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J Clin Microbiol. May 2010; 48(5): 1677–1682.
Published online Mar 10, 2010. doi:  10.1128/JCM.02151-09
PMCID: PMC2863883

New Polymorphic Microsatellite Markers Able To Distinguish among Candida parapsilosis Sensu Stricto Isolates[down-pointing small open triangle]

Abstract

Among the Candida species causing bloodstream infections, Candida parapsilosis is one of the most frequently isolated. The objective of the present work was the identification of new microsatellite loci able to distinguish among C. parapsilosis isolates. DNA sequences with trinucleotide repeats were selected from the C. parapsilosis genome database. PCR primer sets flanking the microsatellite repeats were designed and tested with 20 independent isolates. On the basis of the amplification efficiency, specificity, and observed polymorphism, four of the sequences were selected for strain typing. Two hundred thirty-three independent C. parapsilosis sensu stricto isolates were genotyped by using these markers. The polymorphic loci exhibited from 20 to 42 alleles and 39 to 92 genotypes. In a multiplex analysis, 192 genotypes were obtained and the combined discriminatory power of the four microsatellites was 0.99. Reproducibility was demonstrated by submission of subcultures of 4 isolates each, in triplicate, interspersed with unique numbers among a group of 30 isolates for blind testing. Comparison of the genotypes obtained by microsatellite analysis and those obtained by randomly amplified polymorphic DNA analysis, restriction fragment length polymorphism analysis, and internal transcribed sequence grouping was performed and showed that the microsatellite method could distinguish individual isolates; none of the other methods could do that. Related species, C. orthopsilosis and C. metapsilosis, were not confused with C. parapsilosis sensu stricto. These new microsatellites are a valuable tool for use for the differentiation of C. parapsilosis sensu stricto strains, vital in epidemiology to answer questions of strain relatedness and determine pathways of transmission.

Species of the genus Candida are the agents that are the most frequently implicated in invasive fungal infections, presently ranking as the third most common cause of nosocomial bloodstream infections in the United States (26, 27). In Europe and Latin America, Candida parapsilosis is a predominant species found in patients with systemic infections and causes up to 45% of all cases of candidemia (1, 37). This yeast causes several infections in humans, including a variety of life-threatening conditions triggered by bloodstream infections; and it has been associated with catheters, hyperalimentation, premature birth, and contaminated prosthetic devices (23, 40). C. parapsilosis is particularly isolated from cultures of blood from ill neonates and children (20, 23), surgical intensive care unit patients (17), as well as hematological patients (7, 31). Unlike other Candida species, C. parapsilosis has been found on the hands of health care workers, who install and maintain medical devices, suggesting a potential route for transmission (41). The rapid identification of the strains involved in the infection and the elucidation of their patterns of genetic diversity are issues of great medical relevance since such knowledge can contribute to the development of new strategies for the prevention and treatment of the infections.

Physiologically indistinguishable isolates of C. parapsilosis have been reported to be genetically heterogeneous. Several reports suggested that C. parapsilosis is composed of three distinct groups, groups I, II, and III, on the basis of isoenzyme analysis, random amplification of polymorphic DNA (RAPD) analysis, analysis of the nucleotide sequence of the internal transcribed sequence (ITS) (16, 19, 21, 25), and DNA-DNA hybridization studies (30). Tavanti et al. (36) proposed that the three different C. parapsilosis groups be considered different species on the basis of these observations and the degree of sequence variation observed among groups. Group I isolates maintained the former name, C. parapsilosis (sensu stricto), and groups II and III included the strains to which the new names C. orthopsilosis and C. metapsilosis, respectively, have been proposed. The vast majority of clinical isolates belong to C. parapsilosis group I, characterized by low levels of nucleotide sequence variation, which constitutes a challenge for the development of genotyping techniques.

The aim of the work described here was to identify and describe new polymorphic microsatellite loci in the genome of the pathogenic yeast C. parapsilosis and evaluate their applicability for use as molecular markers for the differentiation of isolates. The performance of this typing method was assessed by comparison of the results to the results obtained by using three previously described typing methods.

MATERIALS AND METHODS

Microsatellite selection and PCR primer design.

A search of C. parapsilosis genome sequences available in databases from the Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/c_parapsilosis) was performed to identify sequences containing more than 20 tri- or tetramicrosatellite repeat units which were expected to have very high degrees of polymorphism. Eleven microsatellites were selected, and primers specific for the nonvariable flanking regions were designed for locus-specific amplification. Primer3 software (available from http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) was used for primer design. On the basis of the results of preliminary studies of amplification efficiency (detailed in Results), species specificity, and the polymorphism observed, 3 of the 11 previously selected sequences were selected for further characterization and for application in strain identification. A microsatellite marker, marker B, previously described for use for C. parapsilosis strain typing (18), was also included in this study. Marker B was chosen from among the previously described microsatellite markers due to its higher degree of polymorphism. The microsatellite sequences selected and their repetitive motifs, the primers used for PCR amplification, and the expected fragment sizes are presented in Table Table11.

