A review of molecular techniques to type Candida glabrata isolates

S. Abbes, I. Amouri, H. Sellami, A. Sellami, F. Makni and A. Ayadi

Laboratoire de biologie mole´culaire parasitaire et fongique, faculte´ de me´decine –Rue magida Boulila, Sfax, Tunisia

Summary

Candida glabrata has emerged as a common cause of fungal infection causing mucosal
and systemic infections. This yeast is of concern because of its reduced antifungal
susceptibility to azole antifungals such as fluconazole. A clear understanding of the
epidemiology of Candida infection and colonisation required a reliable typing system for
the evaluation of strain relatedness. In this study, we discuss the different molecular
approaches for typing C. glabrata isolates. Recent advances in the use of molecular
biology-based techniques have enabled investigators to develop typing systems with
greater sensitivities. Several molecular genotypic approaches have been developed for
fast and accurate identification of C. glabrata in vitro. These techniques have been
widely used to study diverse aspects such as nosocomial transmission. Molecular
typing of C. glabrata could also provide information on strain variation, such as
microvariation and microevolution.

Key words:

Candida glabrata, typing, polymorphism.

Introduction

Candida species are the most common opportunistic
fungal pathogens in humans, with Candida albicans
being the most prevalent pathogen in mucosal and
systemic fungal infections.

1

In addition to C. albicans,

Candida glabrata is now emerging as an important agent
in both mucosal and bloodstream infections. The
prevalence of C. glabrata has increased in the last decade
and this species now ranks as the second or third most
frequently isolated Candida species from all reported
cases of candidiasis.

2,3

To investigate the epidemiology

of this pathogen, it is imperative to develop DNA
fingerprinting methods which can assess genetic dis-
tance between independent isolates in broad epidemio-
logical studies. Different methods have previously been
applied to differentiate C. glabrata isolates, including
random amplification of polymorphic DNA,

4,5

pulsed-

field gel electrophoresis (PFGE),

6,7

multilocus enzyme

electrophoresis (MLEE),

8,9

fingerprinting with complex

DNA

probes,

10,11

multilocus

sequence

typing

(MLST)

6,11

and more recently microsatellite analysis of

markers.

12,13

In this study, we discuss the molecular

tools for genotyping analyses of C. glabrata isolates to
understand the epidemiology of this pathogenic yeast.

Restriction enzymatic electrophoresis

Restriction enzymatic electrophoresis (REA) is one of the
first applied techniques for identification and typing
pathogenic fungus. REA was initially used by Scherer
and Stevens [14], who predicted that this method,
because of its stability and reproducibility, would be
useful for epidemiological studies of Candida albicans.

14–16

This method is straightforward. DNA is extracted from
spheroplasts, digested with one or more endonucleases
and separated by electrophoresis in an agarose gel. The
banding pattern of digested DNA is then visualised by
staining with ethidium bromide. Separation depends
upon the percentage of agarose in the gel, the
electrophoresis time, the voltage and the particular
endonuclease(s) employed. The pattern is based on
different fragment lengths determined by the restriction
sites identified by the particular endonuclease(s) used.
Variations among strains can occur as a result of changes
in restriction site sequences, such as deletions and

Correspondence: A. Ayadi, Laboratoire de biologie mole´culaire parasitaire
et fongique, faculte´ de me´decine, Sfax 3029, Tunisia.
Tel.: [phone]. Fax: [phone]
E-mail: [e-mail]

Accepted for publication 25 May 2009

Review article

 2009 Blackwell Verlag GmbH • Mycoses 53, 463–467

doi:10.1111/j.1439-0507.2009.01753.x

mycoses

Diagnosis,Therapy and Prophylaxis of Fungal Diseases

insertions in the sequences between recognition sites.
REA was widely investigated to know the epidemiology of
nosocomial infection. Vasquez et al. [17] typed isolates
from 24 patients admitted to medical intensive care unit
and bone marrow transplant unit, the typing of three
environmental isolates showed common restriction
profile to five patients. Arif et al. [18] assigned that REA
with HinfI was a more suitable method than other
genotyping systems. Similarly, Vasquez et al. [19] exam-
ined various yeast with different molecular methods, and
suggested that PFGE was more sensitive than REA for
C. lusitaniae, C. parapsilosis, C. tropicalis and for C. glabrata.
It was also suggested that REA was more difficult than
PFGE for strain differentiation. So, the lack of sensitivity
of strain typing and the difficulty of reading complex
pattern made difficult to delineate the epidemiology of C.
glabrata.

