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Journal of Clinical Microbiology, September 2005, p. 4574-4579, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4574-4579.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Microsatellite Marker Analysis as a Typing System for Candida glabrata

F. Foulet,¹ N. Nicolas,¹ O. Eloy,² F. Botterel,¹ J.-C. Gantier,³ J.-M. Costa,^1,4 and S. Bretagne^1,2,3*

Laboratoire de Parasitologie-Mycologie, Hôpital Henri-Mondor-APHP and Université Paris 12, Créteil,¹ Laboratoire de Parasitologie, Hôpital Mignot, Versailles,² Centre National de Référence Mycologie et Antifongiques, Institut Pasteur, Paris,³ Laboratoire de Biologie Moléculaire, Hôpital Américain, Neuilly, France⁴

Received 23 February 2005/ Returned for modification 25 March 2005/ Accepted 7 June 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epidemiological surveillance of candidemia shows that although Candida albicans remains the main species, Candida glabrata is the second leading species recovered from blood cultures in Europe and in the United States (20, 28), especially in intensive care units (9). The same phenomenon is observed for recurrent vaginitis (25). Concern arises about this increase in the incidence of C. glabrata infections because of its frequent decreased sensitivity to azoles compared to C. albicans (14, 22) and the high mortality rate of bloodstream infections (28, 29).

Investigations for C. glabrata have not been as extensive as for C. albicans. The epidemiology is thought to be similar to C. albicans (10). However, few tools are available to study the source of contamination, cross-contamination of patients at risk, and the biology of this yeast, which has, unlike C. albicans, a haploid genome (10). Studies of population structure have begun to emerge using enzymatic and randomly amplified polymorphic DNA typing (6) or sequencing of the cytochrome c oxidase subunit 2 gene (24). Recently, a technique based on the sequencing of six variable-locus genes, or multilocus sequence typing (MLST), has been proposed as a powerful typing method (7). Microsatellites represent another class of genetic markers, defined as short tandem repeats of two to six nucleotides, known to be highly polymorphic (30). Primarily developed for human genetics, they are used for fungi (15) and more specifically for pathogenic fungi such as C. albicans (3-5, 19, 23, 26) and Aspergillus fumigatus (1, 2, 16, 17).

We describe here the development of a genotyping system based on microsatellite markers that has not yet been described for C. glabrata. The polymorphism of microsatellites was evaluated by PCR and allele sizing using fluorescent primers and an automatic sequencer as already reported for C. albicans (3).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolates and DNA extraction. To evaluate the discriminatory power of the microsatellite markers, 138 clinically independent C. glabrata isolates and one reference strain were genotyped. These isolates were collected from different adult patients over a 1-year period from different hospitals and wards. They were collected from the Henri-Mondor Hospital, a 1,000-bed university hospital (85 isolates); from the Versailles Hospital, a 600-bed general hospital (36 isolates); and from outpatients referred to our hospitals (17 isolates). Only one isolate per patient was considered. A total of 67 males and 71 females were sampled. The isolates came from different anatomical sites (blood, 12; abdominal drainage, 8; skin, 7; stool, 33; urine, 46; upper respiratory tract, 32). The isolates came from nine different categories (outpatients, 17; medicine ward, 28; cardiac and neurology surgery, 8; abdominal and urological surgery, 22; intensive care units, 39; hematology ward, 12; nephrology ward, 6; rheumatology ward, 3; human immunodeficiency virus unit, 3). The clinical specimens were cultured on Candida ID (bioMérieux, Marcy l'Etoile, France) at 37°C for 48 h. This chromogenic medium is intended to rapidly discriminate between C. albicans and non-albicans species (31). All blue colonies were identified as C. albicans, and white colonies were phenotyped by using the commercial ATB ID 32C test (API; bioMérieux, Marcy l'Etoile, France) and the Glabrata RTT test (Fumouze Diagnostic, Paris France) (11). The white colonies identified as C. glabrata, one for each specimen, were harvested into plastic tubes and stored at –80°C until further processing. After thawing, the colonies were suspended in 200 µl of tissue lysis buffer (Roche Biochemicals, Meylan, France), and the DNA was extracted using the HP PCR template preparation kit (Roche Biochemicals, Meylan, France) according to the manufacturer's recommendations.

The specificity of the typing method was checked by studying the following reference strains: C. glabrata ATCC 2001, C. albicans B311, Candida kefyr 706 (Pfizer Central Research, Sandwich, United Kingdom), Candida dubliniensis 892 (gift from FC Odds, Aberdeen, United Kingdom), Candida tropicalis ATCC 750, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, and Saccharomyces cerevisiae ATCC 2601.

Multiplex PCR. Three loci containing repeated sequences were simultaneously amplified by multiplex PCR using fluorescently labeled primers. These microsatellite sequences are located upstream of the mitochondrial RNase P precursor (RMP2) gene (unpublished data), the metallothionein I (MTI) gene (21), and the 5,6-sterol desaturase (ERG3) gene (12) of C. glabrata and were therefore named according to the genes they are close to. The sequences of the primers used and their labeling (Proligo, Paris, France) are described in Table 1.

