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Journal of Clinical Microbiology, May 1999, p. 1587-1590, Vol. 37, No. 5
Institut für Hygiene und Mikrobiologie,
Universität Würzburg, 97080 Würzburg,
Germany,1 and Department of Oral
Medicine and Pathology, School of Dental Science and Dublin Dental
Hospital, Trinity College, University of Dublin, Dublin 2, Republic
of Ireland2
Received 1 September 1998/Returned for modification 5 October
1998/Accepted 25 January 1999
The development of a satisfactory means to reliably distinguish
between the two closely related species Candida albicans
and Candida dubliniensis in the clinical mycology
laboratory has proved difficult because these two species are
phenotypically so similar. In this study, we have detected homologues
of the pH-regulated C. albicans PHR1 and PHR2
genes in C. dubliniensis. Restriction fragment length
polymorphism analysis suggests that there are significant sequence
differences between the genes of the two species. In order to exploit
this apparent difference, oligonucleotide primers based on the coding
sequence of the C. albicans PHR1 structural gene were
designed and used in PCR experiments. Use of these primers with
C. albicans template DNA from 17 strains yielded a
predicted 1.6-kb product, while C. dubliniensis template
DNA from 19 strains yielded no product. We therefore propose that PCR
using these primers is a rapid and reliable means of distinguishing the
two germ tube- and chlamydospore-producing species C. albicans and C. dubliniensis.
Opportunistic fungal infections have
gained considerable importance during recent years, and oral candidosis
is among the most common opportunistic infections encountered in human
immunodeficiency virus-infected patients (13). Candida
dubliniensis is a chlamydospore-positive, germ tube-positive
species with many characteristics in common with Candida
albicans. To date, C. dubliniensis isolates have been
primarily recovered from the oral cavities of immunosuppressed individuals (20), although isolates have also been recovered from other anatomic sites (19). It has been shown to be a
low-level constituent of the human oral flora and has the potential to
cause oral candidosis (1, 18). Although C. albicans and C. dubliniensis are phenotypically very
similar, they differ in their carbohydrate assimilation profiles,
growth patterns at elevated temperatures, and intracellular
While analyzing the expression and genomic organization of genes
regulated in response to the ambient pH in C. dubliniensis, we found that the genomic organization of the PHR1 and
PHR2 genes differed between C. dubliniensis and
C. albicans. In C. albicans, the expression of
these functionally homologous genes is regulated by environmental pH
and deletion analysis suggests that they play a role in morphogenesis
and cell survival in vivo (2, 8, 15). The unique
organization of these genes in C. dubliniensis suggested the
opportunity to use these sequences as targets for species-specific
oligonucleotides in PCR experiments to discriminate between C. dubliniensis and C. albicans.
Identification of PHR homologs in C. dubliniensis.
In order to investigate the presence of
PHR1 and PHR2 homologous genes in C. dubliniensis, genomic DNA from reference strains and clinical
isolates (Table 1) was isolated as previously described (16). DNA was digested with the restriction enzymes
EcoRI and HindIII and subsequently used for
Southern blot analysis. The membranes were hybridized by using an
800-bp ClaI fragment of the C. albicans PHR1 gene
or a 1.6-kb HindIII fragment of the C. albicans
PHR2 gene as a probe under stringent conditions (8, 15). Both probes hybridized with the digested C. dubliniensis DNA under these conditions (Fig.
1 and Table
1). However, the sizes of the hybridizing
bands differed between the two species in experiments using
EcoRI or HindIII (Fig. 1 and Table 1). These findings demonstrate that PHR1 and PHR2
homologous genes are present in the C. dubliniensis genome.