19

Recently, genotyping systems based on

restriction enzymatic analysis were less used for typing
C. glabrata isolates,

20

but REA may be useful for the

identification and strain delineation of common and
emerging Candida species.

21

Random amplification of DNA

Random amplification of DNA (RAPD), described by
Williams et al. [22], is one of the most applied techniques
for the survey of the epidemiology of Candida glabrata and
other fungi.

23,24

Using random primers of approximately

10 bases, amplicons throughout the genome are targeted
and amplified. Amplified products are separated on
agarose gel and stained with ethidium bromide. The
genetic variation analysis based on RAPD allows proper
genetic diversity due to its capacity to generate random
markers from the entire genome. Contradictory findings
have been found by some authors.

4,10

Boldo et al. [4],

when analysing 47 clinical isolates from several geo-
graphical origins and diverse body sites, suggested that
the data obtained by five RAPD markers showed no
differentiation among strains (average heterozygosity,
H

0

= 0.372). On the other hand, Lockhart et al. [10]

selected nine RAPD markers from 30 primers that were
able to differentiate among 39 isolates from different
geographical populations. The genetic diversity obtained
for C. glabrata was low

4,25

compared with that reported

by other authors for the diploid species C. albicans.

9,26

However, the RAPD data for C. tropicalis and C. guillier-
mondi showed a discrimination power similar to those
obtained for C. glabrata. For this reason, it was not
possible to associate the low genetic diversity with the
mode of reproduction.

This technique is methodologically easier, less time

consuming and cheaper than the older genomic typing

methods, particularly pulsed gel electrophoresis.

18

How-

ever, the limit of this methodology was the low-stringency
conditions which induce a poor reproducibility of typing
results.

24

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis was based on variation
in electric field vectors that were able to separate large
molecular weight DNA. PFGE has been successfully
applied as a tool for typing Candida strains

19,27,28

and

was used in both identification and typing of C. glabrata
strains.

29,30

This methodology proved to be a reliable

and reproducible method for C. albicans and C. glabrata,
and is more sensitive than DNA enzymatic restriction
for typing C. albicans isolates.

19,31,32

Two electropho-

retic karyotyping systems exist [e.g. contour-clamped
homogeneous electric field gel electrophoresis (CHEF)
and transverse alternating field electrophoresis (TAFE)].
CHEF seems to be more useful than TAFE for C. glabrata
typing system. Using PFGE typing system, several
authors demonstrated that the same C. glabrata strain
remained colonised in the patients for over several
months

Õ time and that resistant and susceptible isolates

remained the same DNA type.

6,33

While leading a

comparative study between PFGE and MLST for 80
superficial C. glabrata isolates collected from 27 inten-
sive care units, Lin et al. [6] suggested that PFGE (DI =
0.99) exhibited more discriminatory power than MLST
(DI = 0.85). PFGE could efficiently divide the groups
defined by MLST. PFGE was also able to show fine
variations of a single strain. Several authors analysed
genotypic variability and azoles susceptibility among
sequential

bloodstream

isolates

by

electrophoretic

karyotyping showed minor karyotypic changes or
microevolution.

33,34

The results of PFGE have shown

a high rate of inter-laboratory agreement

24

and it

continues to be a reliable technique for C. glabrata
infection. So, the major disadvantages of PFGE are the
initial investment cost of the equipment, the inability to
run more than 20 samples at one time and the amount
of time required for the plug preparation and for
electrophoresis.

19

Southern blot hybridisation

To visualise particular fragments in the pattern, we
can probe restriction profile with radiolabelled or
biotinylated DNA sequences that recognise one or more
fragments as a result of sequence homology. Using a
screen for complex genomic fragments containing
moderately repetitive sequences, Lockhart selected two

S. Abbes et al.

464

 2009 Blackwell Verlag GmbH • Mycoses 53, 463–467

probes Cg6 and Cg12 which generate complex South-
ern blot hybridisation patterns with EcoRI-digested
C. glabrata DNA. The capacity and specificity of the
probes to measure genetic distance between indepen-
dent isolates were verified.

