JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010

Feb. 1999, p. 417421 Vol. 37, No. 2

Copyright 1999, American Society for Microbiology. All Rights Reserved.

Molecular and Phenotypic Characterization of GenotypicCandida albicans Subgroups and Comparison with

Candida dubliniensis and Candida stellatoideaMICHAEL J. MCCULLOUGH, KARL V. CLEMONS, AND DAVID A. STEVENS*

Department of Medicine, Division of Infectious Diseases, Santa Clara Valley Medical Center, and California Institutefor 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 (groupsC and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRIenzyme 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 andto 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 differen-tiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiatethe C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIIIdigestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the samegroups as RFLP analysis of the PCR amplicon of the ITS region. C. albicans genotype B isolates have beenshown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensisstrains also have an intron that is larger than that in genotype B C. albicans strains but that is in the samelocation. 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 Cisolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR productidentical to that given by C. dubliniensis. These results indicate that those strains previously designatedC. albicans genotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoideaand C. albicans genotype B strains, and that the C. albicans genotype C strains appear to have the transposableintron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungalsusceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis weresignificantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). Theflucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less sus-ceptible than either C. albicans genotype B or C. albicans genotype C strains. These results indicate that thereis a correlation between the Candida groups and antifungal susceptibility.

There has been an increase in the occurrence of diseasescaused by Candida over the recent past. The majority of thesediseases are caused by Candida albicans (4, 11, 16, 17). Mo-lecular typing methods have been used with increasing fre-quency for epidemiological investigations for the developmentof rational infection control measures (17). One of the earliestmolecular methods for the differentiation of C. albicans strainsused a simple technique of analyzing the restriction fragmentlength polymorphisms (RFLPs) of cellular DNA to divide iso-lates into two large groups on the basis of the position of adimorphic band (group A strains have a band of 3.7 kb andgroup B strains have a band of 4.2 kb) and then subdividedthem into types (19). The method was shown to be reliable andreproducible and was used for a large epidemiological study ofstrains isolated from multiple localities in the United Statesand the United Kingdom (21). In that study it was reported

that there was an association of genotypic group A with in-creased resistance to the antifungal agent flucytosine (21). Re-cent studies by the same methodology have reported on theoccurrence of C. albicans strains with unusual genotypes; thesehave been designated genotype C (strains with both the 3.7-and 4.2-kb bands) and genotype D (strains with neither band)(2, 9, 10). However, no association has been made betweenthese newly described genotypes and antifungal susceptibility.

C. dubliniensis is a recently recognized species that is phe-notypically very closely related to C. albicans. It has beenisolated primarily from the oral cavities of patients infectedwith the human immunodeficiency virus (22). The character-istics and identification of this species have recently been re-viewed (22). That review highlighted the difficulty in differen-tiating this species from C. albicans by routine laboratorymethods (22). C. dubliniensis has been reported to be suscep-tible to the same range of antifungal agents as C. albicans (22).

Recently, a 379-nucleotide insert in the DNA encoding forthe large-subunit rRNA (the 25S rRNA gene [rDNA]) hasbeen shown to be responsible for the larger 4.2-kb band in theC. albicans genotype B isolates, whereas the smaller 3.7-kbband in the genotype A isolates lacks this insert of nucleotides(12). An homologous group 1 intron at the same insertionpoint was shown to be present in strains of C. dubliniensis;

* Corresponding author. Mailing address: Department of Medicine,Division of Infectious Diseases, Santa Clara Valley Medical Center,751 South Bascom Ave., San Jose, CA 95128-2699. Phone: (408) 885-4313. Fax: (408) 885-4306. E-mail: stevens@leland.stanford.edu.

Present address: Victorian Infectious Disease Reference Labora-tory and School of Dental Science, University of Melbourne, Mel-bourne, Australia.

417

however, this intron was approximately 300 nucleotides largerthan that in genotype B C. albicans (1).

The aim of the present study was to use molecular tools tocharacterize recently described new genotypic subgroups ofC. albicans and to compare them with authentic strains ofC. dubliniensis and type I C. stellatoidea. An additional aim wasto assess a large, randomly chosen subset of these isolates fortheir comparative antifungal susceptibilities to fluconazole andflucytosine by the U.S. National Committee for Clinical Lab-oratory Standards (NCCLS) M27-A susceptibility assay (14).