The differences in the restriction fragment lengths point to an
architecture of the PHR1-PHR2 genomic locus in C. dubliniensis different from that in C. albicans. In agreement with the observations of others who suggested that
Candida stellatoidea is a variant or synonym of C. albicans, the C. stellatoidea reference strains tested
were indistinguishable from C. albicans in this study
(11) (Fig. 1 and Table 1). DNA from other
chlamydospore-negative pathogenic or nonpathogenic species either did
not hybridize at all or produced barely detectable signals of different
sizes (Table 1). This may well be due to the existence of
PHR homologous genes in these species. Indeed, using PCR
with degenerate primers deduced from the C. albicans PHR1
and PHR2 sequences, we were able to amplify fragments with
homologies to the PHR1 and PHR2 genes in the
pathogenic yeast Candida glabrata (22).
Furthermore, PHR homologous genes have also been described
for Candida maltosa and Saccharomyces cerevisiae
(9, 21). However, so far C. albicans is the only
species demonstrating strong pH-dependent inverse expression of these
genes.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid PCR Test for Discriminating between
Candida albicans and Candida dubliniensis
Isolates Using Primers Derived from the pH-Regulated PHR1
and PHR2 Genes of C. albicans
ABSTRACT
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-glucosidase activities (1, 12, 14). In addition,
C. dubliniensis possesses a very distinct genomic organization (1, 4, 19). In spite of these differences, rapid discrimination between C. albicans and C. dubliniensis in the clinical mycology laboratory is still
problematic and usually only achieved effectively by using a
combination of standard laboratory procedures (19). The
incidence of C. dubliniensis and its role in disease have
yet to be firmly established. In order to effectively address this
situation, it is important to be able to differentiate this organism
from C. albicans. For this reason, rapid techniques suitable
for the accurate and reliable identification of C. dubliniensis in clinical laboratories are required. Because of the
difficulty in using methods based on phenotypic parameters to
distinguish efficiently between C. dubliniensis and C. albicans, we proposed to develop a rapid and reliable
discriminatory method based on the use of molecular tools.
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FIG. 1.
Southern blot hybridization with the PHR1
gene probe. DNA from C. albicans SC5314 (lanes 1 and 6);
C. dubliniensis CBS 7987 (lanes 2 and 7), CBS 7988 (lanes 3 and 8), and CD 33 (lanes 4 and 9); and C. stellatoidea ATCC
11006 (lanes 5 and 10) digested with EcoRI (lanes 1 to 5) or
HindIII (lanes 6 to 10) was analyzed. The
electrophoretic positions and sizes deduced from DNA standards are
indicated on the left.
TABLE 1.
Reference yeast strains, the sizes of restriction
fragments in Southern blot analysis using PHR1 and
PHR2 probes, and the sizes of PCR products in experiments
using C. albicans PHR1-specific primers
pH-dependent differential expression of the C. dubliniensis PHR homologs. To determine whether the expression of the PHR homologous genes of C. dubliniensis resembles the pattern demonstrated for the C. albicans PHR1 and PHR2 genes, total RNA was isolated from the C. dubliniensis reference strains at pH 4 and pH 8 as previously described (6). Northern blot hybridization was performed with either the PHR1 or the PHR2 probe under stringent conditions. With each of the probes, a transcript with a size comparable to that of the PHR1 or PHR2 mRNA was detected in the C. dubliniensis reference strains (data not shown). Furthermore, the PHR homologous genes of C. dubliniensis exhibited a pH-dependent pattern of expression similar to that of PHR1 and PHR2. Thus, it seems likely that the pH-balanced system of the two functional homologues PHR1 and PHR2 of C. albicans is conserved in C. dubliniensis.
