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
Ó Springer Science+Business Media B.V. 2010
Abstract Candida glabrata is an important human Introduction
pathogen, and an understanding of the genetic
relatedness of its clinical isolates is essential for the The haploid pathogenic yeast Candida glabrata is
prevention and control of fungal infections. In this considered to be the second most commonly isolated
study, we determined the relatedness of 38 Candida Candida species from both bloodstream [1 3] and
glabrata clinical isolates originating from two teach- vaginal infections [4 6]. It is phylogenetically closely
ing hospitals in Slovakia. The 14 different genotypes related to Saccharomyces cerevisiae [7, 8], but is less
were found by using microsatellite marker analysis susceptible to azole antifungals when compared with
(RPM2, MTI and Cg6) and DNA sequencing for Candida albicans [9]. Therefore, the identification,
analysis of the entire ERG11 gene. Subsequent pathogenicity and epidemiology of this species are of
sequencing of amplified DNA fragments of the great importance.
PDR1, NMT1, TRP1 and URA3 loci in ten selected Recently, we have described the susceptibilities to
clinical isolates revealed identical DNA sequence fluconazole, itraconazole and voriconazole, and the
profiles in five of them. They displayed the same molecular mechanisms responsible for azole resis-
microsatellite marker sizes and contained the same tance in a collection of C. glabrata clinical isolates
H576Y amino acid substitution recently described in recovered from patients in two teaching hospitals in
the Pdr1p multidrug resistance transcription factor Slovakia. Two amino acid substitutions, L347F and
responsible for azole resistance. These results dem- H576Y, in the Pdr1p multidrug resistance transcrip-
onstrate the genetic diversity of C. glabrata clinical tion factor were found to be associated with fluco-
isolates in our hospitals and indicate a common nazole resistance. The same amino acid substitution
clonal origin of some drug resistant ones. (H576Y) in Pdr1p has been identified in the 5 isolates
recovered from different patients in the same hospital
Keywords Candida glabrata Drug resistance [10]. The understanding of genetic relatedness of
DNA sequencing Microsatellite marker 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
N. Berila J. Subik (&)
PCR-based methods. Multilocus sequence typing
Department of Microbiology and Virology, Faculty of
[13 15] and microsatellite-based multiple-locus
Natural Sciences, Comenius University in Bratislava,
variable-number tandem-repeat analysis [16, 17]
Mlynska dolina B-2, 842 15 Bratislava 4, Slovak Republic
e-mail: [e-mail] have been increasingly used due to their high
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100 Mycopathologia (2010) 170:99 105
discriminatory power relevant to C. glabrata strain determined by primer elongation with an automated
differentiation. Multilocus sequence typing relies on DNA sequencer (ABI Prism 3100; Applied Biosys-
DNA sequence analysis of nucleotide polymorphisms tems, Foster City, CA). DNA sequencing primers
within selected housekeeping genes [13]. Microsat- were the same as those used for PCR amplification
ellite marker analysis relies on the amplification of and were supplemented with others as follows:
microsatellite sequences defined as repetitive CgERG11-Srev 50-AGGCAAGTTAGGGAAGACG
stretches of two to seven nucleotides in specific A-30, CgPDR1-F3 50-GGTCTTGGTTACTGTGTTC
genes [16, 17]. ACCT-30, CgPDR1-RI 50-GACAATGGAATCGTAA
The aim of this study was to determine the genetic TCGCTC-30, CgPDR1-F6 50-TTTCTGAAGTATG
relatedness of the C. glabrata clinical isolates and CCCTGACC-30 and CgPDR1-R 50-CCGATAAGG-
assess the relationship between their antifungal GAGATGCAGTT-30. Sequence data were compared
susceptibility, molecular basis of azole resistance with genome sequences of the standard strain C. glab-
and gene diversity. rata ATCC 2001 (synonym CBS 138; http://
cbi.labri.fr/Genolevures/elt/CAGL) using the BLAST
program. For DNA fragment length analysis, the PCR
Materials and Methods products were subjected to electrophoresis on a DNA
sequence analyzer (ABI Prism 3100; Applied Biosys-
Microorganisms tems, Foster City, CA) and the data analyzed with the
GeneScan software version 4.0 (Applied Biosystems,
The 38 C. glabrata clinical isolates used in this study Foster City, CA). The strain C. glabrata ATCC 2001,
were recovered from patients treated at University with a known microsatellite pattern [16, 17], was run as
Hospital in Nitra (isolates 1 28) or collected from a control in each experiment. The dendrogram of iso-
vaginal samples of patients in University Hospital in lates was constructed from the matrix of pair-wise
Bratislava (isolates 29 38) in the years 2006 and similarity from the 6,554 bp concatenated DNA
2007. C. glabrata ATCC 2001 (synonym CBS 138) sequences of the ERG11, PDR1, NMT1, TRP1 and
was used as the reference strain. The anatomical sites URA3 loci using the BioNumerics software 4.1
of isolation and azole susceptibilities of isolates [10] (AppliedMath, Sint-Martens-Latem, Belgium).
