Molecular analysis of C.glabrata, Biotechnologia, Oporność wielolekowa drobnoustrojów, Candida glabrata
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Mycopathologia (2010) 170:99–105
DOI 10.1007/s11046-010-9298-1
Molecular analysis of Candida glabrata clinical isolates
Norbert Berila
•
Julius Subik
Received: 28 December 2009 / Accepted: 3 March 2010 / Published online: 17 March 2010
Springer Science+Business Media B.V. 2010
Abstract Candida glabrata is an important human
pathogen, and an understanding of the genetic
relatedness of its clinical isolates is essential for the
prevention and control of fungal infections. In this
study, we determined the relatedness of 38 Candida
glabrata clinical isolates originating from two teach-
ing hospitals in Slovakia. The 14 different genotypes
were found by using microsatellite marker analysis
(RPM2, MTI and Cg6) and DNA sequencing for
analysis of the entire ERG11 gene. Subsequent
sequencing of amplified DNA fragments of the
PDR1, NMT1, TRP1 and URA3 loci in ten selected
clinical isolates revealed identical DNA sequence
profiles in five of them. They displayed the same
microsatellite marker sizes and contained the same
H576Y amino acid substitution recently described in
the Pdr1p multidrug resistance transcription factor
responsible for azole resistance. These results dem-
onstrate the genetic diversity of C. glabrata clinical
isolates in our hospitals and indicate a common
clonal origin of some drug resistant ones.
Introduction
The haploid pathogenic yeast Candida glabrata is
considered to be the second most commonly isolated
Candida species from both bloodstream [
1
–
3
] and
vaginal infections [
4
–
6
]. It is phylogenetically closely
related to Saccharomyces cerevisiae [
7
,
8
], but is less
susceptible to azole antifungals when compared with
Candida albicans [
9
]. Therefore, the identification,
pathogenicity and epidemiology of this species are of
great importance.
Recently, we have described the susceptibilities to
fluconazole, itraconazole and voriconazole, and the
molecular mechanisms responsible for azole resis-
tance in a collection of C. glabrata clinical isolates
recovered from patients in two teaching hospitals in
Slovakia. Two amino acid substitutions, L347F and
H576Y, in the Pdr1p multidrug resistance transcrip-
tion factor were found to be associated with fluco-
nazole resistance. The same amino acid substitution
(H576Y) in Pdr1p has been identified in the 5 isolates
recovered from different patients in the same hospital
[
10
]. The understanding of genetic relatedness of
drug resistant clinical isolates is important for the
prevention and control of fungal infections.
Among a variety of genotyping methods utilized
for the fungal strain delineation, [
11
,
12
] are two
PCR-based methods. Multilocus sequence typing
[
13
–
15
] and microsatellite-based multiple-locus
variable-number tandem-repeat analysis [
16
,
17
]
have been increasingly used due to their high
Keywords Candida glabrata
Drug resistance
DNA sequencing
Microsatellite marker
N. Berila
J. Subik (
&
)
Department of Microbiology and Virology, Faculty of
Natural Sciences, Comenius University in Bratislava,
Mlynska dolina B-2, 842 15 Bratislava 4, Slovak Republic
e-mail: subik@fns.uniba.sk
123
100
Mycopathologia (2010) 170:99–105
discriminatory power relevant to C. glabrata strain
differentiation. Multilocus sequence typing relies on
DNA sequence analysis of nucleotide polymorphisms
within selected housekeeping genes [
13
]. Microsat-
ellite marker analysis relies on the amplification of
microsatellite sequences defined as repetitive
stretches of two to seven nucleotides in specific
genes [
16
,
17
].
