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

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.

Keywords

Candida glabrata

Drug resistance

DNA sequencing

Microsatellite marker

Introduction

The haploid pathogenic yeast Candida glabrata is
considered to be the second most commonly isolated
Candida species from both bloodstream [

–

] and

vaginal infections [

–

]. It is phylogenetically closely

related to Saccharomyces cerevisiae [

], but is less

susceptible to azole antifungals when compared with
Candida albicans [

]. 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
[

]. 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, [

] are two

PCR-based methods. Multilocus sequence typing
[

–

]

and

microsatellite-based

multiple-locus

variable-number tandem-repeat analysis [

]

have been increasingly used due to their high

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

Mycopathologia (2010) 170:99–105
DOI 10.1007/s11046-010-9298-1

discriminatory power relevant to C. glabrata strain
differentiation. Multilocus sequence typing relies on
DNA sequence analysis of nucleotide polymorphisms
within selected housekeeping genes [

]. Microsat-

ellite marker analysis relies on the amplification of
microsatellite

sequences

defined

repetitive

stretches of two to seven nucleotides in specific
genes [

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.

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 [

]

are listed in Table

. The isolates were grown at 30

°C

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 4

°C, subcultured as required

and stored at -80

°C in YEPD broth containing 20%

glycerol.

DNA extraction, PCR Amplification and DNA
Sequencing

Genomic DNA from isolates was extracted [

] 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

Resulting amplicons were purified with a QIA quick
PCR Purification Kit (Qiagen, Hilden, Germany), and
the nucleotide sequences for both strands were

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

-AGGCAAGTTAGGGAAGACG

A-3

, CgPDR1-F3 5

-GGTCTTGGTTACTGTGTTC

ACCT-3

, CgPDR1-RI 5

-GACAATGGAATCGTAA

TCGCTC-3

, CgPDR1-F6 5

-TTTCTGAAGTATG

CCCTGACC-3

and CgPDR1-R 5

-CCGATAAGG-

GAGATGCAGTT-3

. Sequence data were compared

with genome sequences of the standard strain C. glab-
rata ATCC 2001 (synonym CBS 138;

http://

cbi.labri.fr/Genolevures/elt/CAGL

) 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 [

], 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).

Results and Discussion

To assess the genetic relatedness of 38 C. glabrata
clinical isolates listed in Table

, the microsatellite

length polymorphism was determined for 3 markers
RPM2, MTI and Cg6 selected due to their high
discriminatory power [

]. As shown in Table

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

) indicating the

100

Mycopathologia (2010) 170:99–105

123

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 [

], was carried out. Ten different ERG11

alleles were found (Table

). The analysis, using a

Table 1

List of the

C. glabrata clinical isolates
used

S susceptible,
SDD susceptible dose
dependent, R resistant [

]

C. glabrata isolate

Site of isolation

In vitro susceptibility to

Fluconazole

Itraconazole

Endotracheal sputum

Urine

SDD

Tonsil

Urine

Endotracheal sputum

SDD

Tissue

SDD

Endotracheal sputum

SDD

Tongue

Oral cavity

SDD

Trachea

SDD

Vagina

SDD

Endotracheal sputum

SDD

Abscess

SDD

Endotracheal sputum

Tracheal canila

Endotracheal sputum

Tonsil

SDD

Tonsil

SDD

Urine

SDD

Endotracheal sputum

Blood

Endotracheal sputum

Trachea

Urine

Vagina

SDD

Vagina

SDD

Vagina

SDD

Vagina

SDD

Vagina

ATCC 2001

Reference strain

SDD

Mycopathologia (2010) 170:99–105

101

123

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 [

], 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 [

They were supplemented with the ERG11 and PDR1
markers known to be polymorphic particularly in
drug resistant C. albicans and C. glabrata clinical
isolates [

]. Microsatellite marker analysis

revealed two distinct multilocus genotypes (Table

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

). The number of

polymorphic sites per locus was 7 in NMT1, followed
by 5 in PDR1, 4 in ERG11 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.

Table 2

Sequences and features of the primers used for amplification

Gene/
locus

GeneBank
accession no.