10

They found a strong

geographical localisation of C. glabrata strains both
between continents and between cities within a conti-
nent.

10

They demonstrated that both Cg6 and Cg12

probes discriminate microevolution within sequential
isolates of C. glabrata,

10

as observed in clonal population

of C. albicans grown over many generations with
Ca3 probes.

35,36

The probes also contain invariant

sequences which facilitate normalisation in computer-
assisted analysis that can be used in large epidemiolo-
gical studies. When analysing diverse geographical
collection of 107 clinical isolates, Dodgson et al. [11]
demonstrated that Cg6 and Cg12, in contrast to RAPD
and MLST, discriminate between all isolates in all
groups and were the better methods when analysing
microevolution or nosocomial transmission. The major
disadvantage of Southern blot was the radioactive probe
and the complexity of binding patterns needing auto-
matically identification by computer assisted systems.

20

Multilocus enzyme electrophoresis

The MLEE analysis is based on allelic frequency that
assesses isozymes or allozymes. MLEE can discriminate
among the gene products of different alleles for a
number of loci. Meeus et al. [8] have genotyped 63
C. glabrata isolates for 33 putative gene enzymatic loci.
This method was verified for C. albicans by a cluster
analysis of a set of test isolates in which MLEE, RAPD
and Southern blot hybridisation with fingerprinting
probes were compared and parity was demonstrated.

9

However, for C. glabrata, a low genetic differentiation
between distant hospitals (Montpellier and Paris) was
found and no correlation was noted with anatomic
origins or human immunodeficiency virus-positive or
negative (HIV+, HIV

)) patients.

4,8

Boldo et al. [4]

analysed 47 clinical isolates from several geographical
origins and diverse body sites, and estimated for 11
enzymatic loci a high level of genetic relatedness
among isolates and revealed no genetic differentiation
(H

0

= 0.055) among them. No association with their

geographical origin and clinical characteristics was
found. Relationship between resistance to fluconazole
and particular genotype was also searched but no
correlation was found.

8

However it can happen that the

percentage of mutation is so high that it is in association
with several MLEE combinations.

4

MLEE does not show

a significant differentiation among C. glabrata isolates,

reason for which this technique has not been adopted by
the majority of clinicians in addition to the relatively
time consuming to combine the data from at least 10 or
more enzymes that provide variability among isolates.

20

Multilocus sequence typing

To obtain a higher resolution DNA fingerprinting
system based on direct sequence comparison, a MLST
system was developed by Bougnoux et al.[37] to type
C. albicans isolates. The data from alleles at multiple loci
are combined to access genetic relatedness. The study of
genetic variation by MLST based on differences in DNA
sequences is more sensitive than that accessed by
differences in protein mobility.

Dodgson et al. [11] were the first to develop

C. glabrata MLST. They used MLST for two main
objectives. Firstly, the typing was for geographical
repartition of isolates. Analysing three geographically
diverse collections of clinical isolates, they identified five
major clades, three of them exhibited significant
geographical bias.

11

Secondly, MLST was performed to

test the evidence of genetic recombination. Thirty-four
sequence types were defined and fourteen examples of
phylogenetic incompatibility were found. Thus, they
conclude that although C. glabrata has a predominantly
clonal population structure, the multiple phylogenetic
incompatibilities suggested that recombination occurred
during the evolution of C. glabrata, and may infre-
quently occur.

38

Recently, Lin et al. [6] analysed 25 patients with

multiple isolates assume that MLST (DI = 0.85)
exhibited less discriminatory power than PFGE with
BssHII.

This system is highly effective to examine population

structure, but is not suited for studies of nosocomial
infection or microevolution. The C. glabrata MLST
groups revealed a lower variable-site percentage of
1.6%, which is much less than that of C. albicans,

39,40

and then a less discriminatory power was achieved. This
may be due to the haploid nature of C. glabrata.

Microsatellite markers

Genotyping isolates with microsatellite markers has
been recently described for C. glabrata.

