Presentation: A 28-year-old female patient who underwent cystotomy and capitonnage surgery of
left lung due to a hydatid cyst three years ago was admitted to department of thoracic surgery with the complaint of 3-month history of cough with mucoid expectoration, and chest pain. The patient presented no predisposing condition or immunosupression. Chest computed tomography revealed a cavitary lesion located
in the left paracardiac region. After the surgery an abscess specimen which was sent for direct microscopic
examination in the mycology laboratory was stained with Gram and Giemsa and cultured in the Sabouraud
dextrose agar (SDA) medium (Oxoid, UK) with and without an antibiotic (cycloheximide and chloramphenicol). Then, it was incubated at 37 °C and 25 °C. Direct examination and staining revealed a fungal element.
Colonies growth rapidly (except SDA with cycloheximide) and were cottony and white at first, later turned
gray (Figure 1). Slide culture on cornmeal Tween 80 agar showed, single, egg-shaped, truncate conidia (Figure 2). The organism was therefore identified as S. apiospermum. Minimum inhibitor concentration (MIC)
values for amphotericin B, voriconazole, caspofungin, and posaconazole were 0.125µg/mL, 0.016 µg/mL,
0.125 µg/mL and 0.125 µg/mL, respectively. Our patient was immunocompetent and no structural deformities of the lungs that may predispose to scedosporiosis were found.
Conclusion: In conclusion, saprophytic fungus and hydatid cyst coexistence should be considered in individuals with pre- or perioperatively ruptured cysts and ongoing clinical symptoms. Given the fact that early
diagnosis saves lives, specimens should be definitely sent for mycological analysis.
Figure 1. Culture of Scedosporium apiospermum
Figure 2. Conidiophores and conidia of Scedosporium
apiospermum on slide culture
43
Diversity and Barcoding of Medical Fungi: Novel Achievements and Masterclass
A meeting of the ISHAM Working Groups on Barcoding and Scedosporium
Poster
Black yeast habitat choices and species spectrum on high altitude
creosote-treated railway ties
Aylin Döğen1, Macit Ilkit2, G. Sybren de Hoog3,4,5,6,7
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mersin, Mersin, Turkey, 2Division of Mycology, Department of Microbiology,
Faculty of Medicine, University of Çukurova, Adana, Turkey, 3CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, 4Institute for Biodiversity and
Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands, 5Peking University Health Science Center, Research Center for Medical Mycology,
Beijing, China, 6Sun Yat-sen Hospital, Sun Yat-sen University, Guangzhou, China, 7Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military
Medical University, Shanghai, China
1
P
olyextremotolerant black yeast-like fungi thrive in moderately hostile environments where they are
concomitantly subjected to several types of stress, such as toxicity, scarce nutrient availability, and
high or low temperature extremes. Their ability to assimilate alkylbenzenes (toxic environmental
pollutants) enhances their growth in harsh conditions, including on railway ties. Samples were
collected using cotton swabs, premoistened with physiological saline, from 658 oak and concrete railway ties
at six train stations in Turkey at altitudes ranging between 1026 and 1427 m. The samples were inoculated
on malt extract agar supplemented with chloramphenicol, and incubated at 26 °C for 4 weeks. Twenty-four
samples (3.6%), 17 from oak and 7 from concrete (5.6% vs. 2%; P=0.02), tested positive for fungi. Exophiala
crusticola was found to be the most common species (n=13), followed by E. phaeomuriformis (n=7) and
E. heteromorpha (n=4). These results suggest that hydrocarbons, particularly creosote-treated oak woods,
support the growth of black yeasts, some of which are opportunists in humans.
44
.
22-23 April 2014
22042014 23042014

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