0296-05.pdf

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2005, p. 38693876 Vol. 43, No. 80095-1137/05/$08.000 doi:10.1128/JCM.43.8.38693876.2005Copyright 2005, American Society for Microbiology. All Rights Reserved.

New Microsatellite Multiplex PCR for Candida albicans Strain TypingReveals Microevolutionary Changes

Paula Sampaio,1 Leonor Gusmao,2 Alexandra Correia,1 Cntia Alves,2 Acacio G. Rodrigues,2,4Cidalia Pina-Vaz,2,4 Antonio Amorim,2,3 and Celia Pais1*

CBUM-Center of Biology, University of Minho, 4710-057 Braga, Portugal1; IPATIMUP-Institut of Pathology and MolecularImmunology of Porto University, R. Roberto Frias, s/n 4200 Porto, Portugal2; Faculty of Science, University of Porto, Porto,

Portugal3; and Microbiology Department, Faculty of Medicine, University of Porto, Porto, Portugal4

Received 15 February 2005/Returned for modification 28 March 2005/Accepted 18 April 2005

Five new microsatellite loci were described and characterized for use as molecular markers for the identi-fication and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinicalisolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46different genotypes. The discriminatory power obtained by combining the information generated by threemicrosatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. Themultiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the samepatient collected from different body locations and along episodes of vulvovaginal infections. Three differentscenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain,(ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii)isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates fromrecurrent infections indicated that the genotype alterations observed could be the result of events that lead tothe loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, andthis could have been related to exposure to fluconazole, since such strains were exposed to this antifungalduring treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficienttool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of finemicroevolutionary events that could be related to strain microevolution in response to environmental stressconditions.

Candida albicans, the most common fungal pathogen, is acommensal yeast that belongs to the normal microbial popu-lation of the mouth, vagina, and gastrointestinal tract in hu-mans. However, in people with a variety of transient or per-manent immunocompromised conditions, including transplantrecipients, chemotherapy patients, underweight neonates, andhuman immunodeficiency virus-infected individuals, it may be-come an invasive pathogen (30, 56). Infections by opportunisticfungal agents are a major medical problem due to the growingnumber of immunocompromised patients with risk factors forsuch infections. Moreover, this problem is exacerbated by thefact that only a limited array of antifungal drugs is availableand by the growing resistance among clinical isolates (39, 46,55). The development of techniques and strategies that canaccurately differentiate clinical isolates is of great relevance.These techniques should provide the ability to differentiateamong strains responsible for clinical infections, as well as totrace their epidemiological pathways. Several molecular meth-ods have been used to differentiate C. albicans strains, includ-ing electrophoretic karyotyping (2), the use of species-specificprobes such as Ca3 or 27A in restriction enzyme analysis (28,35, 36, 43), PCR-based methods (1, 7, 15, 51), and more re-cently, multilocus sequence typing, which uses nucleotide se-

quences from several genes and offers good discrimination (4,49). Microsatellites or simple tandem repeats (STRs) consist ofstretches of tandemly repeated mono- to hexanucleotide mo-tifs dispersed throughout the genome and have a high level ofpolymorphism compared with those of other molecular mark-ers. In yeast, microsatellite loci have considerable length vari-ations, and this polymorphism quickly made them attractivemarkers for a variety of types of analyses, including straintyping (9, 16, 45), population structure analysis (13, 18, 25),and epidemiological studies (33, 42). Microsatellite polymor-phism is manifested as allelic length differences due to thedifferent numbers of repeated units present in the alleles andis easily assayed by PCR amplification (48).

Several polymorphic microsatellite loci have been identifiedin the C. albicans genome, most of them located near EF3 (5);CDC3 and HIS3 (3); or inside the coding regions ERK1, 2NF1,CCN2, CPH2, and EFG1 (31), although the discriminatorypowers (DPs) calculated for such loci were relatively low (be-tween 0.77 and 0.91). A higher value, 0.97, was estimated bySampaio et al. (42) for microsatellite CAI, located in a non-coding region; and this discriminatory power was identical tothat which had previously been reported for a combination ofthree microsatellites amplified in a multiplex reaction (3). Ahigher polymorphism is expected in microsatellite loci fromnoncoding regions; therefore, in order to obtain a greaterresolution, a set of new microsatellite markers was selectedfrom these regions and characterized for use in a multiplexPCR. This multiplex typing system was applied to C. albicans

* Corresponding author. Mailing address: Departamento de Biolo-gia, Centro de Biologia da Universidade do Minho, Campus de Gual-tar, 4710-057 Braga, Portugal. Phone: (351)253604312. Fax: (351)253678980. E-mail: [email protected].

3869

clinical isolates to test its efficiency for strain differentiation.The potential capacity of this system to identify fine microevo-lutionary events was also evaluated.

