Phenotyping and genotyping of S. prol i f icans isolates from diverse origins

Toward a better knowledge of the Scedosporium species and their relatives

N Gantois 1, F Hernandez 1 * W Meyer 2, S Chen 2, C Hall idays 2, J-P Bouchara 3, F Grenouil let 4, F Botterel 5, T Sorrell 2, E

Viscogliosi 1, E Dei-Cas 1, L Delhaes 1

ADDRESSES: 1 CIIL, EA4547-BDEEP, Pasteur Insti tute of Li l le, Li l le, France, 2Westmead Hospital, Sydney, Austral ia, 3Angers Hospital, GEIHP-University, Angers, France, 4Besançon Hospital, Besançon, France, 5Henri Mondor Hospital, Créteil, France*current address: UNAM, MexicoE-MAILS: nausicaa.gantois@pasteur-l i l le.frlaurence.delhaes@pasteur-l i l le. fr

IntroductionIntroduction

Scedosporium prolif icans 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 / Petriel la/Petriel lopsis -as teleomorphs) and its relat ives supported the opportunistic potential to mammals of Pseudallecheria and S. prolif icans l ineages [Lakner et al. 2011]

S. Prolificans clade

IntroductionIntroductionCompared to the well characterized Scedosporium Pseudallescheria complex, l i t t le is known about the fundamental aspects of S. prolif icans biology, pathogenicity and epidemiology

[from Lakner et al. 2011]

Petriella clade

Petriellopsis clade

Parascedo-sporium clade

Pseudalle-scheria clade

Lophotrichus clade

Graphium clade

S. prolif icans 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 mortalityrate in infected patients

Litt le is known of ecology and pathogenesis

IntroductionIntroduction

[Grenouillet et al. 2009; Porton et al, 2000; Chen et al. 2005]

Our goal was to characterize a large population of S. prolif icans isolates or strains (IHEM: 18755) (n= 52) plus 7 DNA samples)

( i) isolated from animal, human, or environment samples

(i i) in different countries

Aim of the studyAim of the study

France (n=21)

Spain(n=5)

USA (n=1)

Australia

(n=31)

Materials and MethodsMaterials and MethodsStandardized sub-cultures (106 conidia in 30 µl of steri le water) ,

at 30° C on Sabouraud’s agar medium di luted 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, (i i ) beta-tubulin and

(i i i ) ITS1-2 genes.

Phylogenetic trees were constructed with

unambiguously MUSCLE al ignements, and

conducted using MEGA5.

Phenotypic criteria including (i) macroscopic and microscopic features

(morphology)(i i) ATF susceptibi l i t ies

based on E-test® method

Color colony, growth diameter at day 14

Surface of conidia Phialide sizes

In vitro susceptibil it ies to AmB, ITC, PSC,

VRC, and Caspofungine

Results: Phenotype studyResults: Phenotype study

Results: Phenotype studyResults: Phenotype study

[De Hoog et al . 1994]

Results: Antifungal susceptibi l i tyResults: Antifungal susceptibi l i ty

[Lackner et al . 2012]

Results: Molecular analysisResults: Molecular analysis

Phylogenetic analysis by maximum likel ihood method using complete set of data in MEGA5 [Tamura K. et al . 2011]

a

Results: Molecular analysisResults: Molecular analysis

Phylogenetic analysis by maximum likel ihood method using complete set of data in MEGA5 [Tamura K. et al . 2011]

a

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]

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]

No correlation between cl inical-biological characterist ics and genotypic or phenotypic cri teria of S. prol if icans strains was found.

Among our col lect ion composed of 59 isolates/strains (52 cultures isolates plus 7 DNA samples), we identif ied: - 3 dif ferent morphotypes of S. prol if icans with - some genetic polymorphisms: 1.8-2.2% of nucleotide dif ferences between the analyzed sequences.

These low sequence polymorphisms reflect intra-specif ic genetic variat ions, in agreement with previous published data [Delhaes et al. 2008]

Therefore, we hypothesized that S. prol if icans might be stable in space, and apparently insensit ive (or poorly sensit ive) to xenical or environmental factors.

To concludeTo conclude

To concludeTo conclude our results supported the current perception of S.

proli f icans as a unique species and an emerging opportunist ic pathogen, which seems to emerge in recent t imes ( less than 50 mil l ion years ago using MnSOD genes and speciat ion t ime for Pneumocystis to determine the split of S. prolif icans l ineage).

These results wil l be highlighted when our ongoing program on Scedosporium genome sequencing with de novo assemblage wil l 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 dif ferent fungal pathogenesis observed between the species) [Moran et al. Comparative genomics and the evolution of pathogenicity in human pathegenic fungi, Eukaryot Cell 2011],

Thank you for your attention+++

Thank you to the organisers!

We thank al l the colleagues who have submitted cases, isolates, and/or DNA, especially the Australian Scedosporium (AUSCEDO) Study Group and the present W-Group

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 approachIn order to build a l ist of transcripts (a main l ist but probably non

exhaustive)

DNA extraction = done, but not ARN extraction

DNA sequencing might be done next month

Genome sequencing and de novo Genome sequencing and de novo

assemblage assemblage

of S. aurantiacum and P. minutispora of S. aurantiacum and P. minutispora