MATERIALS AND METHODS

The isolates included in this study were made available from previous inves-tigations (2, 3, 8, 9, 21). Authentic strains of C. dubliniensis were made availablefrom D. Sullivan and have been submitted to the National Collection of Patho-genic Fungi (London, United Kingdom) as NCPF 3949 and to the Centraalbu-reau voor Schimmelcultures (Baarn, The Netherlands) as CBS 7987 and CBS7988 (13, 24). Authentic strains of type I C. stellatoidea (B-4257 and B-4404) wereprovided by J. Kwon-Chung (National Institutes of Health, Bethesda, Md.) (5).Authentic strains of other Candida species were obtained from the AmericanType Culture Collection (ATCC). Finally, five strains identified as C. dublinien-sis, including the ex-type strain, were provided by F. Odds; the other four strainswere identified by atypical green color on CHROMagar Candida (CHROMagarMicrobiology, Paris, France), repeated demonstration of the absence of intra-cellular beta-glucosidase activity, and weak hybridization by Southern blottingwith the C. albicans-specific oligonucleotide sequence Ca3 (20).

All C. albicans isolates were identified by germ tube and chlamydosporeformation, and the majority of genotype A, B, and C isolates were confirmed tobe C. albicans with the API 20C system (bioMerieux, Marcy, lEtoile, France).

Cellular DNA was isolated by previously described methods (19).Primers for PCR were designed for three separate areas of the DNA encoding

the rRNA. The first pair of primers, ITS1 (59-TCC GTA GGT GAA CCT GCGG-39) and ITS4 (59-TCC TCC GCT TAT TGA TAT GC-39), have been used inprevious studies (15, 26). These give an expected PCR product extending fromthe 59 end of the 18S rDNA to the 39 end of the 25S rDNA and include bothintergenic transcribed spacer (ITS) regions (ITS1 and ITS2) as well as the entire5.8S rDNA.

The second pair of primers, CA25SV3-L (59-TCT TAA CAG CTT ATC ACCCTG GAA TTG GTT-39) and CA25SV3-R (59-ATT GTG TCA ACA TCA CTTTCT GAC CAT CAC-39), were designed from sequences submitted to GenBank(accession nos. X83718 and X83717). These give an expected PCR product thatspans the V3 region of the 25S rDNA, a region previously used for the taxonomicdifferentiation of C. dubliniensis (24).

The final pair of primers results in an expected PCR product that spans the siteof the transposable intron in the 25S rDNA, as published previously (12). Theprimers CA-INT-L (59-ATA AGG GAA GTC GGC AAA ATA GAT CCGTAA-39) and CA-INT-R (59-CCT TGG CTG TGG TTT CGC TAG ATA GTAGAT-39) were designed from sequences submitted to Genbank (accession nos.Z70663 and X74272).

For all PCRs, DNA was amplified in the buffer supplied by the Taq polymerasemanufacturer (Gibco BRL, Grand Island, N.Y.) in a 50-ml volume containing 1mM primers, 1.5 mM MgCl2, 2.5 U of Taq polymerase, 200 mM dATP, 200 mMdCTP, 200 mM dGTP, and 200 mM dTTP. The reactions were performed with anautomated thermal cycler (GeneMate; Lab-Line Instruments, Melrose Park,Ill.). DNA samples were denatured by incubation for 3 min at 94C before 30cycles of 94C for 1 min, 65C for 1 min, and 72C for 2.5 min. After the PCR,amplified DNA was purified with spin columns (Wizard PCR Preps; Promega,Madison, Wis.).

For RFLP analysis of the first (ITS region) and second (V3 25S rDNA region)PCR amplicons, the purified PCR products were digested with the restrictionenzymes DdeI and HaeIII, respectively (Boehringer Mannheim, Indianapolis,Ind.). Endonuclease digestions were done by overnight incubation (to allow forcomplete digestion of the PCR amplimers) with 10 U of enzyme at the recom-mended temperature and with the corresponding buffer.