are listed in Table 1. The isolates were grown at 30°C
in complete YEPD medium (1% yeast extract, 2%
bacto-peptone and 2% glucose). When grown on Results and Discussion
solid media, 2% agar was added to the medium.
Isolates were stored at 4°C, subcultured as required To assess the genetic relatedness of 38 C. glabrata
and stored at -80°C in YEPD broth containing 20% clinical isolates listed in Table 1, the microsatellite
glycerol. 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,
DNA extraction, PCR Amplification and DNA
the 2, 2 and 4 alleles were found for the RPM2, MTI
Sequencing and Cg6 markers, respectively. Their combination
resulted in 5 different microsatellite marker size
Genomic DNA from isolates was extracted [18] and
patterns that were observed among the isolates and
used as a template for amplification of the CgERG11 the reference strain C. glabrata ATCC 2001. Most of
gene and fragments of the CgPDR1, RPM2, MTI,
the isolates, 30 out of 38, exhibited two marker size
Cg6, NMT1, TRP1 and URA3 loci. PCR was carried patterns. The RPM2, MTI and Cg6, 19 and 11 isolates
out with an Extensor Hi-Fidelity PCR Enzyme Kit
displayed the 128, 237, 320 and 134, 237, 315
(ABgene, Hamburg, Germany). Sequences of primer
microsatellite marker size patterns, respectively.
pairs used and their labeling are described in Table 2. Each of these patterns was found in clinical isolates
Resulting amplicons were purified with a QIA quick
which originated from the two hospitals. The same
PCR Purification Kit (Qiagen, Hilden, Germany), and
patterns were observed among azole susceptible and
the nucleotide sequences for both strands were azole resistant isolates (Tables 1, 3) indicating the
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Mycopathologia (2010) 170:99 105 101
Table 1 List of the
C. glabrata isolate Site of isolation In vitro susceptibility to
C. glabrata clinical isolates
used
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,
38 Vagina S R
SDD susceptible dose
ATCC 2001 Reference strain S SDD
dependent, R resistant [10]
lack of correlation between the marker size patterns sequence analysis of the entire ERG11 gene, known
and azole resistance. to be polymorphic particularly in drug resistant yeast
To further differentiate the isolates displaying the isolates [9, 19], was carried out. Ten different ERG11
same microsatellite marker size patterns, the DNA 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/ GeneBank Primer Orientationa Sequence (50 30) Fluorescence Reference
locus accession no. name labeling
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 50 FAMb [16]
RPM2-REV R ACTTGAACGACTTGAACGCC 
MTI J05133 MTI-FOR F CAGCAATAATAGCTTCTGACTATGAC 50 FAMb [16]
MTI-REV R GACAGGAGCAACCGTTAGGA 
Cg6 BZ298679 Cg6-FOR F AGCAAGAGGGAGGAGGAAACT 50 FAM2 [17]
Cg6-REV R AAATCCGGGGATAGATGAGG 
a
F forward primer, R reverse primer
b
FAM 6-carboxyfluorescein
combination of 4 loci involving 3 microsatellite They were supplemented with the ERG11 and PDR1
markers and the ERG11 gene, resulted in identifica- markers known to be polymorphic particularly in
tion of 15 distinct multilocus genotypes (G1 to G15) drug resistant C. albicans and C. glabrata clinical
among the 38 clinical isolates and the reference strain isolates [9, 10, 20]. Microsatellite marker analysis
ATCC 2001. The 27 clinical isolates from different revealed two distinct multilocus genotypes (Table 4).