MATERIALS AND METHODS

Yeast strains and DNA extraction. A total of 112 clinical isolates of C. albi-cans, obtained from two hospitals and a health care center located in Braga andPorto (northern Portugal), and reference strain WO-1, as well as type strainPYCC 3436 (ATCC 18804), were used in this study. The isolates were obtainedfrom primary cultures, and one colony of each different phenotype was selected.The type strains C. parapsilosis PYCC 2545 (ATCC 22019), C. krusei PYCC 3341(ATCC 6258), C. tropicalis PYCC 3097 (ATCC 750), C. glabrata PYCC 2418(ATCC 2001), C. guilliermondii PYCC 2730 (ATCC 6260), C. lusitaniae PYCC2705 (ATCC 34449), and C. dubliniensis CBS 7987 (ATCC MYA-646) were alsoincluded. All reference strains were obtained from the Portuguese Yeast CultureCollection (PYCC), New University of Lisbon, Lisbon, Portugal, except C. dub-liniensis, which was obtained from Centraalbureau voor Schimmelcultures, Utre-cht, The Netherlands. The isolates were previously identified by PCR finger-printing with primer T3B (7). Prior to DNA isolation, yeast cells were grownovernight on Sabouraud broth medium at 30C. A Zymolyase-based method wasused to extract DNA by following procedures described previously (22). AfterDNA extraction, the cultures were frozen in 30% (wt/wt) glycerol.Microsatellite selection and PCR primers design. A search of the C. albicans

genome sequences, available in databases from Stanfords DNA Sequencing andTechnology Center (http://www-sequence.stanford.edu/group/candida), was per-formed in order to identify sequences containing microsatellite repeats. Thesearch was performed with the aim of identifying microsatellite units of tri-,tetra-, and pentanucleotides according to criteria described previously (42). Thesequences that were obtained and selected for locus specific amplificationare presented in Table 1. Primers for each locus were designed by using thesoftware Primer3, available from http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.

Chromosomal localization of microsatellite markers. The sequences selectedwere searched by BLAST against the latest release (assembly 19) of C. albicansgenome sequence, to give a location to a sequence contig (http://www-sequence.stanford.edu/group/candida).PCR amplification conditions. (i) Singleplex amplification. For all micro-

satelite loci, singleplex PCRs were performed with several different strains inorder to evaluate the locus-specific amplification and to obtain alleles for se-quencing. The locus CAI was amplified by following the conditions described bySampaio et al. (42). The CAIII, CAV, CAVI, and CAVII loci were amplified byusing 25 ng of genomic DNA in a 25-l reaction volume containing 1 PCRbuffer, 0.2 mM of each of the four deoxynucleoside triphosphates, 1.5 mMMgCl2, 0.25 M of each primer, and 1 U of Taq polymerase (GIBCO). After a95C preincubation step of 2 min, PCR amplifications were performed for a totalof 30 cycles by using the following conditions: denaturation at 94C for 30 s,annealing at 60C for 30 s, and extension at 72C for 1 min, with a final extensionstep of 7 min at 72C.(ii) Multiplex amplification. Multiplex PCR was performed by combining 1

PCR buffer (20 mM Tris HCl, pH 8.4, 50 mM KCl), 0.2 mM of each four of thedeoxynucleoside triphosphates, 2 mM MgCl2, 40 ng of genomic DNA, and 2 Uof Taq Gold polymerase (Applied Biosystems) carried in a 25-l final volume.The PCR program consisted of an initial denaturation step at 95C for 10 min,followed by 30 cycles of 30 s at 94C, 30 s at 58C, and 1 min at 72C, with a finalextension step of 60 min at 68C. The multiplex reaction was designed bycombining microsatellites CAI, CAIII, and CAVI; and the primer concentrationsused are depicted in Table 2.DNA sequencing and fragment size determination. All alleles observed for

each locus were sequenced. Before sequencing, the alleles were separated andreamplified as described previously (42). The reamplified DNA fragments werepurified with Microspin S-300 HR columns (Amersham Pharmacia Biotech,Quebec, Canada) and subjected to a dideoxy sequencing reaction with theBigDye terminator cycle sequencing ready reaction kit (Applied Biosystems).Following the sequencing reactions, the products were purified with AutoSeqG-50columns (Amersham Pharmacia Biotech). Finally, the samples were dried, re-

TABLE 1. Microsatellite DNA sequences selected from the database searcha

Microsatellitedesignation Sequence code

b Primer sequencec No. ofrepeated unitsPCR product

size (bp)

CAI 396062C04.sl.seq F-5-ATGCCATTGAGTGGAATTGG-3 41 252R-5-AGTGGCTTGTGTTGGGTTTT-3

CAIII 265080D05.sl.seq F-5-TTGGAATCACTTCACCAGGA-3 11 113R-5-TTTCCGTGGCATCAGTATCA-3

CAIV 265080D05.sl.seq F-5-TGCCAAATCTTGAGATACAAGTG-3 24 263R-5-CTTGCTTCTCTTGCTTTAAATTG-3

CAV 265147C06.yl.seq F-5-TGCCAAATCTTGAGATACAAGTG-3 50 237R-5-CTTGCTTCTCTTGCTTTAAATTG-3

CAVI 385031H11.xl.seq F-5-ACAATTAAAGAAATGGATTTTAGTCAG-3 21 294R-5-TGCTGGTGCTGCTGGTATTA-3

CAVII 396033C06.sl.seq F-5-GGGGATAGAAATGGCATCAA-3 10 222R-5-TGTGAAACAATTCTCTCCTTGC-3

a The sequence codes and primers used for PCR amplification are presented. The number of repeated units and the predicted size of PCR amplification productswere calculated according to the sequence of the strain SC5314 used in the sequencing project.

b Code attributed to the sequence in the Stanford database.c For each primer pair, the letter F indicates the forward primer and the letter R indicates the reverse primer.

TABLE 2. Characteristics of the microsatellite loci selected

STR No. of alleles No. ofgenotypes Size (bp) Repetitive motif DP Chromosome no. Primer concn (M) Dye labela

CAI 27 46 189303 (CAA)2CTG(CAA)n 0.967 4 0.050 FAMCAIII 6 12 95110 (GAA)n 0.853 5 0.050 FAMCAIV b (ATT)n CAV 21 21 102198 (ATT)n 0.574 3CAVI 30 39 224388 (TAAA)n 0.933 2 0.225 HEXCAVII 5 8 200235 (CAAAT)n 0.670 1

a FAM, 6-carboxyfluorescein; HEX, hexachlorofluorescein.b , not determined.