The aim of this study was to determine the genetic
relatedness of the C. glabrata clinical isolates and
assess the relationship between their antifungal
susceptibility, molecular basis of azole resistance
and gene diversity.
determined by primer elongation with an automated
DNA sequencer (ABI Prism 3100; Applied Biosys-
tems, Foster City, CA). DNA sequencing primers
were the same as those used for PCR amplification
and were supplemented with others as follows:
CgERG11-Srev 5
0
-AGGCAAGTTAGGGAAGACG
A-3
0
, CgPDR1-F3 5
0
-GGTCTTGGTTACTGTGTTC
ACCT-3
0
, CgPDR1-RI 5
0
-GACAATGGAATCGTAA
TCGCTC-3
0
, CgPDR1-F6 5
0
-TTTCTGAAGTATG
CCCTGACC-3
0
and CgPDR1-R 5
0
-CCGATAAGG-
GAGATGCAGTT-3
0
. Sequence data were compared
with genome sequences of the standard strain C. glab-
rata ATCC 2001 (synonym CBS 138;
using the BLAST
program. For DNA fragment length analysis, the PCR
products were subjected to electrophoresis on a DNA
sequence analyzer (ABI Prism 3100; Applied Biosys-
tems, Foster City, CA) and the data analyzed with the
GeneScan software version 4.0 (Applied Biosystems,
Foster City, CA). The strain C. glabrata ATCC 2001,
with a known microsatellite pattern [
16
,
17
], was run as
a control in each experiment. The dendrogram of iso-
lates was constructed from the matrix of pair-wise
similarity from the 6,554 bp concatenated DNA
sequences of the ERG11, PDR1, NMT1, TRP1 and
URA3 loci using the BioNumerics software 4.1
(AppliedMath, Sint-Martens-Latem, Belgium).
Materials and Methods
Microorganisms
The 38 C. glabrata clinical isolates used in this study
were recovered from patients treated at University
Hospital in Nitra (isolates 1–28) or collected from
vaginal samples of patients in University Hospital in
Bratislava (isolates 29–38) in the years 2006 and
2007. C. glabrata ATCC 2001 (synonym CBS 138)
was used as the reference strain. The anatomical sites
of isolation and azole susceptibilities of isolates [
10
]
are listed in Table
1
. The isolates were grown at 30C
in complete YEPD medium (1% yeast extract, 2%
bacto-peptone and 2% glucose). When grown on
solid media, 2% agar was added to the medium.
Isolates were stored at 4C, subcultured as required
and stored at -80C in YEPD broth containing 20%
glycerol.
Results and Discussion
To assess the genetic relatedness of 38 C. glabrata
clinical isolates listed in Table
1
, the microsatellite
length polymorphism was determined for 3 markers
RPM2, MTI and Cg6 selected due to their high
discriminatory power [
16
,
17
]. As shown in Table
3
,
the 2, 2 and 4 alleles were found for the RPM2, MTI
and Cg6 markers, respectively. Their combination
resulted in 5 different microsatellite marker size
patterns that were observed among the isolates and
the reference strain C. glabrata ATCC 2001. Most of
the isolates, 30 out of 38, exhibited two marker size
patterns. The RPM2, MTI and Cg6, 19 and 11 isolates
displayed the 128, 237, 320 and 134, 237, 315
microsatellite marker size patterns, respectively.
Each of these patterns was found in clinical isolates
which originated from the two hospitals. The same
patterns were observed among azole susceptible and
azole resistant isolates (Tables
1
,
3
) indicating the
DNA extraction, PCR Amplification and DNA
Sequencing
Genomic DNA from isolates was extracted [
18
] and
used as a template for amplification of the CgERG11
gene and fragments of the CgPDR1, RPM2, MTI,
Cg6, NMT1, TRP1 and URA3 loci. PCR was carried
out with an Extensor Hi-Fidelity PCR Enzyme Kit
(ABgene, Hamburg, Germany). Sequences of primer
pairs used and their labeling are described in Table
2
.