Amplification of PHR1 for differentiation between C. albicans and C. dubliniensis. In order to investigate whether sequence differences between the PHR1 and PHR2 genes of C. albicans and their C. dubliniensis homologues might be useful in terms of differentiation between these two species, we performed PCR with primers derived from the PHR1 sequence of C. albicans. In addition to the strains listed in Table 1, 12 C. albicans and 13 C. dubliniensis fresh clinical isolates from the oral cavities of different human immunodeficiency virus-infected individuals from Ireland were subjected to PCR analysis. Identification of the strains was based on previously described phenotypic criteria (19). The sequence of the forward primer OK3 was 5' ATG TAT TCA TTA ATC AAA TCA 3'. The sequence of the reverse primer OK4 was 5' ATT TAA AAA ACA ACG GAC AT 3'. These primers are capable of amplifying the entire 1,644-bp open reading frame of PHR1. The PCR mixture (total volume, 100 µl) contained 100 ng of genomic template DNA, 0.2 mM each deoxynucleoside triphosphate, 1× AmpliTaq buffer, 1.5 mM MgCl2, 100 pmol of each primer, and 5 U of AmpliTaq polymerase (Perkin-Elmer, Weiterstadt, Germany). Amplification was performed after denaturation at 95°C for 5 min for 30 cycles with a 20-s denaturation at 95°C, primer annealing at 50°C for 60 s, and extension for 90 s at 72°C, followed by a final extension at 72°C for 10 min in a Trio Thermoblock system (Biometra, Göttingen, Germany). Each experiment included negative controls with all reagents except template DNA. As a positive control, plasmid pSMS-24 containing the complete PHR1 gene was used as the template DNA (15). Since assignment of isolates to C. dubliniensis is based on the missing amplification of the PHR1 homologue, proper adjustment of the controls is necessary. Products were separated on an 0.8% (wt/vol) agarose gel, stained with ethidium bromide, and compared to the positive control. Template DNA from all of the 17 C. albicans isolates and both C. stellatoidea strains led to the specific amplification of a fragment with the predicted size of approximately 1.6 kb that was identical to the positive control. When the same conditions were used with 19 C. dubliniensis isolates, no amplimers were detected (Fig. 2 and data not shown). In addition, the PCR test was applied to 109 oral isolates previously classified as C. albicans based on germ tube and chlamydospore formation. Differentiation between C. albicans and C. dubliniensis was then achieved in a blinded fashion based on either phenotypic characteristics or the PCR test. The results obtained by the two methods were shown to correlate (unpublished data). Identical results were obtained when colony material from the C. dubliniensis and C. albicans reference strains was used as a template for the PCR (data not shown). In addition, DNA from reference strains of the chlamydospore-negative species Candida tropicalis, Candida parapsilosis, C. glabrata, Kluyveromyces marxianus (Candida kefyr), Issatchenkia orientalis (Candida krusei), and Pichia guilliermondii (Candida guilliermondii) was isolated and used as a template in this PCR. Similar to the results obtained in the Southern blot analysis, no products were generated or there was amplification of fragments that clearly differed in size from the positive control (Table 1). This, however, does not interfere with the diagnostic value of the procedure since only chlamydospore-positive species are considered.
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Conclusion. C. dubliniensis was only recently identified as a separate species, and since it is difficult to distinguish it from C. albicans in clinical samples there is still very little information available concerning its epidemiology and clinical significance. It has been shown that C. dubliniensis is able to readily develop fluconazole resistance under selective pressure (7). To determine whether this or other factors are implicated in the emergence of C. dubliniensis as a pathogen, and to measure what the true incidence of this species is in humans, novel methods for discriminating between C. dubliniensis and C. albicans are required. Until now, identification of C. dubliniensis was achieved by time-consuming investigation of a combination of potentially variable phenotypic characteristics (12, 17, 19). In this study, we describe for the first time the use of a structural gene, PHR1, as a target for a rapid and reliable PCR-based identification system with the potential to facilitate the analysis of the epidemiology of C. dubliniensis.
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ACKNOWLEDGMENTS |
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This work was supported by grant MU1212/2-1 from the Deutsche Forschungsgemeinschaft (to F.A.M.).
We thank W. A. Fonzi for plasmid pSMS-24. We are grateful to Uwe Gross and Michael Weig for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Hygiene und Mikrobiologie, University Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany. Phone: (0931) 201-3901. Fax: (0931) 201-3445. E-mail: fmuehlschlegel{at}hygiene.uni-wuerzburg.de.
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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