3870 SAMPAIO ET AL. J. CLIN. MICROBIOL.

suspended in 15 l of formamide, and run on an ABI PRISM 310 DNA auto-matic sequencer (Applied Biosystems). The results were analyzed by using Se-quencing 3.7 analysis software. Determination of the fragment sizes of the PCRproducts and the allele sizes were done automatically with GeneScan 3.7 analysissoftware. The alleles have been designated by the number of repeated unitsdetermined after sequencing.Reproducibility and stability. The reproducibility of the method and the

stability of the microsatellite markers were assessed according to the proceduresdescribed previously (42). The reproducibility of the method was also tested bycomparing the results obtained by the singleplex analysis with the ones observedby the multiplex analysis.

RESULTS

Screening and selection of repeat regions in Candida albi-cans sequence database. The search of the genomic DNAdatabase of C. albicans performed in this study provided a totalof 1,086 sequences, 368 obtained with the CAA query, 245obtained with the GAA query, 220 obtained with the ATTquery, 201 obtained with the TAAA query, and 52 obtainedwith the CAAAT query. Selection of the sequences was pri-marily based on the number of simple units repeated and theirlocation in the genome. There should be at least 10 repeats,given that these sequences have a higher probability of showinggreater genetic variability (11, 37), and they should be locatedoutside known coding regions, since a higher polymorphism isexpected in regions less prone to selective forces (32). Subse-quently, a third selection was made for the sequences to allowthe design of primers with an annealing temperature of about60C to ensure specificity and good reproducibility. A total offive sequences were then selected, and specific primers weredesigned for their amplification. Sequence 265080D05.s1.seqpresented two different regions containing microsatellite mo-tifs that were used separately to develop CAIII and CAIV(Table 1). The nomenclature chosen for the new markers wasCA, after C. albicans, followed by a roman numeral notationthat corresponded to the order of the analysis.Microsatellite locus analysis. The microsatellites selected

were used to type 72 unrelated C. albicans strains, isolatedfrom different patients, in order to test their specific amplifi-cation and polymorphism. This analysis revealed that theywere all polymorphic and presented from 5 to 30 alleles and 8to 46 different genotypes (Table 2). Analysis of the CAIV locusrevealed the presence of null alleles; therefore, this marker wasdiscarded.

The different alleles observed at all the loci were sequencedin order to determine the nature of the polymorphisms ob-served. The sequencing results confirmed that the consensussequences of all new microsatellite loci analyzed were in ac-cordance with the ones deposited in Stanfords DNA Sequenc-ing and Technology Center database, confirming the amplifi-cation of the correct loci. CAIII, CAIV, CAV, CAVI, andCAVII were simple STRs with one variable repetitive motif,and only CAI was a compound microsatellite with two differentvariable units (Table 2). For all microsatellites the differencesin the molecular weights of the distinct alleles reflected thedifferences in the number of the repeated motifs, which al-lowed the nomenclature for the alleles to be based upon thenumber of repeated units. This nomenclature prevents prob-lems if further variation is found in the constant stretches ofthe alleles when other C. albicans strain typing studies areperformed (12, 42).

The distribution of genotypes according to the Hardy-Wein-berg equilibrium was investigated for all the microsatellite locianalyzed in the 72 unrelated isolates. A significant departurefrom Hardy-Weinberg equilibrium expectations was found (P 0.001), which supported previous findings that the inheri-tance in infecting C. albicans populations appears to be clonal(15, 25, 50).

The discriminatory power was calculated for each markeraccording to the Simpson index of diversity:

DP 11

NN 1j1

s

njnj 1

where N is the number of strains, s is the total number ofdifferent genotypes, and nj is the number of strains of j geno-type (20). The results indicated that CAI is the microsatellitewith the highest DP value, 0.97, while CAV presented thelowest DP value, 0.57 (Table 2).Stability and specificity. The in vitro stability of the micro-

satellite markers was assessed by growing four independentstrains over approximately 300 generations. For all strainstested, the genotypes were always the same after the 300 gen-erations, suggesting an expected mutation rate of less than 3.33 103 for all microsatellite loci. These markers were alsospecies specific, since no amplification products were obtainedwhen the primers and PCR conditions described above wereused for the amplification of other pathogenic Candida species,namely, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C.guilliermondii, C. lusitaniae, and C. dubliniensis. The specificityregarding C. dubliniensis, which is very closely related to C.albicans and which was just recently recognized as a differentspecies, is noteworthy (47). Similar results were found in pre-vious studies with microsatellites by testing other Candida spe-cies (5, 10, 31, 42).Optimization of multiplex amplification conditions. To in-

crease the discriminatory capacity, simultaneous analysis of thedifferent loci was carried out in a multiplex PCR. The CAI,CAIII, and CAVI loci were the ones selected for the multiplexPCR, since they presented the highest DP values in the single-plex assay and a DP value of 0.998 when analyzed together,while they are located on different chromosomes (Table 2).The differences in the sizes of the alleles amplified at each ofthese loci and the possibility of combining different fluorescentdyes made possible their simultaneous amplification (Fig. 1).For this optimization step, 10 C. albicans strains were used,and the results observed in the multiplex PCRs were alwayscompared with the ones observed in the singleplex assay. Theprimer concentrations used for the multiplex reaction had tobe adjusted, and the best results of multiplex coamplificationwith the three primer pairs were obtained by using the con-centrations described in Table 2. Different DNA template con-centrations were also tested, from 1 ng to 50 ng, and theoptimal DNA concentration needed to coamplify these lociwas 40 ng of template DNA in a final PCR volume of 25 l. Ifa smaller amount of DNA was used, the preferential amplifi-cation of the CAI locus was observed. Comparison of thealleles obtained in the optimized multiplex reaction with theones observed in the corresponding singleplex showed identi-

