Professional Documents
Culture Documents
417421
0095-1137/99/$04.0010
Copyright 1999, American Society for Microbiology. All Rights Reserved.
AND
DAVID A. STEVENS*
Department of Medicine, Division of Infectious Diseases, Santa Clara Valley Medical Center, and California Institute
for Medical Research, San Jose, California 95128, and Department of Medicine, Division of Infectious Diseases and
Geographic Medicine, Stanford University School of Medicine, Stanford, California 94305
Received 30 July 1998/Returned for modification 10 October 1998/Accepted 11 November 1998
There have been increased reports of the isolation of unusual genotypic groups of Candida albicans (groups
C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRI
enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis.
The aim of the present study was to use additional molecular tools to characterize these unusual strains and
to compare them with authentic strains of C. dubliniensis, a recently delineated species, and type I C. stellatoidea.
The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiate
the C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIII
digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same
groups as RFLP analysis of the PCR amplicon of the ITS region. C. albicans genotype B isolates have been
shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensis
strains also have an intron that is larger than that in genotype B C. albicans strains but that is in the same
location. PCR designed to span this region resulted in a single product for C. albicans genotype A (450 bp), B
(840 bp), type 1 C. stellatoidea (840 bp), and C. dubliniensis (1,080 bp), whereas the C. albicans genotype C
isolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR product
identical to that given by C. dubliniensis. These results indicate that those strains previously designated
C. albicans genotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoidea
and C. albicans genotype B strains, and that the C. albicans genotype C strains appear to have the transposable
intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal
susceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis were
significantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). The
flucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less susceptible than either C. albicans genotype B or C. albicans genotype C strains. These results indicate that there
is a correlation between the Candida groups and antifungal susceptibility.
that there was an association of genotypic group A with increased resistance to the antifungal agent flucytosine (21). Recent studies by the same methodology have reported on the
occurrence of C. albicans strains with unusual genotypes; these
have been designated genotype C (strains with both the 3.7and 4.2-kb bands) and genotype D (strains with neither band)
(2, 9, 10). However, no association has been made between
these newly described genotypes and antifungal susceptibility.
C. dubliniensis is a recently recognized species that is phenotypically very closely related to C. albicans. It has been
isolated primarily from the oral cavities of patients infected
with the human immunodeficiency virus (22). The characteristics and identification of this species have recently been reviewed (22). That review highlighted the difficulty in differentiating this species from C. albicans by routine laboratory
methods (22). C. dubliniensis has been reported to be susceptible to the same range of antifungal agents as C. albicans (22).
Recently, a 379-nucleotide insert in the DNA encoding for
the large-subunit rRNA (the 25S rRNA gene [rDNA]) has
been shown to be responsible for the larger 4.2-kb band in the
C. albicans genotype B isolates, whereas the smaller 3.7-kb
band in the genotype A isolates lacks this insert of nucleotides
(12). An homologous group 1 intron at the same insertion
point was shown to be present in strains of C. dubliniensis;
418
MCCULLOUGH ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Order, source, and designation of strains of
the various Candida species and genotypes
Lane no.a
Species/genotypeb
Strain designation
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
C. albicans/A
C. albicans/A
C. albicans/B
C. albicans/B
C. albicans/C
C. albicans/C
C. dubliniensis CD36
C. dubliniensis CM2
C. stellatoidea
C. stellatoidea
C. parapsilosis
C. tropicalis
C. krusei
C. lusitania
C. glabrata
Negative control
ATCC 64124
ATCC 62342
ATCC 38246
ATCC 32354
Singapore 7c
Singapore 8c
NCPF 3949 and CBS 7987
CBS 7988
B-4257
B-4404
ATCC 22019
ATCC 13803
ATCC 6258
ATCC 4125
ATCC 66032
RESULTS
Molecular analysis of Candida spp. A total of 457 strains of
Candida were analyzed by all three PCR methods. Four hundred thirty-nine of these strains derived from a large study of
isolates identified as C. albicans (289 genotype A strains, 85
genotype B strains, 56 genotype C strains and 9 genotype D
strains) obtained over a 20-year period from 15 geographically
diverse areas) (10). Nine authentic strains of different species
of Candida from ATCC, two authentic strains of C. dubliniensis, two authentic strains of type 1 C. stellatoidea, and a further
five strains of C. dubliniensis were analyzed. A list of the authentic strains analyzed is given in Table 1.