sites of isolation recovered from different patients in Only the Cg6 marker was discriminatory, dividing
one hospital were distributed into 10 genotypes the 10 strains into two genotypes involving two
(groups G1, G3, G4, G7-G9, G11-G14). The 11 (isolates 3 and 28) and eight (isolates 1, 7, 21, 22, 27,
vaginal isolates from the other hospital were distrib- 29, 30 and 32) clinical isolates. Therefore, for each of
uted among 7 genotypes (groups G1, G2, G4 G6, the 10 isolates, a total of 6,554 bp from ERG11 and
G10, G13). The G1, G4, and G13 genotypes four additional loci (PDR1, NMT1, TRP1 and URA3)
contained isolates from both hospitals. were also sequenced. Seventeen nucleotide sites were
To assess the genetic relatedness of 10 C. glabrata found to be polymorphic (Table 4). The number of
isolates, investigated recently for the molecular polymorphic sites per locus was 7 in NMT1, followed
mechanisms involved in a decreased susceptibility by 5 in PDR1, 4 in ERG11 and 1 in URA3. The
to azole antifungals [10], the combination of micro- polymorphisms defined were 4 (PDR1), 3 (ERG11), 3
satellite marker analysis and DNA sequence analysis (NMT1) and 2 (URA3) genotypes per locus. No
of five genetic loci (ERG11, NMT1, TRP1, URA3 and polymorphism was observed in TRP1. Among the
PDR1) was used. The 3 genetic loci NMT1, TRP1 and five loci, PDR1 and ERG11 gave the highest
URA3 were recommended for multilocus sequence discrimination ratio yielding 4 and 3 different geno-
typing due to their high sequence variability [13]. types from 5 and 4 polymorphic sites, respectively.
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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 Base substitution in the ERG11 gene Genotype
(bp) 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, 128 237 320 - - - - - ? ? - ? - ? - ? ? G3
17, 18, 19, 21, 22,
25, 27
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, 134 237 315 - - - - - - ? - - ? ? - - ? G13
34, 36, 38
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
Mycopathologia (2010) 170:99 105
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104 Mycopathologia (2010) 170:99 105
The dendrogram indicating the relatedness of the
10 clinical isolates determined by DNA sequence
analysis with amplified fragments of 5 genes is shown
in Fig. 1. Isolates 3 and 28 were differentiated on the
variable sequences in the PDR1 and NMT1 loci
(Table 4). Moreover, isolate 3 was resistant to
fluconazole and itraconazole due to gain-of-function
L347F mutation in Pdr1p and is responsible for
overexpression of multidrug resistance efflux pumps
Cdr1p and Cdr2p [10]. On the other hand, the 5
fluconazole resistant isolates, 1, 7, 21, 22 and 27,
containing the H576Y amino acid substitution in
Pdr1p [10], could not be differentiated even by a
combination of microsatellite marker and DNA
sequence analyses using the genetic loci indicated.
With the exception of isolate 27 recovered from
urine, the other 4 isolates (isolates 1, 7, 21 and 22)
were all recovered from endotracheal sputum of
different patients in a 2-year period. Clinical isolates
7, 21, 22 and 1, 27 were recovered in the years 2006
and 2007, respectively. Additionally, the same azole
resistance patterns, amino acid substitutions in Pdr1p
and Erg11p, DNA sequences in the NMT1 and URA3
loci, and microsatellite marker sizes indicate a
common clonal origin of these five isolates recovered
from patients in the same hospital.