VOL. 43, 2005 MICROSATELLITES REVEAL MICROEVOLUTION IN C. ALBICANS 3871

cal molecular weights, confirming the accuracy and reproduc-ibility of the technique.Use of the multiplex assay for strain differentiation. The

CAI microsatellite was the one presenting the highest discrim-inatory power. However, several independent or unrelated iso-lates presented the same allele combination at this locus.These isolates were analyzed with the multiplex system in or-der to determine if they could indeed be considered the samestrain. The genotypes observed with the multiplex assay indi-cated that, in several cases, the isolates with the same alleliccombination at CAI corresponded to different strains or tostrains undergoing a microevolutionary process (Table 3). Theisolates were defined as identical when the same genotypeswere observed at all loci, including the ones not presented inTable 3 (CAV and CAVII), and this was observed in 10 cases.On the contrary, 14 isolates were considered distinct strainssince they presented different genotypes at several loci. Theremaining three cases could be considered possible microevo-lutionary events, since minor changes in only one locus wereobserved. These results clearly showed the need for multilocusanalysis, even when a highly discriminatory molecular markeris used.

When this multiplex assay was applied to the differentiationof multiple isolates from the same patient, it could be observedthat all isolates showed exactly the same genotypes, confirmingour beliefs that only one strain was present in the infectingpopulation (Table 4). To verify that the infecting populationwas the same at different body locations, isolates were col-lected from the same patient with infections at multiple bodysites. Isolates were collected from patients F, H, I, and J. Theresults showed that the isolates from the upper respiratorytract of patient I were identical strains but were different fromthe urine isolate. The same occurred for patient J, for whomdistinct genotypes were observed for the two isolates, one fromthe vagina and the other from urine. However, isolates frompatients F and H showed the same genotypes regardless oftheir body location. These results show that patients couldhave different clones at different body sites but that the infect-ing population at each body site is monoclonal, with someisolates presenting the capacity of invading different micro-habitats.

Cases of recurrent vaginitis in eight patients were also ana-lyzed with this multiplex system. The results showed that the

infecting C. albicans strains isolated sequentially in differentrelapses of the illness displayed the same genotype for all loci,except in three cases, patients L, N, and P (Table 5). In patientN, a case of strain replacement occurred, since one of the threestrains isolated presented different genotypes at all loci ana-lyzed. In patient P, a microevolutionary event seems to haveoccurred, since the strains presented only a minor change atthe CAVI locus, from genotype 18-21 to genotype 21-21. Anal-ysis of the isolates from patient L showed a change at the CAIlocus, from allele 30 to allele 32. In a previous study we sug-gested that these changes from allele 30 to allele 32 could havebeen due to expansion of the microsatellite (42). However, theanalysis of this case with the remaining microsatellite loci sug-gests the occurrence of genetic changes that could lead to theloss of heterozygosity (LOH). Perepnikhatka et al. (34) studiedthe incubation of C. albicans isolates on fluconazole-containingmedium and observed the loss of one homologue of chromo-some 4 after 7 days of incubation. Interestingly, the CAI mi-crosatellite is located on the same chromosome, and thesepatients were treated with fluconazole; therefore, this hypoth-esis could not be ruled out. The possible loss of chromosome4, or part of it, which includes the CAI locus, would turngenotype 30-32 into genotype 30-30 in only one mutationalstep.

DISCUSSION

Candida albicans is an opportunistic yeast that can causesevere invasive infections, especially in immunocompromisedpatients, thus making the development of methodologies forthe accurate discrimination of strains essential. Knowledge ofthe relatedness of strains involved in infections would be ofextreme relevance to the development and application of thecorrect therapeutic strategy as well as to obtaining a betterunderstanding of the epidemiology of this yeast. Moreover,some strains are capable of invading different body locations,and microevolution may occur as an adaptive response to newenvironments.

Microsatellites are among the most frequent markers usedfor differentiation purposes due to their hypervariability, theease of PCR amplification and interpretation, their codomi-nant profiles, and their potential for use in automated assays.These loci are relatively abundant in C. albicans, and several

FIG. 1. GenScan profiles depicting the results of automatic fragment sizing for the microsatellite multiplex analysis for (A) one strain frompatient E (Table 4) and (B) strain 19C (Table 3). Each marker is represented by a different shade: black, CAI; dark gray, CAVI; light gray, CAIII.


markers have been developed (5, 12, 26). However, the major-ity of microsatellites appear to present low levels of heterozy-gosity, which may be explained by the fact that they are situ-ated in coding regions (12). In these situations, the

discriminatory power is rather low, but it can be compensatedfor by surveying a larger number of loci, facilitated by multi-plexing the PCR. The best approach was obtained by combin-ing three loci, CDC3, EF3, and HIS3, thus yielding a discrim-

TABLE 3. Genotypes observed with microsatellites CAI, CAIII, and CAVI in a selection of strains that showed the same allelecombination with CAI

IsolateGenotype observeda

Institutionb Body locationCAI CAIII CAVI

IGC3436 1723 66 141437M 1723 88 714 HSM Urine3 1723 68 713 HSJ Vaginal exudate

61M 1721 68 1515 HSM Upper respiratory tract24C 1721 68 921 CSC Vaginal exudate1C 1721 68 717 CSC Vaginal exudate

