1. Phenotyping and genotyping of S. prolificans
isolates from diverse origins
Toward a better knowledge of the Scedosporium
species and their relatives
N Gantois1
, F Hernandez1*
W Meyer2
, S Chen2
, C Hallidays2
,
J-P Bouchara3
, F Grenouillet4
, F Botterel5
, T Sorrell2
, E
Viscogliosi1
, E Dei-Cas1
, L Delhaes1
ADDRESSES: 1
CIIL, EA4547-BDEEP, Pasteur Institute of Lille, Lille, France,
2
Westmead Hospital, Sydney, Australia,
3
Angers Hospital, GEIHP-University, Angers, France,
4
Besançon Hospital, Besançon, France,
5
Henri Mondor Hospital, Créteil, France
*current address: UNAM, Mexico
E-MAILS: nausicaa.gantois@pasteur-lille.fr
laurence.delhaes@pasteur-lille.fr

2. IntroductionIntroduction
 Scedosporium prolificans species is able to
colonize and infect Humans and is considered
as emerging/re-emerging pathogen
[Fernandez Guerrero et al. 2011; Lu et al. 2011; Grenouillet et al. 2009]
 A recent study on phylogeny and ecological
trends of Parascedosporium (Scedosporium plus
Graphium -as synanamorph- plus Pseudallescheria /
Petriella/Petriellopsis -as teleomorphs) and its relatives
supported the opportunistic potential to
mammals of Pseudallecheria and S. prolificans
lineages [Lakner et al. 2011]

3. S. Prolificans
clade
IntroductionIntroduction
Compared to the well
characterized
Scedosporium
Pseudallescheria
complex, little is
known about the
fundamental aspects
of S. prolificans
biology, pathogenicity
and epidemiology
[from Lakner et al. 2011]
Petriella clade
Petriellopsis
clade
Parascedo-
sporium clade
Pseudalle-
scheria clade
Lophotrichus
clade
Graphium
clade

4.  S. prolificans
 Previously described as Scedosporium inflatum
 No currently known sexual form
 Ubiquitous distribution in soil
 Local and disseminated infections, both in
immunocompromised and in non-IC patients
 Highly resistant to antifungal drugs, both in
vitro and in vivo → difficult to treat, high mortality
rate in infected patients
 Little is known of ecology and pathogenesis
IntroductionIntroduction
[Grenouillet et al. 2009; Porton et al, 2000; Chen et al. 2005]

5.  Our goal was to characterize a large
population of S. prolificans isolates or strains
(IHEM: 18755) (n= 52) plus 7 DNA samples)
(i) isolated from animal, human, or environment
samples
(ii) in different countries
Aim of the studyAim of the study
France
(n=21)
Spain
(n=5)
USA
(n=1)
Australia
(n=31)

6. Materials and MethodsMaterials and Methods
Standardized sub-cultures (106
conidia in 30 µl of sterile water),
at 30° C on Sabouraud’s agar medium diluted ½ for 1-2
weeks
DNA extraction using
UltraClean Fecal® DNA
kit (MoBio, France).
Genotypic
characterization based on
the analysis of :
(i) Cu/Zn - MnSOD,
(ii) beta-tubulin and
(iii) ITS1-2 genes.
Phylogenetic trees were
constructed with
unambiguously MUSCLE
alignements, and
conducted using MEGA5.
Phenotypic criteria including
(i) macroscopic and
microscopic features
(morphology)
(ii) ATF susceptibilities
based on E-test® method
Color colony, growth
diameter at day 14
Surface of conidia
Phialide sizes
In vitro susceptibilities
to AmB, ITC, PSC,
VRC, and Caspofungine

7. Results: Phenotype studyResults: Phenotype study

8. Results: Phenotype studyResults: Phenotype study
[De Hoog et al . 1994]

9. Results: Antifungal susceptibilityResults: Antifungal susceptibility
[Lackner et al . 2012]

10. Results: Molecular analysisResults: Molecular analysis
 Phylogenetic analysis by
maximum likelihood method
using complete set of data in
MEGA5 [Tamura K. et al . 2011]
a

11. Results: Molecular analysisResults: Molecular analysis
 Phylogenetic analysis by
maximum likelihood method
using complete set of data in
MEGA5 [Tamura K. et al . 2011]
a

12. Results: PhylodatationResults: Phylodatation
 Exploring phylodatation using
molecular analysis of MnSOD
sequences by ML method and
speciation time for
Pneumocystis in MEGA5
[Tamura K. et al . 2011; Scott et al 2003]

13. Results: PhylodatationResults: Phylodatation
 Exploring phylodatation using
molecular analysis of MnSOD
sequences by ML method and
speciation time for
Pneumocystis in MEGA5
[Tamura K. et al . 2011; Scott et al 2003]

14.  No correlation between clinical-biological characteristics
and genotypic or phenotypic criteria of S. prolificans
strains was found.
 Among our collection composed of 59 isolates/strains (52
cultures isolates plus 7 DNA samples), we identified:
- 3 different morphotypes of S. prolificans with
- some genetic polymorphisms: 1.8-2.2% of nucleotide
differences between the analyzed sequences.
 These low sequence polymorphisms reflect intra-specific
genetic variations, in agreement with previous published
data [Delhaes et al. 2008]
 Therefore, we hypothesized that S. prolificans might be
stable in space, and apparently insensitive (or poorly
sensitive) to xenical or environmental factors.
To concludeTo conclude

15. To concludeTo conclude
 our results supported the current perception of S.
prolificans as a unique species and an emerging
opportunistic pathogen, which seems to emerge in
recent times (less than 50 million years ago using MnSOD
genes and speciation time for Pneumocystis to determine the split
of S. prolificans lineage).
 These results will be highlighted when our ongoing
program on Scedosporium genome sequencing with de
novo assemblage will be achieved.
According to a recent study, we might be able to compare
genomes and to search for gene duplication at the
subtelomeric regions or for horizontal transferts between
the species (that might explain or insight the different
fungal pathogenesis observed between the species) [Moran
et al. Comparative genomics and the evolution of pathogenicity in human pathegenic fungi,
Eukaryot Cell 2011],

16.  Thank you for your attention+++
 Thank you to the organisers!
 We thank all the colleagues who have
submitted cases, isolates, and/or DNA, especially
the Australian Scedosporium (AUSCEDO) Study
Group and the present W-Group

17.  High throughput sequencing technology on Ion Torrent /
Life Technologies
 Based on 2 steps
 de novo DNA sequencing of the 2 strains
(expected 1Gb of data per strain, mean size of reads 300
pb)
 plus an ARN expression analysis using RNA-seq approach
In order to build a list of transcripts (a main list but probably non
exhaustive)
 DNA extraction = done, but not ARN extraction
 DNA sequencing might be done next month
Genome sequencing and de novoGenome sequencing and de novo
assemblageassemblage
of S. aurantiacum and P. minutisporaof S. aurantiacum and P. minutispora