Primer
name

Orientation

Sequence (5

–3

)

Fluorescence
labeling

Reference

ERG11 L40389

ERG11-For

GCGATCCCTTCATGTCCATTGTC

–

[

]

ERG11-Rev

GGCTAATGAATCAGCGTATATCCCG

–

PDR1

AY700584.1

PDR1-F2

GTGACTCGGAAGAAAGGGAC

–

[

]

PDR1-Rev

CCGATAAGGGAGATGCAGTT

–

PDR1-F5

CAGAGACATCATATGAGGCAATCAG

–

PDR1-STOP R

GATATATGAATTCTCATTCAGAATCGAAGGG –

NMT1

AF073886

NMT1-For

GCCGGTGTGGTGTTGCCTGCTC

–

[

]

NMT1-Rev

CGTTACTGCGGTGCTCGGTGTCG

–

TRP1

U31471

TRP1-For

AATTGTTCCAGCGTTTTTGT

–

[

]

TRP1-Rev

GACCAGTCCAGCTCTTTCAC

–

URA3

L13661

URA3-For

AGCGAATTGTTGAAGTTGGTTGA

–

[

]

URA3-Rev

AATTCGGTTGTAAGATGATGTTGC

–

RPM2

AF338039

RPM2-FOR

ATCTCCCAACTTCTCGTAGCC

FAM

[

]

RPM2-REV

ACTTGAACGACTTGAACGCC

–

MTI

J05133

MTI-FOR

CAGCAATAATAGCTTCTGACTATGAC

FAM

[

]

MTI-REV

GACAGGAGCAACCGTTAGGA

–

Cg6

BZ298679

Cg6-FOR

AGCAAGAGGGAGGAGGAAACT

FAM

[

]

Cg6-REV

AAATCCGGGGATAGATGAGG

–

F forward primer, R reverse primer

FAM 6-carboxyfluorescein

102

Mycopathologia (2010) 170:99–105

123

Table

Genotypes

glabrata

clinical

isolates

and

the

reference

strain

ATCC

2001

based

the

microsatellite

marker

analysis

and

the

ERG11

gene

sequencing

glabrata

isolate

Marker

allele

size

(bp)

Base

substitution

the

ERG11

gene

Genotype

group

RPM2

MTI

Cg6

G87A

G90A

C192T

G296A

C539A

C588T

T768C

T834C

C918T

G927A

A1023G

T1275C

A1505T

T1557A

128

237

315

128

237

315

14,

15,

16,

17,

18,

19,

21,

22,

25,

128

237

320

11,

29,

30,

128

237

320

128

237

320

128

237

320

128

237

322

128

237

322

128

237

322

128

237

322

G10

134

237

315

G11

134

237

315

G12

10,

13,

26,

34,

36,

134

237

315

G13

134

237

315

??-

G14

ATCC

2001

128

228

326

G15

base

substitution

present;

base

substitution

absent

Mycopathologia (2010) 170:99–105

103

123

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.

. Isolates 3 and 28 were differentiated on the

variable sequences in the PDR1 and NMT1 loci
(Table

). 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 [

]. On the other hand, the 5

fluconazole resistant isolates, 1, 7, 21, 22 and 27,
containing the H576Y amino acid substitution in
Pdr1p [

], 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
[

]. 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

). This suggests that they may harbor the same

Table

Polymorphisms

the

RPM2,

MTI,

Cg6,

PDR1,

ERG11,

NMT1

and

URA3

loci

the

azole

resistant

glabrata

clinical

isolates

glabrata

isolate

Marker

allele

size

(bp)

The

position

the

polymorphism

the

coding

sequence

the

locus

PDR1

ERG11

NMT1

URA3

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

21,

22,

128

237

320

TTG

128

237

315

GAT

128

237

315

29,

30,

128

237

320

The

polymorphisms

indicated

bold

result

the

L347T,

H576Y

and

E502

amino

acid

substitutions

Pdr1p

and

Erg11p,

respectively

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

104

Mycopathologia (2010) 170:99–105

123

gain-of-function H576Y mutation in Pdr1p associated
with azole resistance. Along with these fluconazole
resistant isolates, the genotype group G3 also con-
tained clinical isolates displaying fluconazole sensi-
tivity (isolate 15) and fluconazole dose dependence
(isolates 14, 18 and 19; Table

) [

]. The microevo-

lution of C. glabrata isolates from sensitive to
fluconazole resistant ones and their clonal prolifera-
tion in one of the teaching hospitals cannot be ruled out
as the source of the recovered isolates. To our
knowledge, this is the first study presenting the genetic
relatedness of C. glabrata clinical isolates for which
the molecular mechanisms of multidrug resistance
were found to be associated with mutations in the
CgPDR1 gene.

Acknowledgments

We thank H. Drahovska for help with

BioNumerics software and D. Hanson for careful reading of the
manuscript. This work was supported by grants from the
Slovak Research and Developmental Agency (LPP-0022-06,
LPP-0011-07, VVCE-0064-07) and the Slovak Grant Agency
of Science (VEGA 1/0001/09).

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Molecular analysis of Candida glabrata clinical isolates

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