39M 1818 66 77 HSM Upper respiratory tract77M 1818 66 77 HSM Catheter5C 1818 66 77 CSC Vaginal exudate

36C 1118 68 642 CSC Vaginal exsudate45C 1118 68 642 CSC Vaginal exudate

WO1 1627 66 7727C 1627 611 921 CSC Vaginal exudate

26M 2028 69 3545 HSM Upper respiratory tract39C 2028 69 3944 CSC Vaginal exudate88M 2028 69 4244 HSM Urine74M 2028 69 2147 HSM Urine

31 2126 89 710 HSJ Vaginal exudate6C 2126 89 710 CSC Vaginal exudate49C 2126 67 915 CSC Vaginal exudate16C 2126 67 916 CSC Vaginal exudate

69M 2125 67 915 HSM Upper respiratory tract8M 2125 67 916 HSM Urine1M 2125 67 916 HSM Urine31C 2125 67 917 CSC Vaginal exudate11C 2125 77 1116 CSC Vaginal exudate

17 2121 67 815 HSJ Vaginal exudate52M 2121 67 77 HSM Urine

39 2122 67 77 HSJ Vaginal exudateSER5 2122 67 77 HSJ41M 2122 77 77 HSM Urine

H58 2222 67 77 HSJ64M 2222 67 77 HSM Upper respiratory tract20 2626 66 77 HSJ Vaginal exudate4M 2626 66 77 HSM Peritoneal exudate

H37 2527 68 1622 HSJ Upper respiratory tract63M 2527 66 917 HSM Urine

22 2327 68 2121 HSJ Vaginal exudate41 2327 68 1820 HSJ Vaginal exudate

35 2525 77 915 HSJ Vaginal exudate45 2525 77 915 HSJ Vaginal exudate

19C 2727 611 921 CSC Vaginal exudate46C 2727 68 2224 CSC Vaginal exudate

a Cases of microevolution are marked in boldface.b HSM, Hospital S. Marcos; HSJ, Hospital S. Joao; CSC, Centro de Saude do Caranda.


inatory power of 0.97 (3). In the present work, a multiplexstrategy was developed with three new microsatellite markers,located in noncoding regions, which presented the highest DPobserved for C. albicans differentiation, 0.99.

The analysis of 72 independent isolates with CAI identified16 different genotypes shared by two or more isolates. Appli-cation of this multiplex assay to assessment of the geneticrelatedness of those isolates allowed the discrimination of 14 of27 (51.8%) strains that otherwise would have been consideredidentical. It also allowed the confirmation of the identities of10 isolates, which were, in fact, the same strain, and three casesof microevolution. Microevolution resulted from minorchanges of the strain genotypes, i.e., a change at only one locusthat could be explained by a single mutational step. Overall,

the improved discriminatory potential of the methodology de-scribed here revealed the presence of three basic scenarios: (i)isolates that were the same strain, (ii) isolates that were thesame strain(s) but that were apparently undergoing a processof microevolution, and (iii) isolates that corresponded to dis-tinct strains. These results clearly indicate the need for mul-tilocus analysis even when isolates are studied with a highlydiscriminatory molecular marker. Application of this method-ology to the analysis of isolates from recurrent infections alsorevealed the same set of basic scenarios described previously(23, 24, 44). Of the 15 recurrent cases analyzed, 12 were due tothe same strain, 2 were due to the same strain which wasundergoing microevolution, and only 1 was due to strain re-placement. This multiplex system demonstrated that C. albi-cans strains can undergo microevolutionary events at body sitesof carriage during colonization between recurrent episodes ofinfection and that these events seem to be relatively morefrequent than strain replacement (6, 23, 24).

Analysis of the microevolutionary events observed in thisstudy suggested that two different mutational events could beoccurring: strand slippage of the DNA polymerase in the mi-crosatellite region and LOH as the result of chromosomalrearrangements; both of these mutational events take place atsimilar rates. The reported rates for chromosomal rearrange-ments were from 1.2 103 to 3.0 103 (8, 41), while formicrosatellite loci the values varied between 102 and 106

(14, 19, 53, 54). Chromosomal alterations have been widelydocumented in C. albicans, with some being associated withthe loss of entire or parts of chromosomes (27, 40, 41, 50, 52).Several conditions are known to induce LOH by chromosomalalterations, i.e., heat shock (17); exposure to different carbonsources, such as L-sorbose and D-arabinose (21, 41); and expo-sure to fluconazole (34). Metzgar et al. (31) also reportedchanges in microsatellites in vivo between the pretreatmentisolate and the isolate obtained after treatment with differentdoses of fluconazole. The association of highly polymorphicSTRs to different chromosomes and subchromosomal loca-tions could be of great use for the evaluation of the loss of theentire chromosome or parts of chromosomes in C. albicans.Although LOH was observed at CAI, additional experimentsare needed to confirm if it can be associated with rearrange-ments at chromosome 4 in response to fluconazole pressure.

Several studies have supported the concept that C. albicanscontains a source of potentially beneficial genes that are acti-vated by changes in chromosome number and that this elabo-rate mechanism regulates the utilization of food supplies andpossibly other important functions in response to environmen-tal stress. As C. albicans clinical populations are usually clonal,we suggest that in the absence of genetic exchange throughsexual reproduction, microevolution could conceivably confera selective advantage under adverse environmental conditions.

