Genetic variation analysis and relationships among environmental strains of Scedosporium apiospermum sensu stricto in Bangkok, Thailand
Genetic variation analysis and relationships among environmental strains of Scedosporium apiospermum sensu stricto in Bangkok, Thailand
Thanwa Wongsuk 0 1
Potjaman Pumeesat 0 1
Natthanej Luplertlop 0 1
0 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University , Bangkok , Thailand , 2 Department of Clinical Pathology, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University , Bangkok , Thailand , 3 Department of Medical Technology, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University , Bangkok , Thailand
1 Editor: Craig Eliot Coleman, Brigham Young University , UNITED STATES
The Scedosporium apiospermum species complex is an emerging filamentous fungi that has been isolated from environment. It can cause a wide range of infections in both immunocompetent and immunocompromised individuals. We aimed to study the genetic variation and relationships between 48 strains of S. apiospermum sensu stricto isolated from soil in Bangkok, Thailand. For PCR, sequencing and phylogenetic analysis, we used the following genes: actin; calmodulin exons 3 and 4; the second largest subunit of the RNA polymerase II; û-tubulin exon 2±4; manganese superoxide dismutase; internal transcribed spacer; transcription elongation factor 1α; and beta-tubulin exons 5 and 6. The present study is the first phylogenetic analysis of relationships among S. apiospermum sensu stricto in Thailand and South-east Asia. This result provides useful information for future epidemiological study and may be correlated to clinical manifestation.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This study was supported by Health
System Research Institute (HSRI) 2016, National
Research Council of Thailand (NRCT) 2016 and
Tropical Medicine grant 2014, Faculty of Tropical
Medicine, Mahidol University. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
Competing interests: The authors have declared that no competing interests exist.
level is important as epidemiology, clinical outcomes, and antifungal susceptibility are
In order to overcome the difficulties in identifying Scedosporium species by routine
microbiological methods, several molecular techniques have been proposed such as quantitative
real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated
isothermal amplification (LAMP) [
]. Additionally, globally standardized genotyping of S.
apiospermum and S. boydii, the Multi-Locus Sequences Typing (MLST) scheme, was developed by
Bernhardt et al. . The MLST scheme amplifies sequences at five genetic loci±actin (ACT),
calmodulin exons 3 and 4 (CAL), the second largest subunit of RNA polymerase II gene
(RPB2), û-tubulin exons 2±4 (BT2), and manganese superoxide dismutase (SOD2) and has
been found to have strong repeatability [13±15]. The allele types (ATs) and sequences types
(STs) numbers of the consensus MLST scheme can be obtained through the Fungal MLST
Database (http://mlst.mycologylab.org/). In our previous study, we investigated the spatial
distribution of the S. apiospermum species complex in soil in Bangkok, Thailand. We found that
the S. apiospermum species complex is widespread in soil across Bangkok and detected
predominance of S. apiospermum sensu stricto (72%) [
Here, we continue the study by considering the genetic variation and relationships among
S. apiospermum sensu stricto isolated from soil in Bangkok. The data may be used for further
epidemiological research, for which it is important to recognize different strains and
Materials and methods
We used 48 isolates of S. apiospermum sensu stricto from our stock collection. Each isolate
originated from soil and had previously been typed by PCR of the beta-tubulin gene (exons 5
and 6) [
]. Each isolate was labeled as TMMI (Department of Microbiology and
Immunology, Faculty of Tropical Medicine, Mahidol University yeast and moulds culture collection)
with a unique identification number (Table 1).
DNA was extracted with an E.Z.N.A. Fungal DNA mini kit (Omega Bio-tek). The DNA
samples were quantified and the quality was checked with a NanoDrop 2000 spectrophotometer
(Thermo Fisher, Wilmington, DE, USA) and stored at −20ÊC until further use.
Molecular biology technique
For PCR, sequencing and phylogenetic analysis we used eight genes: ACT, CAL, RPB2, BT2,
SOD2, internal transcribed spacer (ITS), transcription elongation factor 1α (TEF-1α), and 28S
large subunit ribosomal RNA (LSU). PCR amplification of eight gene regions was carried out
with the specific primer pairs listed in Table 2. Each PCR reaction mixture was performed in
25-μl final volume containing: 0.5 μM of each primer, KAPA 2G Fast HS ReadyMix PCR kit
with loading dye (KAPA Biosystems, USA), nuclease-free water and genomic DNA. PCR
amplifications were carried out in a T100 Thermal Cycler (Bio-Rad) according to the following
protocol: an initial step of 96ÊC for 6 min, followed by 35 cycles of 94ÊC for 1 min, an
annealing temperature that specific with each gene (Table 2) for 1 min and 72ÊC for 45 s and a final
extension step of 72ÊC for 10 min. Then, 5 μL of the PCR products were electrophoresed in a
1.5% agarose gel containing SERVA DNA Stain G (SERVA Electrophoresis GmbH, Germany)
in 1X TBE buffer, and photographed using a Gel Doc XR+ system (Bio-Rad).