TABLE 1.
Microsatellite DNA sequences selected, sequence codes, and primers used for PCR amplificationa

Yeast strains and DNA extraction.

A total of 236 independent C. parapsilosis isolates, including 100 from blood cultures, 9 from the respiratory tract, 27 from skin and nails, 58 from other body sources, 39 from the environment (including hospital air and surfaces), type strain ATCC 22019, and reference strains PNAEQ008-1/2004 and NQ7356/1850 (clinical isolates used for quality control purposes), were selected for use in this study. Most of them were collected from different places in Portugal, but collections of isolates that included those from a variety of geographical origins were also analyzed. These collections were obtained from France, Spain, the United States, and Peru. The type strains of C. albicans (ATCC 18804), C. krusei (ATCC 6258), C. tropicalis (ATCC 750), C. glabrata (ATCC 2001), C. bracarensis (NCYC D3853), C. guilliermondii (ATCC 6260), C. lusitaniae (ATCC 34449), C. dubliniensis (CBS 7987), C. orthopsilosis (ATCC 96139), C. metapsilosis (ATCC 96144), and Lodderomyces elongisporus (ATCC 22688) were also tested. One isolate of Aspergillus fumigatus as well as isolates of the bacteria Escherichia coli and Staphylococcus aureus, obtained from the culture collection of our laboratory, were also included in this study. All C. parapsilosis isolates were identified by using ID 32C strips or Vitek YBC identification cards (bioMérieux, SA, Marcy l'Étoile, France) and by PCR fingerprinting with primer T3B by using the methodology described previously (6). Stock cultures were maintained on Sabouraud glucose agar medium at 4°C. Prior to DNA isolation, yeast cells were grown for 24 h on Sabouraud glucose agar medium at 37°C. DNA extraction was performed by using a High Pure PCR template kit (Roche Diagnostics Corp., Indianapolis, IN), according to the manufacturer's instructions.

PCR amplification conditions.

For all microsatellite loci, PCRs were performed with several different strains to evaluate the locus-specific amplification. Loci CP1, CP4, and CP6 (their selection is described in Results) were amplified by using 25 ng of genomic DNA in a 25-μl reaction volume containing 1× PCR buffer, 0.2 mM each four deoxynucleoside triphosphates, 1.5 mM MgCl2, 0.25 μM each primer, and 1 U of Taq polymerase (Applied Biosystems, Inc., Foster City, CA). After a 95°C preincubation step for 2 min, PCR amplifications were performed in a total of 28 cycles under the following conditions: denaturation at 94°C for 30 s, annealing at 54 to 60°C (depending on the locus) for 30 s, and extension at 72°C for 1 min and with a final extension step of 7 min at 72°C.

Fragment size determination and DNA sequence analysis.

Following PCR, the amplification products were diluted 1:10 with distilled water and a 1-μl aliquot was added to 13.7 μl of formamide and 0.3 μl of the internal size standard (GenScan 500 6-carboxytetramethylrhodamine [TAMRA]; Applied Biosystems, Inc.). The samples were denatured at 95°C for 5 min and rapidly chilled on ice. The samples were run on an ABI 310 genetic analyzer (Applied Biosystems). For accurate allele size determination, the forward primers were fluorescently labeled with tetrachloro-6-carboxyfluorescein (TET), 6-carboxyhexafluorescein (HEX), or 6-carboxyfluorescein (FAM) (Table (Table1)1) and the sizes of the PCR products were determined by using GeneScan (version 3.7) analysis software. The alleles have been designated by their sizes (in base pairs).

For DNA sequence analysis, only homozygous strains were selected. After PCR amplification, the products were purified with Microspin S-300 HR columns (Pharmacia). The purified products were subjected to a dideoxy cycle sequencing reaction with a BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems). Sequence analysis was performed on an ABI 310 genetic analyzer.

RFLP, RAPD, and ITS sequencing.

RFLP, RAPD, and ITS analyses were performed as described previously (38).

Stability.

Three different clinical isolates and the reference strains were grown in 1-liter Erlenmeyer flasks containing 500 ml of Sabouraud medium and incubated at 30°C in an orbital shaker (160 rpm). At the end of the exponential phase, a 1/10 dilution with new medium was made in order to allow the continuation of cell duplication. This procedure went on for 4 weeks, until the completion of about 300 generations. The cells were harvested at the end of approximately 100, 200, and 300 generations; and the DNA was extracted for amplification.

