Analysis of Microsatellite Markers of Candida albicans Used for Rapid Typing

Abstract

To obtain a rapid genotyping method of Candida albicans, three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, called CDC3, EF3, and HIS3, were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci CDC3, EF3, and HIS3, respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for C. albicans. An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans.Among the yeasts that have emerged as major fungal pathogens in recent years (3), the commensal Candida albicans is the most prevalent and acts as an opportunistic agent in immunocompromised patients. The ability to discriminate strains of the organism has been developed for a better understanding of the epidemiology of this yeast. Thus, the route of acquisition (21), nosocomial transmission (16), or the emergence of antifungalresistant strains (6, 25) can be identified by using DNA-based methods already used for typing C. albicans. Strain-typing techniques such as restriction length polymorphic DNA (RFLP) with hybridization with a C. albicans-specific probe and the random amplified polymorphic DNA (RAPD) have been recently reviewed (23). The RFLP technique is very informative but is time-consuming since Southern blots are needed. The PCR-based RAPD technique is rapid but poorly reproducible, especially between laboratories. Other authors have developed allele-specific oligonucleotide probes in Southern hybridizations with PCR-amplified DNA regions (6). Another PCRbased method is the analysis of microsatellites, defined as tandemly repetitive stretches of two to five nucleotides. Since most microsatellites show a substantial level of polymorphism between individuals, microsatellites are extensively used for physical mapping in humans (28). Moreover, since microsatellites test the presence of different alleles at a given locus, distinguishing heterozygotes in diploid organisms such as C. albicans is possible in contrast to the RFLP and RAPD methods. Several studies have already reported the application of this technique for the genotyping of C. albicans (4,7,10,13,22).One microsatellite marker in the EF3 promoter sequence...

Microsatellite Typing of Catheter-Associated Candida albicans Strains

“…The DP of EF3 is 0.86, the DP of CDC3 is 0.77, and the DP of HIS3 is 0.91 (Botterel et al, 2001). The combined DP of all three markers was 0.97.…”

“…For DNA extraction the PrepManTM Ultra Kit (Applied Biosystems [AB], Foster City, California) was used. For the microsatellite typing three different primer pairs were used (Botterel et al, 2001). The unmarked primers were HIS3R, CDC3R, and EF3 (Invitrogen, Lofer, Austria).…”

“…These different multilocus genotypes are used to distinguish strains within a single species (Applied Biosystems [AB], 2005). Microsatellite markers provide the possibility to discriminate strains of the same species and to trace their epidemiological pathways (Botterel et al, 2001;Sampaio et al, 2005).…”

“…The combined DP of all three markers was 0.97. In order to get reliable results, this index has to be greater than 0.90 (Botterel et al, 2001). …”

“…For this study, pairs for three loci (CDC3, EF3, and HIS3) on three different chromosomes developed by Botterel et al (2001) were used to compare the C. albicans strains which were found to produce a biofilm, with those strains which did not produce a biofilm on the investigated catheter material. The differentiation of biofilm and non-biofilm forming 4 strains was based on scanning electron microscopical findings (Paulitsch et al, 2009).…”