12,13

The poly-

morphism of microsatellites was evaluated by PCR using
fluorescent primers and an automatic sequencer as
already reported for C. albicans. Microsatellites represent
another class of genotyping defined as short tandem
repeats of two to six nucleotides known to be highly
polymorphic.

Molecular methods for typing C. glabrata isolates

 2009 Blackwell Verlag GmbH • Mycoses 53, 463–467

465

Foulet et al. [12] adopted three polymorphic micro-

satellite markers, RPM2, MTI and ERG3, for rapid
typing of 138 isolates of C. glabrata and found a
discriminatory power equal to 0.84. Irregular distribu-
tion of C. glabrata population was observed which was
similar to that observed with MLST. Twenty-four per
cent of the isolates belonged to the same sequence
type.

40

Recently, a multiple-locus variable-number

tandem-repeat analysis (MLVA) using six microsatellite
markers was assessed in 127 C. glabrata isolates. Thirty-
seven different genotypes, stable both in vitro and in vivo,
were observed leading to discriminatory power (DI =
0.902).

13

It seems that an ecological advantage for

some genotypes exists.

12

Microsatellite marker system for DNA fingerprinting

was easy to assay, adaptable to large series and can be
used as a typing system to investigate clinical issues
such as nosocomial transmission of isolates or the origin
of infective strains.

12,13

However, the necessity to use

fluorescently labelled primers with elevated cost and an
automatic sequencer limits the utilisation of this
technology.

Conclusion

Following the widespread use of antifungal therapy
and the increased number of immunocompromised
patients, C. glabrata has emerged as an important
opportunistic pathogen in superficial and invasive
infections. The typing of C. glabrata isolates has been
developed mainly to detect the origin of nosocomial
transmission.

17,41,42

Then subsequently several objec-

tives have been added, such as analyse variability
between geographically distant isolates; recently, char-
acterisation of resistant strains and determination if
resistance is the result of a selection or an induction of
initially sensitive strains. Some genotyping systems
such as RFLP (due to the difficulties in interpretation)
and RAPD (due to the non-reproducibility) are not
used anymore. On the other hand, fine variations
between strains have been searched by a polymor-
phism of lengths of microsatellite sequences. The
principal reason for typing was to show that the
population of C. glabrata is mainly clonal and that a
particular profile for the resistant strains does not exist.
Southern blot with Cg6 and Cg12, karyotyping anal-
yses and microsatellite marker analysis were the most
adequate techniques for typing C. glabrata strains.
However, PFGE is not easy to achieve; therefore, it
was not chosen for epidemiological issues such as
nosocomial transmission or the origin of infective
strains.

References

1 Safdar A, Chaturvedi V, Koll BS, Larone DH, Perlin DS,

Armstrong D. Prospective, multicenter surveillance study
of Candida glabrata: fluconazole and itraconazole suscepti-
bility profiles in bloodstream, invasive and colonizing
strains and differences between isolates from Three Urban
teaching Hospitals in New York City. Antimicrob Agents
Chemother 2002; 46: 3268–72.

2 Richardson MD. Changing patterns and trends in systemic

fungal infections. Antimicrob Agents Chemother 2005; 56:
5–11.

3 Malani A, Hmoud J, Chui L, Carver PL, Bielaczyc A,

Kauffman CA. Candida glabrata fungemia: experience in
a tertiary care center. J Clin Microbiol 2005; 41: 975–
81.

4 Boldo XM, Villa-Tanaca L, Zuniga G, Hernandez-Rodri-

guez C. Genetic diversity among clinical isolates of Candida
glabrata analysed by randomly amplified polymorphic
DNA and multilocus enzyme electrophoresis analyses.
J Clin Microbiol 2003; 41: 4799–804.

5 Song X, Sun J, Store G, Eribe ERK, Hansen BF, Olsen I.

Genotypic relatedness of yeast in thrush and denture
stomatis. Oral Microbiol Immunol 2006; 21: 301–8.

6 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.

7 Redding SW, Kirkpatrick WR, Saville S, Coco BJ, White W,

Fothergill A. Multiple patterns of resistance to fluconazole
in Candida glabrata isolates from a patient with oropha-
ryngeal candidiasis receiving head and neck radiation.
J Clin Microbiol 2003; 41: 619–22.