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GenBank accession numbers
ITS LSU ACT CAL SOD2
KY122868 KY122916 KY122964 KY123012 KY123060
KY122869 KY122917 KY122965 KY123013 KY123061
KY122870 KY122918 KY122966 KY123014 KY123062
Each PCR product was purified using a FavorPrepTM GEL/PCR Purification Mini Kit
(Favorgen Biotech Corporation, Taiwan) and sequenced using gene-specific both forward and
reverse primers at the AITbiotech Pty Ltd (Singapore). The retrieved sequence files were
analyses using BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and compared
with existing sequences in GenBank using BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Genotyping variation and relationship analysis of eight housekeeping
Nucleotide sequences of seven genes (ACT, CAL, RPB2, BT2, SOD2, ITS, TEF-1α) were
used for genetic variation and relationship analysis together with û-tubulin exons 5 and 6
gene (TUB). The following TUB sequences were downloaded from GenBank: KU533691,
KU533702, KU533711, KU533712, KU533691, KU533656, KU533658, KU533655, KU533649,
KU533673, KU533713, KU533674, KU533675, KU533695, KU533676, KU533703, KU533723,
KU533704, KU533692, KU533693, KU533694, KU533677, KU533705, KU533678, KU533659,
KU533679, KU533696, KU533680, KU533681, KU533682, KU533683, KU533707, KU533660,
KU533684, KU533708, KU533706, KU533666, KU533697, KU533685, KU533650, KU533651,
KU533652, KU533709, KU533710, KU533657, KU533716, KU533698, and KU533717. Each
sequence was trimmed to the correct length with the start and the end of each gene (shown in
Table 3). Genotype variation of each gene was analyzed by MLSTest v126.96.36.199 software, which
was downloaded from http://ipe.unsa.edu.ar/software [
]. The ATs number was created by
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using the same software and allele profiles were used to assign the STs number. A phylogenetic
network of each loci was performed with the neighbor-net algorithm by SplitsTree4 and
downloaded from http://www.splitstree.org/ [
Sequences were concatenated by the following order respectively; ACT, CAL, RPB2, BT2,
SOD2, ITS, TEF-1α and TUB. The best model of evolution for concatenated data set were
selected from the Bayesian Information Criterion (BIC) in MEGA7 [
]. Model with the
lowest BIC score was chosen to construct a maximum likelihood phylogenetic tree. A phylogenetic
tree of the 48 aligned sequences excluding gaps and missing data for the heuristic search was
obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated
using the maximum likelihood approach based on the best model in MEGA7 [
]. The tree is
drawn to scale, with branch lengths measured in the number of substitutions per site. Codon
positions included were 1st+2nd+3rd+Noncoding. There were a total of 4,841 positions in
the final dataset. A bootstrap analysis was conducted with 1000 replications and bootstrap
values 50% were shown above branches.
Phylogenetic analysis of the concatenated sequences of ACT, CAL,
RPB2, BT2 and SOD2 genes
To assess evolutionary relationships among isolates, all 48 generated concatenated of ACT,
CAL, RPB2, BT2 and SOD2 genes sequences as well as 27 concatenated sequences of S.
apiospermum sensu stricto (isolate IHEM 15555, IHEM 15553, IHEM 15552, IHEM 15551, IHEM
15148, IHEM 15146, IHEM 14764, IHEM 14763, IHEM 14762, IHEM 14465, IHEM 14463,
IHEM 14462, IHEM 15151, IHEM 15149, IHEM 15643, IHEM 14276, IHEM 14275, IHEM
14273, IHEM 14270, IHEM 14269, IHEM 14268), BMU07462, BMU04729, BMU04111,
BMU03882, BMU01117 and BMU00491) from other regions of the world and three S. boydii
(isolate IHEM 14362, IHEM 14638 and IHEM 14457) concatenated sequences downloaded
from GenBank were multiply-aligned using BioEdit. The concatenated sequences of S.
aurantiacum isolate IHEM 15458, S. angusta isolate BMU01115, Pseudallescheria fusoidea isolate
BMU01297 and Pseudallescheria ellipsoidea isolate BMU01118 were included in the
phylogenetic analysis. GenBank accession numbers are listed in S1 Table.
] was used to selected the best model of evolution and a phylogenetic tree was
constructed as described above. The analysis involved 82 nucleotides sequences. All position
containing gaps and missing data were eliminated. There were a total of 2,958 positions in
the final dataset. A bootstrap analysis was conducted with 1000 replications and bootstrap
values 50% were shown above branches also.