Ten microliters of the PCR products, with and without endonuclease diges-tion, were analyzed by electrophoresis through a 3% (wt/vol) agarose gel (2%Nusieve GTG, 1% SeaKem Gold; FMC BioProducts, Philadelphia, Pa.) in TAEbuffer (40 mM Tris-acetate, 0.2 mM EDTA) for 2 h at 10 V/cm. Bands werevisualized by Uv transillumination at 302 nm after ethidium bromide staining.

The testing of a representative sample of 105 strains for their susceptibilitiesto both fluconazole and flucytosine was done by the NCCLS M27-A method (14).The strains included in this analysis were randomly selected from a variety ofgeographically diverse sites and included 30 genotype A strains, 30 genotype Bstrains, 30 genotype C strains, and 15 genotype D strains (10). Statistical analysisof these antifungal susceptibility test results was done by transforming the re-sultant susceptibility level by log2. Initially, the transformed results were com-pared for all groups by a multiway analysis of variance (ANOVA). Wheredifferences were observed at the level of a P value of ,0.05, pairs of groups werecompared by a two-sample t test with the assumption of unequal variances.

Differences between groups were assumed to be significant when the probability(P) was #0.05.

RESULTS

Molecular analysis of Candida spp. A total of 457 strains ofCandida were analyzed by all three PCR methods. Four hun-dred thirty-nine of these strains derived from a large study ofisolates identified as C. albicans (289 genotype A strains, 85genotype B strains, 56 genotype C strains and 9 genotype Dstrains) obtained over a 20-year period from 15 geographicallydiverse areas) (10). Nine authentic strains of different speciesof Candida from ATCC, two authentic strains of C. dublinien-sis, two authentic strains of type 1 C. stellatoidea, and a furtherfive strains of C. dubliniensis were analyzed. A list of the au-thentic strains analyzed is given in Table 1.

The RFLPs generated by DdeI digestion of the PCR prod-ucts from the ITS region did not differentiate among C. albi-cans genotypes A, B, and C and type I C. stellatoidea (Fig. 1).However, this method did differentiate the C. albicans geno-type D strains from C. albicans genotype A, B, and C strains(Fig. 1). The profiles of the C. albicans genotype D strains wereidentical to those of the C. dubliniensis strains.

The RFLPs generated by HaeIII digestion of the PCR prod-ucts of the V3 region of the 25S rDNA could differentiate thesame groups as the RFLP analysis of the PCR amplicon of theITS region (Fig. 2). The profiles of the C. albicans genotype Dstrains were identical to those of the C. dubliniensis strains.

PCR designed to span the region that included the site of thetransposable intron in genotype B C. albicans and C. dublini-ensis strains (1, 12) resulted in a single product for C. albicansgenotypes A (;450 bp) and B (;840 bp), C. stellatoidea (;840bp), and C. dubliniensis (;1,080 bp) (Fig. 3). All of the C. al-bicans genotype C isolates had two PCR products (;450 and;840 bp) that were identical in size to the respective productsfrom C. albicans genotypes A and B (Fig. 3). All C. albicansgenotype D isolates gave a PCR product identical in size tothat of C. dubliniensis.

To assess if the C. albicans genotypic subgroup C strainswere not a mixed culture of strains, two randomly chosenisolates were subcultured on yeast potato dextrose (YPD) agarfor 48 h. Twenty-five individual colonies were chosen and

TABLE 1. Order, source, and designation of strains ofthe various Candida species and genotypes

Lane no.a Species/genotypeb Strain designation

1 C. albicans/A ATCC 641242 C. albicans/A ATCC 623423 C. albicans/B ATCC 382464 C. albicans/B ATCC 323545 C. albicans/C Singapore 7c

6 C. albicans/C Singapore 8c

7 C. dubliniensis CD36 NCPF 3949 and CBS 79878 C. dubliniensis CM2 CBS 79889 C. stellatoidea B-425710 C. stellatoidea B-440411 C. parapsilosis ATCC 2201912 C. tropicalis ATCC 1380313 C. krusei ATCC 625814 C. lusitania ATCC 412515 C. glabrata ATCC 6603216 Negative control

a Lane numbers in Fig. 1 to 3.b The C. dubliniensis strains were made available by D. Sullivan (23); the

C. stellatoidea strains were made available by K. J. Kwon-Chung (6).c The Singapore strains were of genotype C (2).

418 MCCULLOUGH ET AL. J. CLIN. MICROBIOL.

grown in YPD broth for 24 h, and their DNAs were extractedand assessed for the presence of the intron in the 25S rDNA.All 25 colonies gave identical results, exhibiting DNA bandstypical of those of genotype A and B strains; this result there-fore indicated that the intron was partially present throughouttheir genomes.