Resulting amplicons were purified with a QIA quick
PCR Purification Kit (Qiagen, Hilden, Germany), and
the nucleotide sequences for both strands were
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Mycopathologia (2010) 170:99–105
101
Table 1 List of the
C. glabrata clinical isolates
used
C. glabrata isolate
Site of isolation
In vitro susceptibility to
Fluconazole
Itraconazole
1
Endotracheal sputum
R
R
2
Urine
S
SDD
3
Tonsil
R
R
4
Urine
S
S
5
Endotracheal sputum
S
SDD
6
Tissue
S
SDD
7
Endotracheal sputum
R
R
8
Endotracheal sputum
S
SDD
9
Tongue
S
S
10
Oral cavity
S
SDD
11
Trachea
SDD
S
12
Vagina
S
SDD
13
Endotracheal sputum
SDD
SDD
14
Abscess
SDD
R
15
Endotracheal sputum
S
R
16
Tracheal canila
R
R
17
Endotracheal sputum
R
R
18
Tonsil
SDD
R
19
Tonsil
SDD
R
20
Urine
R
SDD
21
Endotracheal sputum
R
R
22
Endotracheal sputum
R
R
23
Blood
R
R
24
Endotracheal sputum
S
R
25
Trachea
R
R
26
Urine
S
R
27
Urine
R
R
28
Vagina
S
R
29
Vagina
S
R
30
Vagina
S
R
31
Vagina
SDD
R
32
Vagina
S
R
33
Vagina
SDD
R
34
Vagina
S
R
35
Vagina
SDD
R
36
Vagina
SDD
SDD
37
Vagina
S
R
S susceptible,
SDD susceptible dose
dependent, R resistant [
10
]
38
Vagina
S
R
ATCC 2001
Reference strain
S
SDD
lack of correlation between the marker size patterns
and azole resistance.
To further differentiate the isolates displaying the
same microsatellite marker size patterns, the DNA
sequence analysis of the entire ERG11 gene, known
to be polymorphic particularly in drug resistant yeast
isolates [
9
,
19
], was carried out. Ten different ERG11
alleles were found (Table
3
). The analysis, using a
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102
Mycopathologia (2010) 170:99–105
Table 2 Sequences and features of the primers used for amplification
Gene/
locus
GeneBank
accession no.
Primer
name
Orientation
a
Sequence (5
0
–3
0
)
Fluorescence
labeling
Reference
ERG11 L40389
ERG11-For F
GCGATCCCTTCATGTCCATTGTC
–
[
10
]
ERG11-Rev R
GGCTAATGAATCAGCGTATATCCCG
–
PDR1 AY700584.1 PDR1-F2
F
GTGACTCGGAAGAAAGGGAC
–
[
10
]
PDR1-Rev
R
CCGATAAGGGAGATGCAGTT
–
PDR1-F5
F
CAGAGACATCATATGAGGCAATCAG
–
PDR1-STOP R
GATATATGAATTCTCATTCAGAATCGAAGGG –
NMT1 AF073886
NMT1-For
F
GCCGGTGTGGTGTTGCCTGCTC
–
[
13
]
NMT1-Rev R
CGTTACTGCGGTGCTCGGTGTCG
–
TRP1 U31471
TRP1-For
F
AATTGTTCCAGCGTTTTTGT
–
[
13
]
TRP1-Rev
R
GACCAGTCCAGCTCTTTCAC
–
URA3 L13661
URA3-For
F
AGCGAATTGTTGAAGTTGGTTGA
–
[
13
]
URA3-Rev R
AATTCGGTTGTAAGATGATGTTGC
–
RPM2 AF338039
RPM2-FOR F
ATCTCCCAACTTCTCGTAGCC
5
0
FAM
b
[
16
]
RPM2-REV R
ACTTGAACGACTTGAACGCC
–
MTI
J05133
MTI-FOR
F
CAGCAATAATAGCTTCTGACTATGAC
5
0
FAM
b
[
16
]
MTI-REV
R
GACAGGAGCAACCGTTAGGA
–
Cg6
BZ298679
Cg6-FOR
F
AGCAAGAGGGAGGAGGAAACT
5
0
FAM
2
[
17
]
Cg6-REV
R
AAATCCGGGGATAGATGAGG
–
a
F forward primer, R reverse primer
b
FAM 6-carboxyfluorescein
combination of 4 loci involving 3 microsatellite
markers and the ERG11 gene, resulted in identifica-
tion of 15 distinct multilocus genotypes (G1 to G15)
among the 38 clinical isolates and the reference strain
ATCC 2001. The 27 clinical isolates from different
sites of isolation recovered from different patients in
one hospital were distributed into 10 genotypes
(groups G1, G3, G4, G7-G9, G11-G14). The 11
vaginal isolates from the other hospital were distrib-
uted among 7 genotypes (groups G1, G2, G4–G6,
G10, G13). The G1, G4, and G13 genotypes
contained isolates from both hospitals.