Reproducibility.

The reproducibility of the microsatellite method was assessed systematically by including a reference strain as a control in each run. In addition, samples were included in some runs in coded replicates, prepared by coinvestigators not involved in that microsatellite run (blind testing; see Results).

Statistical analysis.

Allelic and genotypic frequencies were determined by using ARLEQUIN (version 2.000) software, and the discriminatory power of the markers was calculated as described by Hunter and Gaston (15).

RESULTS

Screening and selection of repeat regions in C. parapsilosis genome sequence database.

The search of the genomic DNA database for C. parapsilosis sequences that was performed provided ~1,000 sequences when AAC, AAG, AAT, CCA, CCG, CTT, CAAA, and AACA queries were used. Selection of the sequences was primarily based on the number of simple units repeated. The criteria defined were that the sequence should be at least 20 repeats, given that these sequences have a higher probability of showing greater genetic variability, and they should be located outside known coding regions, since a higher degree of polymorphism is expected in these regions less prone to selective forces (12, 24, 28). Subsequently, another selection was made for sequences that allowed the design of primers with an annealing temperature above 54°C to ensure specificity and good reproducibility. Eleven sequences met our criteria and were selected for locus-specific primer design and amplification in studies with a set of independent C. parapsilosis strains. The nomenclature chosen for the new markers was CP, after C. parapsilosis, followed by a number that corresponded to the order in which they were studied in the pilot experiments. On the basis of the results of the amplification of DNA from 23 selected C. parapsilosis strains, seven markers did not show suitable amplification efficiencies and two markers, although they were chosen from different sequences, turned out to be the same locus. The remaining three, CP1, CP4, and CP6, gave satisfactory results with regard to the selection criteria and were used for further characterization. Table Table11 shows the chosen loci and the respective repetitive motifs, the numbers of repetitions, as well as the expected PCR product size, according to the database sequence.

Sequence analysis.

Several representative alleles have been sequenced, and sequencing analysis of the amplified fragments revealed that the consensus sequences were in accordance with those originally published (Table (Table1),1), confirming the locus-specific amplification and structure of the alleles. A direct correlation between the fragment size and the number of microsatellite repeats was found for all the markers, with the differences in fragment sizes being consistent with the variation in the number of repetitions.

Specificity and stability.

These markers were revealed to be species specific, since no amplification products were obtained when the primers and PCR conditions described were used for the screening of other pathogenic Candida species, namely, C. albicans, C. krusei, C. tropicalis, C. glabrata, C. bracarensis, C. guilliermondii, C. lusitaniae, C. dubliniensis, and C. metapsilosis. Faint amplification was detected with microsatellite CP6 by analysis with GeneScan software when strains of C. orthopsilosis were tested. Raising the PCR annealing temperature to 60°C for the amplification of marker CP6 eliminated the amplicons of the C. orthopsilosis strains. This alteration did not interfere with the amplification of the C. parapsilosis isolates.

No PCR products were obtained when DNA from Lodderomyces elongisporus, a species closely related to and recently described in cases of bloodstream infections misidentified as C. parapsilosis, was amplified (22). Likewise, no amplification was obtained with the fungus Aspergillus fumigatus or with the bacteria Escherichia coli and Staphylococcus aureus.

The in vitro stability of the microsatellites was followed over 300 generations. For all the strains tested, the genotypes were the same after these generations, suggesting that their expected mutation rate is less than 3.33 × 10−3. Similar results have previously been reported for C. albicans microsatellites (32, 33).

Microsatellite locus analysis.

The microsatellites selected were used to type 236 independent C. parapsilosis isolates, including clinical isolates from different origins and environmental strains, in order to evaluate their specific amplifications and polymorphisms. One or two PCR fragments per locus were obtained for each strain, and since C. parapsilosis is a diploid species (5), each fragment was assigned to an allele. The strains showing two PCR products were typed as heterozygous, while strains presenting a single amplification product were considered homozygous. Less intense stutter bands were frequently present, reflecting polymerase slippage during the PCR, but they did not present any problem with the identification of the correct fragment since they were of a lower intensity. No PCR products were obtained with the new microsatellite markers from three bloodstream isolates assigned to the species C. parapsilosis. These isolates were further analyzed by sequencing of the ITS and D1/D2 regions of the 26S rDNA. This showed that two of the isolates were indeed C. metapsilosis strains and that the other was a strain of C. orthopsilosis. PCR products were obtained for all the other 233 C. parapsilosis isolates at all three loci, resulting in 100% typeability.