Correlation of species, DNA type, and antifungal suscepti-bility. Descriptive statistics of the MICs obtained by antifungalsusceptibility testing of the randomly chosen 105 isolates(30 C. albicans genotype A strains, 30 C. albicans genotype Bstrains, 30 C. albicans genotype C strains, and 15 C. dubliniensisstrains) are presented in Table 2.

Multiway ANOVA for the comparative fluconazole suscep-tibilities of the four groups gave a P value of 0.003. Analysis ofeach pair of groups by a two-sample t test assuming unequalvariance showed that the C. dubliniensis strains were signifi-cantly more susceptible than each of the C. albicans genotypeA, B, and C strains (P 5 0.018, 0.002, and 0.004, respectively).No other statistically significant comparative differences werefound for the results of fluconazole susceptibility testing.

Multiway ANOVA for the comparative flucytosine suscep-tibilities of the four groups gave a P value of 0.021. Analysis ofeach pair of groups by a two-sample t test assuming unequalvariance showed that the C. albicans genotype A strains weresignificantly less flucytosine susceptible than either C. albicansgenotype B or C. albicans genotype C (P 5 0.004 and 0.003,respectively). No other significant differences were noted forthe results of flucytosine susceptibility testing.

DISCUSSION

The results presented here indicate that those strains previ-ously designated C. albicans genotype D (2, 9, 10) should beassigned to the species C. dubliniensis. All molecular methodsfor the differentiation of isolates to the species level describedhere used PCR techniques. The method that detects the pres-ence and the size of the intron in the 25S rDNA is particularlyeasily adapted for use in reference laboratories for the rapididentification of large numbers of isolates. This simple PCRmethod has the additional advantage of differentiating strainsof 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. 1. Ethidium bromide-stained, UV-transilluminated PCR products (A)and the same PCR products obtained after digestion with DdeI (B) obtained byPCR with the primers for the ITS region. The photograph was obtained afterelectrophoresis in a 3% agarose gel. The DNA from the PCR (A) had first beenpurified with the Wizard PCR Preps purification system prior to overnightdigestion with 10 U of the restriction endonuclease DdeI (B). Molecular sizemarkers 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.

FIG. 2. Ethidium bromide-stained, UV-transilluminated PCR products (A)and the same PCR products obtained after digestion with HaeIII (B) obtained byPCR with the primers for the V3 region of the 25S rDNA. The photograph wasobtained after electrophoresis in a 3% agarose gel. The DNA from the PCR (A)had first been purified with the Wizard PCR Preps purification system prior toovernight digestion with 10 U of the restriction endonuclease HaeIII (B). Mo-lecular size markers are in the lanes marked M, and their corresponding sizes (inbase pairs) are given on the left. The isolate in each lane is specified in Table 1.

FIG. 3. Ethidium bromide-stained, UV-transilluminated PCR products ob-tained 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. Molec-ular size markers are in the lanes marked M, and their corresponding sizes (inbase pairs) are given on the left. The isolate in each lane is specified in Table 1.

