Molecular analysis of C.glabrata.pdf

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

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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; 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 [ 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

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

S susceptible,

SDD susceptible dose

dependent, R resistant [ 10 ]

Vagina

ATCC 2001

Reference strain

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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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

GTGACTCGGAAGAAAGGGAC

–

[ 10 ]

PDR1-Rev

CCGATAAGGGAGATGCAGTT

–

PDR1-F5

CAGAGACATCATATGAGGCAATCAG

–

PDR1-STOP R

GATATATGAATTCTCATTCAGAATCGAAGGG –

NMT1 AF073886

NMT1-For

GCCGGTGTGGTGTTGCCTGCTC

–

[ 13 ]

NMT1-Rev R

CGTTACTGCGGTGCTCGGTGTCG

–

TRP1 U31471

TRP1-For

AATTGTTCCAGCGTTTTTGT

–

[ 13 ]

TRP1-Rev

GACCAGTCCAGCTCTTTCAC

–

URA3 L13661

URA3-For

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

CAGCAATAATAGCTTCTGACTATGAC

5 0

FAM b

[ 16 ]

MTI-REV

GACAGGAGCAACCGTTAGGA

–

Cg6

BZ298679

Cg6-FOR

AGCAAGAGGGAGGAGGAAACT

5 0

FAM 2

[ 17 ]

Cg6-REV

AAATCCGGGGATAGATGAGG

–

F forward primer, R reverse primer

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.

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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

(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

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

128

237 320 ---- - -?--? ? - - ? G5

128

237 320 ---- - -??-- ? - - ? G6

2, 20

128

237 322 ?-?- - -??-- ? - - ? G7

128

237 322 ---- - -??-- ? - - ? G8

128

237 322 ---- - -?--- ? ? - ? G9

128

237 322 ---- - -?--? ? - - ? G10

134

237 315 -?-- - -?--- ? - - ? G11

134

237 315 -?-? ? -?--? ? - - ? G12

8, 9, 10, 13, 26,

34, 36, 38

134

237 315 ---- - -?--? ? - - ? G13

134

237 315 ---- - ??--- ? - - ? G14

ATCC 2001

128

228 326 G

G15

?, base substitution present; -, base substitution absent

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