8 de Meeus T, Renaud F, Mouveroux E, Reynes J, Galeazzi G,

Mallie´ M. Genetic structure of Candida glabrata populations
in AIDS and non-AIDS patients. J Clin Microbiol 2002;
40: 2199–206.

9 Pujol C, Joly S, Shawn R et al. Parity among the

randomly amplified polymorphic DNA method, multilo-
cus enzyme electrophoresis, and Southern blot hybrid-
ization with the moderately repetitive DNA probe Ca3
for fingerprinting Candida albicans. J Clin Microbiol 1997;
35: 2348–58.

10 Lockhart SR, Joly S, Pujol C, Sobel JD, Pfaller MA, Soll DR.

Development and verification of fingerprinting probes for
Candida glabrata. Microbiology 1997; 143: 3733–46.

11 Dodgson AR, Pujol C, Denning DW, Soll DR, Fox AJ.

Multilocus sequence typing of Candida glabrata reveals
geographically enriched clades. J Clin Microbiol 2003;
41: 5709–17.

12 Foulet F, Nicolas N, Eloy O et al. Microsatellite Marker

analysis as a typing system for Candida glabrata. J Clin
Microbiol 2005; 43: 4574–9.

13 Grenouillet F, Millon L, Bart JM et al. Multiple-locus

variable-number tandem-repeat analysis for rapid
typing of Candida glabrata. J Clin Microbiol 2007;
45: 3781–4.

S. Abbes et al.

466

 2009 Blackwell Verlag GmbH • Mycoses 53, 463–467

14 Scherer S, Stevens DA. Application of DNA typing meth-

ods to epidemiology and taxonomy of Candida Species.
J Clin Microbiol 1987; 25: 675–9.

15 Delabesse EB, Boiron P, Carlotti A, Dupont B. Candida

albicans genotyping in studies with patients with AIDS
developing resistance to Fluconazole. J Clin Microbiol
1993; 31: 2933–7.

16 Clemons KV, Feroze F, Holmbreg K, Stevens DA. Com-

parative analyses of genetic variability among Candida
albicans isolates from different geographic locales by three
genotypic methods. J Clin Microbiol 1997; 35: 1332–6.

17 Vasquez JA, Dembry LM, Sanchez V. Nosocomial Candida

glabrata colonization: an Epifemiologic Study. J Clin
Microbiol 1998; 36: 421–6.

18 Arif S, Barkham T, Power EG, Howell SA. Techniques for

investigation of an apparent Outbreak of infections with
Candida glabrata. J Clin Microbiol 1996; 34: 2205–9.

19 Vasquez JA, Beckley A, Donabedian S, Sobel JD, Zervos MJ.

Comparaison of restriction enzyme analysis vs. pulsed field
gradient gel electrophoresis as a typing system for Torul-
opsis glabrata and Candida species other than C.albicans.
J Clin Microbiol 1993; 31: 2021–30.

20 Soll DR. The ins and outs of DNA fingerprinting. Clin

Microbiol Rev 2000; 13: 332–70.

21 Fujita S, Hashimoto T. DNA fingerprinting patterns of

Candida species using HinfI endonuclease. Int J Syst Evol
Microbiol 2000; 50: 1381–9.

22 Williams JH, Kubelik AR, Rafalski JA, Tingey SV. DNA

polymorphisms amplified by arbitrary primers are
useful as genetis markers. Nucleic Acids Res 1990;
18: 6531–5.

23 Robert F, Lebreton F, Bougnoux ME et al. Use of random

amplified polymorphic DNA as a typing method for
Candida albicans in epidemiological surveillance of burn
unit. J Clin Microbiol 1995; 33: 2366–71.

24 Becker K, Badehorn D, Deiwick S, Peters G, Fegeler W.

Molecular genotyping of Candida species with special
respect to C. glabrata strains by arbitrarily primers PCR.
J Med Microbiol 2000; 49: 575–81.

25 Schwab U, Chernomas F, Larcon L, Weems J. Molecular

typing and fluconazole susceptibility of urinary Candida
glabrata isolates from hospitalized patients. Diagn Microbiol
Infect Dis 1997; 29: 11–7.