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Nucleotide sequences accession numbers
The generated nucleotide sequences were deposited in GenBank under accession numbers
and listed in Table 1.
PCR amplification of the eight genes was successful for all strains, with a single band
investigated on gels after electrophoresis. The BLASTn algorithm was used for sequence similarity
searching in the NCBI database. Sequence-based identities with a cutoff of 99% were
considered significant [
Genotypic variation profiling of S. apiospermum sensu stricto was generated and showed
37 good different STs from 48 strains (Table 4). The numbers of ATs variation at each loci
were 8 (ACT), 7 (CAL), 11 (RPB2), 12 (BT2), 18 (SOD2), 15 (ITS), 6 (TEF-1α), and 8 (TUB)
(Table 5). The number of polymorphisms, typing efficacy, and power of discrimination (95%
confidential interval) were calculated (Table 3).
We found diverse genetic relationships among the genotyped variants of 48 S. apiospermum
sensu stricto after analyzing each gene with a neighbor-net algorithm (Fig 1). The best model
for concatenated data set (ACT, CAL, RPB2, BT2, SOD2, ITS, TEF-1α and TUB, respectively)
analyses was HKY+G+I (HKY: Hasegawa-Kishino-Yano; +G: Gamma distribution; +I:
invariable sites) and the BIC score was 18004.65987. Therefore, the maximum likelihood
phylogenetic tree of concatenated data set was created based on the HKY model [
]. A discrete
Gamma distribution was used to model evolutionary rate differences among sites (5 categories
(+G, parameter = 0.0500). The rate variation model allowed for some sites to be evolutionarily
invariable ([+I], 48.7606% sites) (Fig 2).
To compare the genetic relationship of strains in Thailand with other regions of the world,
the ACT, CAL, RPB2, BT2 and SOD2 sequences from previous studies in France, China and
Japan were downloaded from GenBank. Data for these five genes were concatenated. TN93+G
+I model (TN93: Tamura-Nei) was chosen according the best model of evolution analyses (the
BIC score was 17282.89945). Then, a phylogenetic tree constructed by maximum likelihood
analysis based on the TN93 model [
]. A discrete Gamma distribution was used to model
evolutionary rate differences among sites (5 categories (+G, parameter = 0.1587)). The rate
variation model allowed for some sites to be evolutionarily invariable ([+I], 42.7147% sites). As
a tree (Fig 3), 82 nucleotide sequences comprised 2,958 positions were involved. S.
apiospermum sensu stricto was strongly clustered together among Thai, French, Chinese and Japanese
isolates (strongly supported by bootstrap value of 100%) and were subdivided into two groups
(Group I and Group II).
The Scedosporium apiospermum species complex contains important opportunistic species.
Giraud and Bouchara [
] and the European Confederation of Medical Mycology (ECMM)/
International Society for Human and Animal Mycology (ISHAM) classify the novel
nomenclature of the S. apiospermum species complex as comprising five species: S. apiospermum sensu
stricto, S. boydii (= Pseudallescheria boydii), S. aurantiacum, S. dehoogii, and S. minutispora. In
contrast, a recent study [
] defined the S. apiospermum species complex as only S.
apiospermum, S. boydii, and S. angusta (= Pseudalleschelia angusta) because phylogenetic analysis of
βtubulin (BT2), γ-actin, transcriptional elongation factor 1α (TEF-1α), and internal transcribed
spacer of the small ribosomal protein 60sS L10 (L1) distinguished S. minutispora, S.
aurantiacum, and S. dehoogii from these three species. These days, there are numerous molecular
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Gene ATs Frequency
Gene ATs Frequency
techniques to determine genetic diversity that are robust and reproducible. MLST has been
proposed as one of the best tools for genotypic and evolutionary studies.
In the fungal research field, four working groups have established public MLST schemes:
(1) ISHAM established an MLST scheme for Cryptococcus gattii, Cryptococcus neoformans,
S. apiospermum, S. boydii, S. aurantiacum, Bipolaris australiensis, Bipolaris. hawaiiensis,
Bipolaris spicifera (http://mlst.mycologylab.org/); (2) Westerdijk Fungal Biodiversity Institute
(CBS-KNAW Fungal Biodiversity Centre) established an MLST scheme for Fusarium spp.
(http://www.westerdijkinstitute.nl/fusarium/); (3) Imperial College established an MLST
scheme for Candida albicans and Candida glabrata (http://www.mlst.net/); and (4) Oxford
University established an MLST scheme for Aspergillus fumigatus, Pichia kudriavzevii
(Candida krusei) and Candida tropicalis (http://pubmlst.org/). MLST is clearly a powerful method
for typing and studying genetic variation in microorganisms.