The analysis of the 233 isolates showed that all the microsatellite loci were polymorphic, presenting between 20 and 42 alleles and from 39 to 92 different genotypes. The size ranges of the alleles as well as the corresponding number of repeats are shown in Table Table2.2. A significant departure from Hardy-Weinberg equilibrium expectations was found (P < 0.001), supporting the suggestion that inheritance in C. parapsilosis is mainly clonal, as has been inferred by other authors (36).

TABLE 2.
Characteristics of the microsatellite loci selected

The discriminatory power (DP) was calculated for each marker according to the Simpson index:

equation M1

where N is the number of strains, s is the total number of different genotypes, and nj is the number of strains of genotype j (15). The results showed that CP6 was the microsatellite with the highest DP value (0.96), while CP1 presented the lowest DP value (0.85). Microsatellite marker B produced 22 different alleles and 40 genotypes, resulting in a discriminatory power of 0.86 (18). The total number of different alleles and genotypes and the respective frequencies obtained for all microsatellite markers are presented in Table Table22.

In the multiplex analysis with markers CP1, CP4, CP6, and B, a total of 192 multilocus genotypes were obtained and the combined discriminatory power was 0.99. An example of a multilocus GeneScan profile is shown in Fig. Fig.1.1. The great majority of the multilocus genotypes were found only once (79%). The most frequently detected multilocus genotype was shared by 11 independent widespread strains, and the 13 most frequently detected multilocus genotype accounted for 21% of the isolates (Table (Table33).

FIG. 1.
GenScan profile obtained by multilocus analysis with the markers CP1 (green), CP4 (black), CP6 (blue), and B (blue). The strain analyzed is homozygous for markers CP4 and B and heterozygous for markers CP1 and CP6.
TABLE 3.
Most common multilocus genotypes found among the isolates analyzed and their respective frequencies

Comparison of results of different C. parapsilosis typing methods.

Thirty isolates of the C. parapsilosis family typed by ITS sequencing, RFLP analysis, and RAPD analysis were selected for genotyping with the microsatellite markers described above so that the results of the microsatellite marker genotyping method could be compared with those of the other typing methods. Ten isolates of C. orthopsilosis and C. metapsilosis strains (including one C. metapsilosis isolate and one C. orthopsilosis isolate, each included in triplicate as a blind reproducibility test) were correctly identified as not C. parapsilosis sensu stricto (these would be RAPD groups B and C and RFLP groups other than VII-1). The results of microsatellite genotyping of the isolates determined to be C. parapsilosis sensu stricto are shown in Table Table4.4. Fourteen different multilocus genotypes were obtained. Two isolates of C. parapsilosis sensu stricto (RAPD group A, RFLP group VII-1) were included in triplicate (isolates IMR013, IMR160, and IMR339 and isolates IMR073, IMR320, and IMR 327, respectively) as a blind reproducibility test, and the isolates in each triplet were correctly identified to be identical.

TABLE 4.
Correlation of RLFP, RAPD, and ITS1 typing results with results of microsatellite analysis

Thus, from 15 truly unique isolates, 14 different multilocus genotypes were obtained. Two C. parapsilosis sensu stricto isolates, isolates IMR649 and IMR013 (and blind replicate isolates of IMR013, isolates IMR160 and IMR339) could not be distinguished by microsatellite typing (nor could they be distinguished by RFLP, RAPD, or ITS1 typing). These two isolates originally came from two different sites (the mouth and the vagina) from patients in two different cities in England (Leeds and London) (although the overall collection studied [36, 38] came from a broad geographic background) in the early 1970s (and were possibly collected in the same year). The two isolates came from two different sets of isolates gathered by the same researcher in both places and presumably came from two different patients. Although one cannot completely exclude the possibility that they were the same patient who may have been involved in two different studies, the best explanation is that two patients were infected with the same genotype, and whether there was any connection between the patients is unknown.

Two unique isolates are special cases. Both had discrepant RAPD results in two laboratories (36, 38), with one laboratory grouping them with C. parapsilosis sensu stricto and the other not. These were not RFLP type VII-1, which is generally synonymous with C. parapsilosis sensu stricto (38), and were more consistent with C. orthopsilosis types, as delineated by RFLP analysis, whereas they were grouped with C. parapsilosis sensu stricto by ITS1 sequencing. Thus, it was tentatively concluded (38) either that RFLP analysis can differentiate a few types of C. parapsilosis sensu stricto that ITS typing cannot or that these isolates represented some hybrid group sharing some characteristics with C. parapsilosis sensu stricto and C. orthopsilosis. By microsatellite typing, one of these two isolates was determined not to be C. parapsilosis sensu stricto, tentatively grouping the isolate with C. orthopsilosis and C. metapsilosis (data not shown). The other isolate (isolate IMR082 in Table Table4)4) was determined to be C. parapsilosis sensu stricto by microsatellite typing.