It is clear that the microsatellite multiplex PCR-based sys-tem described here enables high-speed typing, which makes ituseful in large epidemiological studies. Furthermore, its capac-ity to detect microevolutionary events can make it useful forthe detection of strain microevolution in response to environ-mental stress conditions, providing support for therapeutic ad-justments, especially in patients with recurrent infections.

The standardization of microsatellite typing systems, includ-ing the primers and the separation techniques used and the

TABLE 4. Characteristics of multiple strains isolated from thesame patient and cultured simultaneously

Patienta No. ofisolates Body locationGenotype observed

CAI CAIII CAVI

A 3 Urine 2125 67 916B 4 Peritoneal exudate 2626 66 77C 2 Upper respiratory tract 1717 68 714D 2 Urine 2121 67 77E 5 Urine 2122 77 77F 1 Urine 1847 611 919F 1 Upper respiratory tract 1847 611 919G 2 Urine 3636 611 641H 1 Upper respiratory tract 2125 67 915H 2 Urine 2125 67 915I 2 Upper respiratory tract 2222 67 77I 1 Urine 2028 69 4244J 1 Vaginal exudate 2324 66 1627J 1 Urine 1723 88 714R 2 Vaginal exudate 2125 67 917

a Each patient is referred to as a separate letter (A to J and R).

TABLE 5. Genotypes of sequential isolates from vulvovaginalrecurrent infections

Patienta No. of recurrentstrains

Genotype observedb

CAI CAIII CAVI

K 4 1723 68 713

L 3 3032 66 19233032 66 19233030 66 1923

M 5 1825 66 77

N 3 2121 67 8152029 69 39462029 69 3946

O 2 2626 66 77

P 2 2327 68 18212327 68 2121

Q 2 2223 68 2424

S 2 2020 99 3137

a Each patient is referred to as a separate letter (K to S).b Cases of microevolution are marked in boldface.


allele nomenclature, is an issue that should be accomplished toallow interlaboratory comparisons. The creation of public da-tabases that would make microsatellite allele data availableworldwide, similar to those already in use for human micro-satellites (29, 38), is another essential topic that deserves at-tention.

REFERENCES

1. Barchiesi, F., L. F. Di Francesco, P. Compagnucci, D. Arzeni, O. Cirioni, andG. Scalise. 1997. Genotypic identification of sequential Candida albicansisolates from AIDS patients by polymerase chain reaction techniques. Eur.J. Clin. Microbiol. Infect. Dis. 16:601605.

2. Barchiesi, F., R. J. Hollis, M. Del Poeta, D. A. McGough, G. Scalise, M. G.Rinaldi, and M. A. Pfaller. 1995. Transmission of fluconazole-resistant Can-dida albicans between patients with AIDS and oropharyngeal candidiasisdocumented by pulsed-field gel electrophoresis. Clin. Infect. Dis. 21:561564.

3. Botterel, F., C. Cesterke, C. Costa, and S. Bretagne. 2001. Analysis ofmicrosatellite markers of Candida albicans used for rapid typing. J. Clin.Microbiol. 39:40764081.

4. Bougnoux, M. E., S. Morand, and C. dEnfert. 2002. Usefulness of multilocussequence typing for characterization of clinical isolates of Candida albicans.J. Clin. Microbiol. 40:12901297.

5. Bretagne, S., J. M. Costa, C. Besmond, R. Carsique, and R. Calderone. 1997.Microsatellite polymorphism in the promoter sequence of the elongationfactor 3 gene of Candida albicans as the basis for a typing system. J. Clin.Microbiol. 35:17771780.

6. Chong, P. P., Y. L. Lee, B. C. Tan, and K. P. Ng. 2003. Genetic relatednessof Candida strains isolated from women with vaginal candidiasis in Malaysia.J. Med. Microbiol. 52:657666.

7. Correia, A., P. Sampaio, J. Almeida, and C. Pais. 2004. Study of molecularepidemiology of candidiasis in Portugal by PCR fingerprinting of Candidaclinical isolates. J. Clin. Microbiol. 42:58995903.

8. Cowen, L. E., D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, andL. M. Kohn. 2000. Evolution of drug resistance in experimental populationsof Candida albicans. J. Bacteriol. 182:15151522.

9. Dalle, F., N. Franco, J. Lopez, O. Vagner, D. Caillot, P. Chavanet, B.Cuisenier, S. Aho, S. Lizard, and A. Bonnin. 2000. Comparative genotypingof Candida albicans bloodstream and nonbloodstream isolates at a polymor-phic microsatellite locus. J. Clin. Microbiol. 38:45544559.

10. Field, D., and C. Wills. 1996. Long polymorphic microsatellite in simpleorganisms. Proc. R. Soc. London B Biol. Sci. 263:209215.

11. Field, D., and C. Wills. 1998. Abundant microsatellite polymorphism inSaccharomyces cerevisiae, and the different distributions of microsatellites ineight prokaryotes and S. cerevisiae, result from strong mutation pressuresand a variety of selective forces. Proc. Natl. Acad. Sci. USA 95:16471652.

12. Field, D., L. Eggert, D. Metzgar, R. Rose, and C. Wills. 1996. Use ofpolymorphic short and clustered coding-region microsatellites to distinguishstrains of Candida albicans. FEMS Immunol. Med. Microbiol. 15:7379.

13. Fundyga, R. E., T. J. Lott, and J. Arnold. 2002. Population structure ofCandida albicans, a member of the human flora, as determined by micro-satellite loci. Infect. Genet. Evol. 2:5768.