Isolates 16, 17 and 25 were recovered from trachea
in the same hospital and also displayed cross-resis-
tance to fluconazole, itraconazole and voriconazole
[10]. Based on the results of microsatellite marker
analysis and the ERG11 gene sequencing, they belong
to the same genotype as isolates 1, 7, 21, 22 and 27
(Table 3). This suggests that they may harbor the same
Fig. 1 Dendrogram showing the relatedness of ten C. glabrata
clinical isolates taking into account the concatenated sequences
from the ERG11, PDR1, NMT1, TRP1 and URA3 loci. Isolates
1, 3, 7, 21, 22 and 27 were resistant to fluconazole; isolates 28,
29, 30 and 32 were fluconazole sensitive
123
URA3
C
A
NMT1
C
A
T
G
ERG11
PDR1
128
237
315
T
T
CTCC
A
G
G
GATA
128
237
315
T
C
C
A
T
C
C
A
A
G
T
G
G
G
T
T
A
128
237
320
C
C
C
A
T
T
T
G
A
A
C
A
A
C
A
C
G
RPM2
MTI
Cg6
837
1,039
1,726
2,319
2,578
588
918
927
1,505
681
744
822
955
1,221
1,238
1,246
601
1, 7, 21, 22, 27
128
237
320
C
C
T
T
C
TTGT
A
A
CACG
3
28
29, 30, 32
Table 4 Polymorphisms of the RPM2, MTI, Cg6, PDR1, ERG11, NMT1 and URA3 loci in the 10 azole resistant C. glabrata clinical isolates
C. glabrata isolate
Marker allele size (bp)
The position of the polymorphism in the coding sequence of the locus
The polymorphisms indicated by bold result in the L347T, H576Y and E502 V amino acid substitutions in Pdr1p and Erg11p, respectively
Mycopathologia (2010) 170:99 105 105
7. Bialkova A, Subik J. Biology of the pathogenic yeast
gain-of-function H576Y mutation in Pdr1p associated
Candida glabrata. Folia Microbiol. 2006;51:3 20.
with azole resistance. Along with these fluconazole
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9. Sanglard D. Genomic view on antifungal resistance
tivity (isolate 15) and fluconazole dose dependence
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d Enfert Ch, Hube B, editors. Candida: comparative and
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2007. p. 359 82.
10. Berila N, Borecka S, Dzugasova V, Bojnansky J, Subik J.
tion in one of the teaching hospitals cannot be ruled out
Mutations in CgPDR1 and CgERG11 genes in azole-
as the source of the recovered isolates. To our
resistant Candida glabrata clinical isolates from Slovakia.
knowledge, this is the first study presenting the genetic
Int J Antimicrob Agents. 2009;33:574 8.
relatedness of C. glabrata clinical isolates for which
11. Dassanayake RS, Samaranayake LP. Amplification-based
nucleic acid scanning techniques to assess genetic poly-
the molecular mechanisms of multidrug resistance
morphism in Candida. Crit Rev Microbiol. 2003;29:1 24.
were found to be associated with mutations in the
12. Abbes S, Amouri I, Sellami H, Sellami A, Makni F,
CgPDR1 gene.
Ayadi A. A review of molecular techniques to type
Candida glabrata isolates. Mycoses. 2010. doi:10.1111/
Acknowledgments We thank H. Drahovska for help with
j.1439-0507.2009.01753.x.
BioNumerics software and D. Hanson for careful reading of the
13. Dodgson AR, Pujol C, Denning DW, Soll DR, Fox AJ.
manuscript. This work was supported by grants from the
Multilocus sequence typing of Candida glabrata reveals
Slovak Research and Developmental Agency (LPP-0022-06,
geographically enriched clades. J Clin Microbiol. 2003;
LPP-0011-07, VVCE-0064-07) and the Slovak Grant Agency
41:5709 17.
of Science (VEGA 1/0001/09).
14. Lin CY, Chen YC, Lo HJ, Chen KW, Li SY. Assessment
of Candida glabrata strain relatedness by pulsed-field gel
electrophoresis and multilocus sequence typing. J Clin
Microbiol. 2007;45:2452 9.
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