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TABLE 1. Sequences and features of the primers used in this study

Amplification was carried out using a GeneAmp 9700 thermal cycler (Applied Biosystems, Courtaboeuf, France) in a 20-µl volume containing 2 µl of C. glabrata DNA. The composition of the PCR mixture was as follows: 1x PCR buffer II, 2 mM MgCl₂, 0.2 mM each deoxynucleoside triphosphate, 5 pmol each of the RPM2 and ERG3 primers, 20 pmol each of the MTI primers, and 1.25 U of AmpliTaq Gold (Applied Biosystems, Courtaboeuf, France). After an initial step of 10 min at 95°C, the PCR included 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by an additional step of 5 min at 72°C.

Two microliters of the PCR mixture was then added to 20 µl of formamide containing 0.5 µl of the Genescan 500 carboxytetramethylrhodamine-labeled standard (Applied Biosystems, Courtaboeuf, France) and denatured for 2 min at 95°C. The PCR products were subjected to electrophoresis on an ABI 310 sequence analyzer and the data analyzed with the Genescan software (Applied Biosystems).

Sequence analysis. To verify that the differences observed between the different alleles were due to the number of microsatellite sequence repeats, one allele of each length was sequenced after amplification using unlabeled primers. The PCR products were purified using the HP PCR product purification kit (Roche Biochemicals, Meylan, France) and submitted to bidirectional cycle sequencing with the Big Dye terminator v3.0 reaction kit. The sequencing reaction mixtures were analyzed by using an ABI 310 sequence analyzer with the Sequence Analysis software (Applied Biosystems). For the ERG3 locus, as no regular microsatellite sequence was obtained, we decided to sequence all PCR products of the 139 C. glabrata isolates.

Statistical analysis. The clinical data mentioned above (source, sex, ward, and anatomical sites) were coded to generate a contingency table. We performed a factorial correspondence analysis with StatITCF (Institut Technique des Céréales et des Fourages, Paris, France). Only the first three axes were considered for explaining the contingency table variability. Because no clinical data were available for reference strain ATCC 2001, this strain was excluded from the analysis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Specificity and polymorphism of three microsatellite markers were studied using one reference strain and 138 independent C. glabrata isolates. The other seven Candida strains (C. albicans, C. kefyr, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, and S. cerevisiae) were not amplified. For all C. glabrata isolates and for a given locus, only one peak was observed, as expected, since C. glabrata is haploid. For convenience, the alleles were named after the length in base pairs calculated by the automatic sequencer. The stability of the markers was checked for the C. glabrata reference strain after 25 subcultures, corresponding to around 300 generations. Five, 7, and 15 alleles were found for the RPM2, MTI, and ERG3 loci, respectively (Table 2). Overall, a total of 21 distinct multilocus genotypes could be identified using a combination of the three markers (Table 3). This led to an index of discrimination of 0.84 according to the following formula (13):

where N is the number of isolates, s is the number of groups (s = 21), and x_j is the number of isolates falling into the jth group.

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TABLE 2. Allelic frequency of the three markers for the 138 C. glabrata isolates studied and C. glabrata ATCC 2001

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TABLE 3. Characteristics and frequency of the 21 multilocus genotypes determined in this study

Two genotypes were overrepresented, with 34 (24.5%) and 37 (26.6%) of the isolates for genotype numbers 10 and 18, respectively (Table 3). Interestingly, the reference strain C. glabrata ATCC 2001 had its own group (genotype number 5, Table 3). The factorial correspondence analysis showed a very low explanation of the total variance with the three first axes (axis 1 = 7%, axis 2 = 6%, and axis 3 = 6%). The cumulated percentage of the variance was 19%. No correlation was evident between the multilocus genotypes and the clinical data.

To demonstrate that the variability in the size of the PCR products corresponded to a variable number of repeat motifs, one strain representative of each different allele was sequenced for the RPM2 and MTI loci. The difference in length for the RPM2 locus was due to a four- to eightfold repeat of a hexanucleotide (Fig. 1). For the MTI locus, the differences were due to an association of three microsatellite sequences (Fig. 2). Regarding the ERG3 locus, a complex rearrangement was observed instead of an expected simple tandem repeated sequence (Fig. 3). To determine whether sequencing of the PCR products could provide a more polymorphic marker than the length of the PCR product, we sequenced the PCR products of the ERG3 locus for all isolates. We found only two alleles, alleles 181 and 213 (Fig. 3), with little sequence variation. Allele 181 (Table 2) was represented by three C. glabrata isolates which were already classified as unique genotypes (genotypes 2, 5, and 12; see Table 3). Five point variations were observed among the three sequences. For allele 213 (Table 2), four of the five isolates had the same DNA sequence and were classified in genotype 14 (Table 3) while the fifth one had two point variations and was already classified as a unique genotype (genotype 12; see Table 3). Therefore, these differences in sequence did not change the discriminatory power of the analysis. For these eight isolates, the classification depended on the MTI marker.