In this study, we used the MLST tool to study S. apiospermum sensu stricto previously
isolated from soil samples. We chose eight housekeeping gene loci (ACT, BT2, CAL, ITS, RPB2,
SOD2, TEF-1α, LSU) coupled with the TUB gene from our previous study to analyze the
genetic variation and relationships among S. apiospermum sensu stricto strains, which are
currently unknown and have not been previously analyzed genetically in Thailand or South-east
Asia. We successfully sequenced all selected loci, but only the LSU sequences showed no
polymorphisms (data not show). We identified 37 STs after combining the 8 genes (except LSU).
In each gene fragment, the number of alleles varied; 8, 12, 8, 15, 11, 18, 6, 8 for ACT, BT2,
CAL, ITS, RPB2, SOD2, TEF-1α, and TUB, respectively. The number of polymorphisms for
each gene fragment varied from 6 (TEF-1α) to 41 (BT2). The sequences of the 48 S.
apiospermum sensu stricto strains were accessed by a neighbor-net algorithm in SplitsTree4.
SplitsTree4 graphs of ACT, CAL, RPB2, BT2, SOD2, ITS, TEF-1α, and TUB sequence data showed
parallelograms that implied the character is shared by a set of species.
In terms of discriminatory power (DP) evaluation, SOD2 showed the highest DP (0.932);
TUB showed the lowest DP, which was 0.688. Additionally, there were 37 STs, which could be
grouped; each isolate had one ST type except ST3, ST9, ST11, ST13, ST18, and ST21, which
had 2, 5, 2, 4, 2, and 2 ST types, respectively. In our study, SOD2 provided the highest number
of alleles (18), and another study showed a similar high number of alleles for SOD2 [
Moreover, we combined five loci (ACT, CAL, RPB2, BT2 and SOD2) for objective genetic
relationship analysis between Thailand (representing South-east Asia), China and Japan (Asia), and
France (Europe): (i) to assess the relationship between clinical and environmental strains of
S. apiospermum; and (ii) to assess the global variation between S. apiospermum strains. Our
results detected a close relationship between the environmental strains from Thailand and
the clinical strains from France [
], China and Japan [
]. Therefore, French, Chinese and
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Fig 1. Phylogenetic network (A±H). SplitTree decomposition analysis using the neighbor-net algorithm of each of the
eight genes i.e. A. ACT, B. CAL, C. RPB2, D. BT2, E. SOD2, F. ITS, G. TEF-1α and H. TUB (in blanket to show the number
of the collection name of each strain).
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Fig 2. Molecular phylogenetic maximum likelihood analysis of the concatenated sequences of ACT,
CAL, RPB2, BT2, SOD2, ITS, TEF-1α, and TUB genes. The tree with the highest log likelihood (-8390.7037)
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Fig 3. Molecular phylogenetic maximum likelihood analysis of the concatenated sequences of ACT,
CAL, RPB2, BT2 and SOD2 genes. The tree with the highest log likelihood (-1105.3764) is shown. The black
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circle represents the strains from Thailand (environmental isolates), the blue circle represents the strains from
France (clinical isolates), the green circle represents the strains from China (clinical isolates) and the pink
circle represents the strains from Japan (clinical isolates).
Japan isolates originated in Thailand also. These data suggest that S. apiospermum sensu stricto
isolates retrieved from different regions and different countries shared a close genetic
relatedness. One limitation of our study was a lack of clinical isolates from Thailand to compare
with our environmental strains. We hope that our data may be useful for other researchers in
future study. Interestingly, TEF-1α used as a marker in an MLST scheme for S. aurantiacum
(http://mlst.mycologylab.org/) including other filamentous fungi such as Fusarium [
not use S. apiospermum or S. boydii. In our data, TEF-1α presented the lowest number of alleles
(6) and the DP was also quite low (0.688). This result may explain why TEF-1α was not used
for S. apiospermum or S. boydii.
In summary, we here present the first phylogenetic analysis of relationships among S.
apiospermum sensu stricto in Thailand and the South-east Asian region. The results provide
valuable knowledge to assist future study and perhaps link the relationships of species in clinical
CAL, RPB2, BT2 and SOD2.)
S1 Table. Strains, specimens, countries and GenBank accession numbers of 34 sequences.
(All sequences were using for phylogenetic analysis of the concatenated sequences of ACT,
We thank Dr. Ana Alastruey-Izquierdo (National Centre for Microbiology, Instituto de Salud
Carlos III, Majadahonda 228220, España) for her valuable assistance.
Conceptualization: Natthanej Luplertlop.
Data curation: Natthanej Luplertlop.
Formal analysis: Natthanej Luplertlop.
Investigation: Thanwa Wongsuk, Potjaman Pumeesat.
Methodology: Thanwa Wongsuk, Potjaman Pumeesat, Natthanej Luplertlop.
Writing ± original draft: Thanwa Wongsuk, Potjaman Pumeesat.
Writing ± review & editing: Natthanej Luplertlop.
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