26 Bougnoux ME, Robert F, Beria S, Cassinat B, Nicolas MH,

Dupoy-Camet J. Typage mole´culaire d

Õisolats de Candida

par «Random Amplified Polymorphic DNA». J Mycol Med
1994; 4: 3–8.

27 Chen KW, Lo HJ, Lin YH, Li SY. Comparison of four

molecular typing methods to assess genetic relatedness of
Candida albicans clinical isolates in Taiwan. J Med Microbiol
2005; 54: 249–58.

28 Di Francesco LF, Barchiesi F, Caselli F, Cirioni O, Scalise G.

Comparison of four methods for DNA typing of clinical
isolates of Candida glabrata. J Med Microbiol 1999; 48:
955–63.

29 Koc¸ AN, Kocagoz S, Erdem F, Gundez Z. Outbreak of

nosocomial fungemia caused by C.glabrata. Mycoses 2002;
45: 11–2.

30 Cormican MG, Hollis RJ, Pfaller MA. DNA macrorestric-

tion profiles and antifungal susceptibility of C. glabrata.
Diagn Microbiol Infect Dis 1996; 25: 83–7.

31 Barrchiesi F, Falconi Di Francesco L, Arzeni D, caselli F,

Gallo D, Scalise G. Electrophoretic karyotyping and triaz-
ole susceptibility of Candida glabrata clinical isolates. Eur
J Clin Microbiol Infect Dis 1999; 18: 184–7.

32 Bennett JE, Izumikawa K, Marr KA. Mechanism of

increased fluconazole resistance in Candida glabrata during
prophylaxis. Antimicrob Agents Chemother 2004; 48:
1773–7.

33 Jong HS, Myung JC, Jeong WS, et al. Changes in karyotype

and azole susceptibility of sequential bloodstream isolates
from patients with Candida glabrata candidemia. J Clin
MIcrobiol 2007; 45: 2385–91.

34 Klempp-Selb B, Rimek D, Kappe R. Karyotyping of C.

albicans and C. glabrata from patients with Candida sepsis.
Mycoses 2000; 43: 159–63.

35 Lockhart SR, Fritch JJ, Sturdevant MA et al. Colonis-

ing populations of Candida albicans are clonal in origin
but undergo microevolution through C1 fragment
reorganisation as demonstrated by DNA fingerprinting
and C1 sequencing. J Clin Microbiol 1995; 33:
1501–9.

36 Schmid J, Voss E, Soll DR. Computer-Assisted Methods for

assessing strain relatedness in Candida albicans by finger-
printing with the moderately repetitive sequence Ca3.
J Clin Microbiol 1990; 28: 1236–43.

37 Bougnoux ME, Morand S, d

Õenfert C. Usefulness of

multilocus sequence typing for characterization of clinical
isolates of Candida albicans. J Clin Microbiol 2002; 40:
1290–7.

38 Dodgson AR, Pujol C, Pfaller MA, Denning DW, Soll DR.

Evidence for recombination in Candida glabrata. Fungal
Genet Biol 2005; 42: 233–43.

39 Bougnoux M, Tavanti A, Bouchier C et al. Collaborative

consensus for optimised multilocus sequence typing
of Candida albicans. J Clin Microbiol 2003; 41: 5265–6.

40 Tavanti A, Neil ARG, Maiden MCJ, Odds FC, Shaw JD.

Genetic evidence for recombination in Candida albicans
based on haplotype analysis. Fungal genet biol 2004; 41:
553–62.

41 Hamel P, Kappe R, Rimek D. Rate of transmission and

endogenous origin of Candida albicans and Candida glabrata
on adult intensive care units studied by pulsed field gel
electrophoresis. J Hosp Infect 2001; 49: 37–42.

42 Bougnoux ME, Cac G, Aegepter P, d

Õenfert C, Fagon JY,

candiRea study group. Candidemia and candiduria in
critically ill patients admitted to intensive care units
in France: incidence, molecular diversity, management
and outcome. Intensive Care Med 2008; 34: 292–9.

Molecular methods for typing C. glabrata isolates

 2009 Blackwell Verlag GmbH • Mycoses 53, 463–467

467