To assess the genetic relatedness of 10 C. glabrata
isolates, investigated recently for the molecular
mechanisms involved in a decreased susceptibility
to azole antifungals [
10
], the combination of micro-
satellite marker analysis and DNA sequence analysis
of five genetic loci (ERG11, NMT1, TRP1, URA3 and
PDR1) was used. The 3 genetic loci NMT1, TRP1 and
URA3 were recommended for multilocus sequence
typing due to their high sequence variability [
13
].
They were supplemented with the ERG11 and PDR1
markers known to be polymorphic particularly in
drug resistant C. albicans and C. glabrata clinical
isolates [
9
,
10
,
20
]. Microsatellite marker analysis
revealed two distinct multilocus genotypes (Table
4
).
Only the Cg6 marker was discriminatory, dividing
the 10 strains into two genotypes involving two
(isolates 3 and 28) and eight (isolates 1, 7, 21, 22, 27,
29, 30 and 32) clinical isolates. Therefore, for each of
the 10 isolates, a total of 6,554 bp from ERG11 and
four additional loci (PDR1, NMT1, TRP1 and URA3)
were also sequenced. Seventeen nucleotide sites were
found to be polymorphic (Table
4
). The number of
polymorphic sites per locus was 7 in NMT1, followed
by 5 in PDR1,4inERG11 and 1 in URA3. The
polymorphisms defined were 4 (PDR1), 3 (ERG11), 3
(NMT1) and 2 (URA3) genotypes per locus. No
polymorphism was observed in TRP1. Among the
five loci, PDR1 and ERG11 gave the highest
discrimination ratio yielding 4 and 3 different geno-
types from 5 and 4 polymorphic sites, respectively.
123
Table 3 Genotypes of C. glabrata clinical isolates and the reference strain ATCC 2001 based on the microsatellite marker analysis and the ERG11 gene sequencing
C. glabrata isolate
Marker allele size
(bp)
Base substitution in the ERG11 gene
Genotype
group
RPM2 MTI Cg6 G87A G90A C192T G296A C539A C588T T768C T834C C918T G927A A1023G T1275C A1505T T1557A
3, 28
128
237 315 ---- - -?--? ? - - ? G1
37
128
237 315 ---- - -??-- ? - - ? G2
1, 7, 14, 15, 16,
17, 18, 19, 21, 22,
25, 27
128
237 320 ---- - ??-?- ? - ? ? G3
4, 11, 29, 30, 32
128
237 320 ---- - ??-?- ? - - ? G4
31
128
237 320 ---- - -?--? ? - - ? G5
33
128
237 320 ---- - -??-- ? - - ? G6
2, 20
128
237 322 ?-?- - -??-- ? - - ? G7
12
128
237 322 ---- - -??-- ? - - ? G8
23
128
237 322 ---- - -?--- ? ? - ? G9
35
128
237 322 ---- - -?--? ? - - ? G10
5
134
237 315 -?-- - -?--- ? - - ? G11
6
134
237 315 -?-? ? -?--? ? - - ? G12
8, 9, 10, 13, 26,
34, 36, 38
134
237 315 ---- - -?--? ? - - ? G13
24
134
237 315 ---- - ??--- ? - - ? G14
ATCC 2001
128
228 326 G
G
C
G
C
C
T
T
C
G
A
T
A
T
G15
?, base substitution present; -, base substitution absent
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