The RFLPs generated by DdeI digestion of the PCR products from the ITS region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea (Fig. 1).
However, this method did differentiate the C. albicans genotype D strains from C. albicans genotype A, B, and C strains
(Fig. 1). The profiles of the C. albicans genotype D strains were
identical to those of the C. dubliniensis strains.
The RFLPs generated by HaeIII digestion of the PCR products of the V3 region of the 25S rDNA could differentiate the
same groups as the RFLP analysis of the PCR amplicon of the
ITS region (Fig. 2). The profiles of the C. albicans genotype D
strains were identical to those of the C. dubliniensis strains.
PCR designed to span the region that included the site of the
transposable intron in genotype B C. albicans and C. dubliniensis strains (1, 12) resulted in a single product for C. albicans
genotypes A (;450 bp) and B (;840 bp), C. stellatoidea (;840
bp), and C. dubliniensis (;1,080 bp) (Fig. 3). All of the C. albicans genotype C isolates had two PCR products (;450 and
;840 bp) that were identical in size to the respective products
from C. albicans genotypes A and B (Fig. 3). All C. albicans
genotype D isolates gave a PCR product identical in size to
that of C. dubliniensis.
To assess if the C. albicans genotypic subgroup C strains
were not a mixed culture of strains, two randomly chosen
isolates were subcultured on yeast potato dextrose (YPD) agar
for 48 h. Twenty-five individual colonies were chosen and
419
DISCUSSION
The results presented here indicate that those strains previously designated C. albicans genotype D (2, 9, 10) should be
assigned to the species C. dubliniensis. All molecular methods
for the differentiation of isolates to the species level described
here used PCR techniques. The method that detects the presence and the size of the intron in the 25S rDNA is particularly
easily adapted for use in reference laboratories for the rapid
identification of large numbers of isolates. This simple PCR
method has the additional advantage of differentiating strains
of C. albicans into the described genotypic subgroups.
C. stellatoidea has previously been divided into two types,
types I and II, with type II isolates shown to be sucrose-
FIG. 3. Ethidium bromide-stained, UV-transilluminated PCR products obtained by PCR with the primers that span the intron position in the 25S rDNA.
The photograph was obtained after electrophoresis in a 3% agarose gel. Molecular size markers are in the lanes marked M, and their corresponding sizes (in
base pairs) are given on the left. The isolate in each lane is specified in Table 1.
420
MCCULLOUGH ET AL.
J. CLIN. MICROBIOL.
TABLE 2. Descriptive statistics of the MICs obtained by testing 105 strains their susceptibilities to both fluconazole and flucytosine
Fluconazolea
Flucytosineb
C. albicans
genotype A
C. albicans
genotype B
C. albicans
genotype C
C. dubliniensis
C. albicans
genotype A
C. albicans
genotype B
C. albicans
genotype C
C. dubliniensis
22.8
29.8
4.2
30
11.1
33.4
29.1
10.3
30
10.8
32.3
30.5
8.9
30
11.4
4.7
9.3
1.1
15
5.2
6.9
3.5
0.54
30
7.2
0.26
0.07
0.18
30
0.14
2.3
2.1
0.22
30
4.3
8.7
5.8
0.32
15
12.4
a
ANOVA for the fluconazole susceptibility results of the four groups gave a P value of 0.003. Analysis of each pair of groups by a two-sample t test assuming unequal
variance showed that the results for C. dubliniensis were significantly different from those for each of C. albicans genotypes A, B, and C (P 5 0.018, 0.002, and 0.004,
respectively). No other statistically significant differences were observed.
b
ANOVA for the flucytosine susceptibility results for the four groups gave a P value of 0.021. Analysis of each pair of groups by a two-sample t test assuming unequal
variance showed that the results for the C. albicans genotype A strains were significantly different from those for each of C. albicans genotypes B and C (P 5 0.004
and 0.003, respectively). No other statistically significant differences were observed.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
421