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FIG. 1. Parts of the sequences of the five different alleles of the RPM2 marker showing the numbers of microsatellite repeats. For allele numbers and frequencies, refer to Table 2.

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FIG. 2. Parts of the sequences of the seven different alleles of the MTI marker showing the numbers of microsatellite repeats. For allele numbers and frequencies, refer to Table 2.

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FIG. 3. Parts of the aligned sequences of the 15 different alleles of the ERG3 marker. For allele numbers and frequencies, refer to Table 2.

The sequencing of all alleles allowed us to determine that the length of the PCR products calculated on the basis of the internal size marker was close to but not the same as the physical length. For instance, allele 181 of the ERG3 marker is composed of 183 bp according to the sequencing and to what was previously reported (12). For this reason, the reference strain was assayed regularly to check that no drift in the automatic calculation occurred.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microsatellite markers are a highly effective method for DNA fingerprinting of mammals and eukaryotic microorganisms such as pathogenic fungi (15). The method is discriminant, reproducible, and easy to perform, and the results remain stable over many generations. However, it is noteworthy that sequencing of the PCR products did not give the length calculated on the basis of the size markers by the software (GenScan). Therefore, it is important to regularly check the reproducibility of the calculation with a reference strain, as already mentioned for C. albicans (3).

Here, we have characterized for the first time three polymorphic markers from C. glabrata. Two of them, RPM2 (Fig. 1) and MTI (Fig. 2), are perfect microsatellites, with different repeats of the same motif. Sequencing of an example of each allele confirmed that the length polymorphism was due to the number of nucleotide motif repeats for the first two of them. For the ERG3 marker, we did not find any regular motif and we decided to sequence all the PCR products to search for additional information. The ERG3 sequences were not strictly identical for 2 out of the 15 alleles. However, this did not change the discriminatory power as the isolates were already found to be classified in different multilocus genotypes with the other markers. However, this homoplasy, i.e., identical lengths but different DNA sequences, could have some deleterious consequences for taxonomy as an allele could be deemed identical although different. Therefore, microsatellite markers must be used cautiously for phylogenetic studies (27). In contrast, for some clinical issues such as whether two clinical isolates are different, this is not of the utmost importance if the isolates are already different using the other microsatellite markers.

The polymorphism of each marker was the same, as 5, 7, and 15 alleles were found for the RPM2, MTI, and ERG3 loci, respectively. These three markers combined gave 21 multilocus genotypes with a discriminatory index of 0.84. The index of discrimination is based on the probability that two unrelated strains sampled from the test population will be placed into different typing groups (13). A regular distribution of the 139 isolates among the 21 multilocus genotypes would have led to a higher discriminatory index. As two multilocus genotypes represented 24.5% and 26.6% of the isolates, this leads to a discriminatory power below the desirable 0.90 if the typing results are to be interpreted with confidence (13). Therefore, one should be cautious before saying that two isolates are identical in clinical studies using the present markers. The discriminatory index might be improved by investigating other loci. Indeed, this is what we obtained when we investigated microsatellite markers from C. albicans. We increased the discriminatory power of 0.88 when testing one microsatellite marker (4) to 0.96 when testing three microsatellite markers (3). When we initiated our work, the whole genome of C. glabrata had not been sequenced. This has been recently achieved (8), and the available sequences can be used to search for additional markers.

The irregular distribution of the C. glabrata population observed with the present genetic markers has already been observed using MLST as 24% (26/109) of the isolates belonged to the same sequence type (7). These authors showed that neither the MLST method nor the randomly amplified polymorphic DNA method discriminated between the great majority of the isolates of the biggest group. Only Southern blot hybridization with the Cg6/Cg12 probe was the superior method (18). Therefore, the fact that some clusters are more prominent could mean that some genotypes have a selective ecological advantage. The fact that the reference strain ATCC 2001 has a unique multilocus genotype might mean that this reference strain is not representative of the general population of C. glabrata.

Another major finding of our study is that no multilocus genotype was associated with any of the clinical data recorded. Therefore, it does not seem that a specific multilocus genotype is associated with a given pathology or a given anatomical site. Additionally, an initial study of 10 pairs of isolates collected at the same time from different anatomical sites, including six blood cultures, showed that in all of the pairs the genotype was the same. These results suggest that the patients were infected with their own colonizing strain, as already shown for C. albicans (26).

In summary, we have developed a microsatellite marker system for DNA fingerprinting of the yeast pathogen C. glabrata. These markers are stable, easy to assay, adaptable to large series, and discriminatory enough to be used as a typing system to investigate clinical issues, such as the nosocomial transmission of isolates or the origin of the infective strains. Our results suggest that some C. glabrata populations are more prominent than others. Further investigations are needed to confirm this hypothesis.

 
We thank Richard Calderone of Georgetown University, Washington, D.C., for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Hôpital Henri-Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France. Phone: 33 1 49 81 36 41. Fax: 33 1 49 81 36 01. E-mail: bretagne{at}univ-paris12.fr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, September 2005, p. 4574-4579, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4574-4579.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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