14. Gardner, M. G., C. M. Bull, S. J. B. Cooper, and G. A. Duffield. 2000.Microsatellite mutations in litters of the Australian lizard, Egernia stokesii. J.Evol. Biol. 13:551560.

15. Graser, Y., M. Volovsek, J. Arrington, G. Schonian, W. Presber, T. G.Mitchell, and R. Vilgalys. 1996. Molecular markers reveal the populationstructure of the human pathogen Candida albicans exhibits both clonalityand recombination. Proc. Natl. Acad. Sci. USA 93:1247312477.

16. Hennequin, C., A. Thierry, G. F. Richard, G. Lecointre, H. V. Nguyen, C.Gaillardin, and B. Dujon. 2001. Microsatellite typing as a new tool foridentification of Saccharomyces cerevisiae strains. J. Clin. Microbiol. 39:551559.

17. Hilton, C., D. Markie, B. Corner, E. Rikkerink, and R. Poulter. 1985. Heatshock induces chromosome loss in the yeast Candida albicans. Mol. Gen.Genet. 200:162168.

18. Howell, K. S., E. J. Bartowsky, G. H. Fleet, and P. A. Henschke. 2004.Microsatellite PCR profiling of Saccharomyces cerevisiae strains during winefermentation. Lett. Appl. Microbiol. 38:315320.

19. Huang, Q. Y., F. H. Xu, H. Shen, H. Y. Deng, Y. J. Liu, Y. Z. Liu, J. L. Li,R. R. Recker, and H. W. Deng. 2002. Mutation patterns at dinucleotidemicrosatellite loci in humans. Am. J. Hum. Genet. 70:625634.

20. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discrimina-tory ability of typing systems: an application of Simpsons index of diversity.J. Clin. Microbiol. 26:24652466.

21. Janbon, G., F. Sherman, and E. Rustchenko. 1999. Appearance and prop-erties of L-sorbose-utilizing mutants of Candida albicans obtained on a se-lective plate. Genetics 153:653664.

22. Kaiser, C., S. Michaelis, and A. Mitchell. 1994. Methods in yeast genetics.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

23. Lockhart, S. R., B. D. Reed, C. L. Pierson, and D. R. Soll. 1996. Mostfrequent scenario for recurrent Candida vaginitis is strain maintenance withsubshuffling: demonstration by sequential DNA fingerprinting with probesCa3, C1, and CARE2. J. Clin. Microbiol. 34:767777.

24. Lockhart, S. R., J. J. Fritch, A. S. Meier, K. Schroppel, T. Srikantha, R.Galask, and D. R. Soll. 1995. Colonizing populations of Candida albicans areclonal in origin but undergo microevolution through C1 fragment reorgani-zation as demonstrated by DNA fingerprinting and C1 sequencing. J. Clin.Microbiol. 33:15011509.

25. Lott, T. J., R. E. Fundyga, M. E. Brandt, L. H. Harrison, A. N. Sofair, R. A.Hajjeh, and D. W. Warnock. 2003. Stability of allelic frequencies and distri-butions of Candida albicans microsatellite loci from U.S. population-basedsurveillance isolates. J. Clin. Microbiol. 41:13161321.

26. Lunel, F. V., L. Licciardello, S. Stefani, H. A. Verbrugh, W. J. Melchers, J. F.Meis, S. Scherer, and A. van Belkum. 1998. Lack of consistent short se-quence repeat polymorphisms in genetically homologous colonising andinvasive Candida albicans strains. J. Bacteriol. 180:37713778.

27. Magee, B. B., and P. T. Magee. 2000. Induction of mating in Candida albicansby construction of MTLa and MTLalpha strains. Science 289:310313.

28. Magee, B. B., T. M. Souza, and P. T. Magee. 1987. Strain and speciesidentification by restriction length polymorphism in the ribosomal DNArepeats of Candida species. J. Bacteriol. 169:16391643.

29. Martin, P. D., H. Schmitter, and P. M. Schneider. 2001. A brief history of theformation of DNA databases in forensic science within Europe. Forensic Sci.Int. 119:225231.

30. Matthews, R. C. 1994. Pathogenicity determinants of Candida albicans: po-tential targets for immunotherapy? Microbiology 140:15051511.

31. Metzgar, D., A. van Belkum, D. Field, R. Haubrich, and C. Wills. 1998.Random amplification of polymorphic DNA and microsatellite genotypingof pre- and posttreatment isolates of Candida spp. from human immunode-ficiency virus-infected patients on different fluconazole regimens. J. Clin.Microbiol. 36:23082313.

32. Metzgar, D., J. Bytof, and C. Wills. 2000. Selection against frameshift mu-tations limits expansion in coding DNA. Genome Res. 10:7280.

33. Meyer, W., K. Marszewska, M. Amirmostofian, R. P. Igreja, C. Hardtke, K.Methling, M. A. Viviani, A. Chindamporn, S. Sukroongreung, M. A. John,D. H. Ellis, and T. C. Sorrell. 1999. Molecular typing of global isolates ofCryptococcus neoformans var. neoformans by polymerase chain reaction fin-gerprinting and randomly amplified polymorphic DNAa pilot study tostandardize techniques on which to base a detailed epidemiological survey.Electrophoresis 20:17901799.

34. Perepnikhatka, V., F. J. Fischer, M. Niimi, R. A. Baker, R. D. Cannon, Y. K.Wang, F. Sherman, and E. Rustchenko. 1999. Specific chromosome alter-ations in fluconazole-resistant mutants of Candida albicans. J. Bacteriol.181:40414049.

