AFLP diversity in

1
AFLP diversity in Pseudallescheria boydii
W.H.J. Becker1,2, J.S. Zeng1, S.M. Reiffert2,
A.H.G. Gerrits van den Ende1, G.S. de Hoog1 and
R. Horré2,3
1 Centraalbureau voor Schimmelcultures, KNAW,
Utrecht, The Netherlands 2 Institute for Medical
Microbiology, Immunology and Parasitology, Uni
Bonn, Germany 3 Federal Institute for Drugs and
Medical Products (BfArM), Bonn, Germany
2
Methods

• Strains
• - Grown 2-4 weeks on MEA (Malt Extract Agar)
• DNA-extraction
• Minipestles, silica-mix
• Purification with chloroform, amylalcohol,
  ammoniumacetate
• Without phenol
• Results of optimisation (Data not shown here)
• - RNA Digestion with RNAse not necessary
• - Further purifications e.g. with colums not
  necessary
• modified according Moeller E.M., Bahnweg G,
  Sandermann H, Geiger H.H.
• A simple and efficient protocol for isolation of
  high molecular weight DNA
• from filamentous fungi, fruit bodies and infected
  plant tissues
• Nucleic Acids Research 1992, Vol 20, No. 22, 6115
  - 6116

3
Methods
AFLP The primers of the last selective
PCR-reaction are marked with fluorescent
colours Green Blue Yellow AG Joe AC
6-Fam AT Ned Standard Red ROX Gene
Scan 500 Modified after Vos et al.
AFLP, a new technique for DNA fingerprinting,
Nucleic Acids Research 1995, Vol. 23 4407-4414.
4
Methods

5
Methods

6
Methods
Filter

Data processing ABI Prism 310 Genetic Analyser
7
Method Data Processing

8
Wards averaging
N 58 strains
Pseudallescheria boydii
B1
Cut-off 85 90 (Ward and UPGMA) dependent on
quality of AFLP-pattern
B
B2
A1
A2
A3
A
A4
A5
Scedosporium prolificans
9
UPGMA
Pseudallescheria boydii
B
A2
A3
A1
A
A4
A5
Scedosporium prolificans
10
Wards averaging
Pseudallescheria boydii
B11
B1
B12
B13
B14
B
B21
B2
B22
B23
A
11
How to define groups ?

• Main groups (A,B)
• Clusters in Ward
• UPGMA distance above 50
• Subgroups (e.g. A4, B22)
• Groups are recognized in
• Wards Averaging and UPGMA

12
DNA-DNA-Reassociation
E. Guého, G.S. de Hoog Taxonomy of the medical
species of Pseudallescheria and Scedosporium, J.
Mycol. Méd., 1991, 1 3-9
Pseudallescheria_at_yahoo.de
13
Wards averaging
Wards averaging
Wards averaging
Pseudallescheria boydii
Pseudallescheria boydii
Pseudallescheria boydii
B11
B1
B12
IP 1742.88 CBS 116894, Group 3
B13
B14
B
CBS 108.54 , Group 3
B21
CBS 101.22 , Group 1
B2
B22
B23
A1
CBS 297.38 , Group 2
A2
A3
A
A4
A5
Scedosporium prolificans
14
Results 1 Groups

• 3 Maingroups in AFLP A, B1, B2

15
Results 1 Groups

• 3 Maingroups in AFLP A, B1, B2
• AFLP-Groups confirm the
• DNA-DNA-Reassociation-Groups

16
Results 1 Groups

• 3 Maingroups in AFLP A, B1, B2
• AFLP-Groups confirm the
• DNA-DNA-Reassociation-Groups
• More genetic variability in group B1/B2 than in
  group A

17
Results 1 Groups

• 3 Maingroups in AFLP A, B1, B2
• AFLP-Groups confirm the
• DNA-DNA-Reassociation-Groups
• More genetic variability in group B1/B2 than in
  group A
• More invasive strains in group A (63 ) than in
  group B (21 )

18
Results 1 Groups

• 3 Maingroups in AFLP A, B1, B2
• AFLP-Groups confirm the
• DNA-DNA-Reassociation-Groups
• More genetic variability in group B1/B2 than in
  group A
• More invasive strains in group A (63 ) than in
  group B (21 )
• All 3 marker strains with growth max at 36C are
  found in group A

19
Conclusions 1 Groups

• 3 Maingroups in AFLP A, B1, B2
• AFLP-Groups confirm the
• DNA-DNA-Reassociation-Groups
• More genetic variability in group B1/B2 than in
  group A ? restricted sexuality in group A ?
• More invasive strains in group A (63 ) than in
  group B (21 )
• All 3 marker strains with growth max at 36C are
  found in group A

20
Wards averaging
Pseudallescheria boydii
B11
B1
B12
B13
B14
B
B21
Clinical Case Near drowing, Patient BAL 2
different (?) strains of the same group (A3), But
in watersample a complete different genotype was
found (B12)
B2
B22
B23
More than one strain in one patient ?
A1
A2
A3
A
A4
A5
Scedosporium prolificans
21
Wards averaging
Pseudallescheria boydii
B11
B1
B12
Two similar strains Both group B11, Both
otomycosis, but one from Canada and one from NL !
B13
B14
B
B21
B2
B22
B23
A1
A2
A3
A
A4
A5
Scedosporium prolificans
22
Wards averaging
Pseudallescheria boydii
B11
B1
B12
B13
B14
B
B21
B2
B13 Similar strains from North America, Africa
and Europe
B22
B23
A3 Similar strains from South America and Europe
A1
A2
A3
A
A4
Worldwide distribution, No local clusters seen so
far.
A5
Scedosporium prolificans
23
Wards averaging
Pseudallescheria boydii
B11
B1
B12
B13
B14
B
B21
B2
B22
B23
A1
Mycetoma strains can be found in group B22 and
A3, A4, A5 but inside this groups the genotypes
are very similar.
A2
A3
A
A4
A5
Scedosporium prolificans
24
Results 2 Strains

• More than one strain per patient ?

25
Results 2 Strains

• More than one strain per patient ?
• Sometimes clustering in clinical pictures
• (e.g. otitis, mycetom)

26
Results 2 Strains

• More than one strain per patient ?
• Sometimes clustering in clinical pictures
• (e.g. otitis, mycetom)
• No local clusters seen so far

27
Conclusions 2 Strains

• More than one strain per patient
• ? very sufficient mode of infection
• Sometimes clustering in clinical pictures
• (e.g. otitis, mycetom)
• No local clusters seen so far

28
Conclusions 2 Strains

• More than one strain per patient
• ? very sufficient mode of infection
• Sometimes clustering in clinical pictures
• (e.g. otitis, mycetom)
• ? similar strains may share the same
• factors of pathogenicity
• No local clusters seen so far

29
Conclusions 2 Strains

• More than one strain per patient
• ? very sufficient mode of infection
• Sometimes clustering in clinical pictures
• (e.g. otitis, mycetom)
• ? similar strains may share the same
• factors of pathogenicity
• No local clusters seen so far
• ? worldwide distribution of same genotypes

30
Pseudallescheria_at_yahoo.de
Method Gel