VOL. 37, 1999 CHARACTERIZATION OF THREE CANDIDA SPECIES 419

negative mutants of C. albicans (5). There has been extensivedebate over the taxonomic relationship between type I C. stel-latoidea and C. albicans. Genetic traits that have been used todifferentiate these two species have relied on electrophoretickaryotyping (6), mitochondrial DNA restriction fragment pat-terns (6), cellular DNA restriction fragment length polymor-phisms (7), or hybridization of cellular DNA with midrepeatprobes (6). Recently, evidence that shows that it is not possibleto support the differentiation of type I C. stellatoidea fromC. albicans by a variety of molecular and nonmolecular meth-ods has been accumulating (1, 18, 23). A recent study (24)which examined the homology of the rRNA V3 regions andalso sequencing data concluded that C. stellatoidea types I andII are identical to each other and differ from C. albicans by only1 or 2 bases. These results support the previous contention thattype I C. stellatoidea does not merit species status (25). One ofthe earliest reports of the use of molecular methods for thedifferentiation of C. stellatoidea from C. albicans assessed therDNA differences among 15 isolates of C. albicans and 2 iso-lates of C. stellatoidea (7). We note that all 15 of the C. albicansisolates assessed in this previous study were genotypic sub-group A C. albicans strains, because all strains had the 3.7-kbrDNA band, whereas the 2 C. stellatoidea isolates were geno-typic subgroup B C. albicans strains (7). The strains of C. stel-latoidea investigated in the present study were indistinguish-able from the C. albicans genotype B strains by all methods bywhich the strains were assessed. Thus, our results indicate thattype I C. stellatoidea is synonymous with C. albicans genotypeB.

The results of the antifungal susceptibility testing for flucon-azole showed that C. dubliniensis is more susceptible thanC. albicans (genotypes A, B, and C). In early reports there weresome differences in the antifungal susceptibilities of this spe-cies (8, 13, 24). However, the previous studies did not use theaccepted NCCLS method for susceptibility testing. The re-cently reported development of stable fluconazole resistance athigh frequency in vitro in C. dubliniensis (13) was not assessedin the present study, yet it may go a long way toward explainingthe reported increasing occurrence of this species, particularlyin patients with long-term exposure to prophylactic fluconazole(22).

The C. albicans genotype A strains studied in the presentinvestigation showed increased levels of resistance to the an-tifungal agent flucytosine. This finding is consistent with pre-vious data (12, 21), and it has been postulated that there is adirect causal relationship between the presence of the group 1intron in the 25S rDNA (the presence of which determines thatthe strain should be classified as genotype B) and a decrease in

the level of resistance to flucytosine (12). The results presentedhere indicate that this group 1 intron is only partially presentthroughout the rDNA repeats in the genomes of C. albicansgenotype C strains. It may be postulated that we are observingthese strains during a period when this intron is being lost andthey are moving from a genotype B strain to a genotype Astrain and concurrently developing an increased level of resis-tance to flucytosine. Such an hypothesis would partly explainthe increase in the occurrence of genotype C strains worldwide(10), particularly in areas outside the United States wherethere may well be an increasing rate of use of flucytosine.

An alternative hypothesis for the present observations maybe that the genotype C strains represent the genotype A iso-lates that have acquired the intron and that are becominggenotype B strains. The mechanisms by which this acquisitionof genetic material occurs is as yet unknown, but an interestingpossibility would be that genotype C strains represent the prog-eny of the sexual union between a genotype A strain with agenotype B (or type I C. stellatoidea) strain. Further research isrequired to elucidate whether or not the genotype C isolateshave stable genotypes or whether these strains are in the pro-cess of losing or acquiring the group 1 intron in the 25S rDNA.

ACKNOWLEDGMENTS

We thank Lynda Treat-Clemons, diaDexus Corp., Santa Clara, Cal-if., for advice on the statistical analysis and PilSang Park for laboratoryassistance.

This research was funded in part by a fellowship from the Common-wealth AIDS Research Grants Committee of the National Health andMedical Research Council of the Australian Federal Government.

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TABLE 2. Descriptive statistics of the MICs obtained by testing 105 strains their susceptibilities to both fluconazole and flucytosine

Fluconazolea Flucytosineb

C. albicansgenotype A

C. albicansgenotype B

C. albicansgenotype C C. dubliniensis

C. albicansgenotype A

C. albicansgenotype B

C. albicansgenotype C C. dubliniensis

Mean MIC (mg/ml) 22.8 33.4 32.3 4.7 6.9 0.26 2.3 8.7SD MIC (mg/ml) 29.8 29.1 30.5 9.3 3.5 0.07 2.1 5.8Geometric mean MIC (mg/ml) 4.2 10.3 8.9 1.1 0.54 0.18 0.22 0.32No. of isolates 30 30 30 15 30 30 30 15Confidence level (95.0%) 11.1 10.8 11.4 5.2 7.2 0.14 4.3 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 unequalvariance 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.

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420 MCCULLOUGH ET AL. J. CLIN. MICROBIOL.

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