DISCUSSION

A variety of strain typing methods have been used for the differentiation of the C. parapsilosis family, including electrophoretic karyotype (EK) analysis (9, 35), DNA fingerprinting with probe Cp3-13 (11), RAPD analysis (10, 42), RFLP analysis (38, 39), multilocus sequence typing (MLST) (36), and pyrosequencing (2). van Asbeck et al. (38) compared three of these molecular typing methods, ITS sequencing, RAPD analysis, and RFLP analysis, and indicated that ITS1 typing and RFLP analysis appear to have similar potentials to differentiate C. parapsilosis and both have discriminatory abilities superior to the discriminatory ability of RAPD analysis. This inferiority of RAPD analysis was also true for other methods, which subdivide the C. parapsilosis sensu lato complex into only three or a few more groups, as ITS sequencing and RFLP analysis are able to subtype RAPD type B/group II/C. orthopsilosis. However, the inability of ITS1 typing and RFLP analysis to subtype the dominant group, group I/C. parapsilosis sensu stricto, has been an obstacle.

The only previously reported method able to distinguish isolates within the C. parapsilosis sensu stricto group is a microsatellite method based on dinucleotide repeats, but the combined DP of 0.97 achieved for all seven loci was not optimal (18). Furthermore, the typing of dinucleotide microsatellites presents limitations for accurate allele identification, even in automatic systems, owing to the high frequency of dinucleotide repeat slippage.

Analysis of microsatellite loci has an advantage over other commonly used typing methods because microsatellite loci behave as codominant markers, evolve rapidly in the genome, and may be able to distinguish between isolates and detect microevolutionary variations (32). The microsatellite genotyping of several yeast species, namely, Saccharomyces cerevisiae (14), C. albicans (3, 4, 33), C. krusei (34), and C. glabrata (13), has been described.

In the present report, we described new microsatellite markers for C. parapsilosis sensu stricto. In a multilocus analysis, 192 genotypes were found among 233 isolates, resulting in a DP of 0.99, much higher than that published previously. This microsatellite method would therefore be a more robust method for epidemiologic purposes than RFLP analysis, RAPD analysis, MLST, or ITS1 sequencing. None of those methods can distinguish isolates, possibly with rare exceptions (38), coincident with the most common group encountered in patients or the environment, C. parapsilosis sensu stricto. The reproducibility of this microsatellite method was demonstrated by the inclusion of replicates in blind testing. Isolates of the related species C. orthopsilosis, C. metapsilosis, and L. elongisporus, isolates of other Candida species, and isolates of other fungal and bacterial species were not confused with C. parapsilosis sensu stricto.

The high degree of polymorphism observed could be correlated with a high mutation rate, which would eventually limit the use of these markers for strain identification. However, our results demonstrate that these microsatellites are relatively stable in laboratory culture, with the estimated mutation rate being less than 3 × 10−3. Mutation rates for microsatellites can range from 10−2 to 10−6, while point mutations are known to accumulate more slowly, at approximately 10−9 mutations per generation (8, 29). Therefore, for outbreak investigations, which usually span short time intervals, we believe that the microsatellites are suitable markers.

In conclusion, these new microsatellites are a valuable tool for use for the differentiation of C. parapsilosis strains and for application in studies that must distinguish epidemiologically related isolates, study nosocomial cross-transmission, and study the kinetics of the colonization-to-infection process. This method is easy to perform, discriminatory, and highly reproducible, constituting the ideal method for use in large-scale epidemiological studies. The standardization of the microsatellite typing systems and the creation of public databases that would make microsatellite allele data available worldwide are essential issues that deserve attention.

Acknowledgments

We are indebted to Frédéric Grenouillet, Guillermo Garcia-Effron, Zélia Videira, and Maria da Luz Martins for kindly providing C. parapsilosis strains. We thank Eveline van Asbeck for collaboration with the RFLP and RAPD studies and Magda Graça for the sequencing analysis.

This research was supported by the Fundação para a Ciência e Tecnologia (FCT), Portugal, through a multiyear contract with the Centre of Molecular and Environmental Biology (CBMA), University of Minho. Raquel Sabino received a fellowship from FCT, Portugal (contract BD/22100/2005).

Footnotes

[down-pointing small open triangle]Published ahead of print on 10 March 2010.

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