35. Pfaller, M. A., S. R. Lockhart, C. Pujol, J. A. Swails-Wenger, S. A. Messer,M. B. Edmond, R. N. Jones, R. P. Wenzel, and D. R. Soll. 1998. Hospitalspecificity, region specificity and fluconazole resistance of Candida albicansbloodstream isolates. J. Clin. Microbiol. 36:15181529.

36. Pujol, C., S. Joly, S. R. Lockhart, S. Noel, M. Tibayrenc, and D. R. Soll. 1997.Parity among the randomly amplified polymorphic DNA method, multilocusenzyme electrophoresis, and Southern blot hybridization with the moder-ately repetitive DNA probe Ca3 for Candida albicans. J. Clin. Microbiol.35:23482358.

37. Pupko, T., and D. Graur. 1999. Evolution of microsatellites in the yeastSaccharomyces cerevisiae: role of length and number of repeated units. J.Mol. Evol. 48:313316.

38. Roewer, L., M. Krawczak, S. Willuweit, M. Nagy, C. Alves, A. Amorim, K.Anslinger, C. Augustin, A. Betz, E. Bosch, A. Caglia, A. Carracedo, D.Corach, T. Dobosz, B. M. Dupuy, S. Furedi, C. Gehrig, L. Gusmao, J. Henke,L. Henke, M. Hidding, C. Hohoff, B. Hoste, M. A. Jobling, H. J. Kargel, P.de Knijff, R. Lessig, E. Liebeherr, M. Lorente, B. Martnez-Jarreta, P.Nievas, M. Nowak, W. Parson, V. L. Pascali, G. Penacino, R. Ploski, B. Rolf,A. Sala, U. Schmidt, C. Schmitt, P. M. Schneider, R. Szibor, J. Teifel-Greding, and M. Kayser. 2001. Online reference database of EuropeanY-chromosomal short tandem repeat (STR) haplotypes. Forensic Sci. Int.118:106113.

39. Ruhnke, M., A. Eigler, I. Tennagen, B. Geiseler, E. Engelmann, and M.Trautmann. 1994. Emergence of fluconazole-resistant strains of Candidaalbicans in patients with recurrent oropharyngeal candidosis and humanimmunodeficiency virus infection. J. Clin. Microbiol. 32:20922098.

40. Rustchenko, E. P. 1991. Variations of Candida albicans electrophoretickaryotypes. J. Bacteriol. 173:65866596.

41. Rustchenko, E. P., D. H. Howard, and F. Sherman. 1994. Chromosomalalterations of Candida albicans are associated with the gain and loss ofassimilating functions. J. Bacteriol. 176:32313241.

42. Sampaio, P., L. Gusmao, C. Alves, C. Pina-Vaz, A. Amorim, and C. Pais.2003. Highly polymorphic microsatellite for identification of Candida albi-cans strains. J. Clin. Microbiol. 41:552557.

43. Schmid, J., S. Herd, P. R. Hunter, R. D. Cannon, M. S. Yasin, S. Samad, M.Carr, D. Parr, W. McKinney, M. Schousboe, B. Harris, R. Ikram, M. Harris,A. Restrepo, G. Hoyos, and K. P. Singh. 1999. Evidence for a general


purpose genotype in Candida albicans, highly prevalent in multiple geo-graphic regions, patient types and types of infection. Microbiology 145:24052413.

44. Schroppel, K., M. Rotman, R. Galask, K. Mac, and D. R. Soll. 1994. Evo-lution and replacement of Candida albicans strains during recurrent vaginitisdemonstrated by DNA fingerprinting. J. Clin. Microbiol. 32:26462654.

45. Shemer, R., Z. Weissman, N. Hashman, and D. Kornitzer. 2001. A highlypolymorphic degenerate microsatellite for molecular strain typing of Can-dida krusei. Microbiology 147:20212028.

46. Sternberg, S. 1994. The emerging fungal threat. Science 266:16321634.47. Sullivan, D. J., T. J. Westerneng, K. A. Haynes, D. E. Bennett, and D. C.

Coleman. 1995. Candida dubliniensis sp. nov.: phenotypic and molecularcharacterisation of a novel species associated with oral candidiasis in HIV-infected individuals. Microbiology 141:15071521.

48. Tautz, D. 1998. Evolutionary biology. Debatable homologies. Nature 395:1719.

49. Tavanti, A., N. A. Gow, S. Senesi, M. C. Maiden, and F. C. Odds. 2003.Optimization and validation of multilocus sequence typing for Candida al-bicans. J. Clin. Microbiol. 41:37653776.

50. Tavanti, A., N. A. Gow, M. C. Maiden, F. C. Odds, and D. J.Shaw. 2004.

Genetic evidence for recombination in Candida albicans based on haplotypeanalysis. Fungal Genet. Biol. 41:553562.

51. Thanos, M., G. Schonian, W. Meyer, C. Schweynoch, Y. Graser, T. G.Mitchell, W. Presber, and H. J. Tietz. 1996. Rapid identification of Candidaspecies by DNA fingerprinting with PCR. J. Clin. Microbiol. 34:615621.

52. Tsang, P. W., B. Cao, P. Y. Siu, and J. Wang. 1999. Loss of heterozygosity,by mitotic gene conversion and crossing over, causes strain-specific adeninemutants in constitutive diploid Candida albicans. Microbiology 145:16231629.

53. Vazquez, J. F., T. Perez, J. Albornoz, and A. Domnguez. 2000. Estimation ofmicrosatellite mutation rates in Drosophila melanogaster. Genet. Res. 76:323326.

54. Weber, J. L., and C. Wong. 1993. Mutation of human short tandem repeats.Hum. Mol. Genet. 2:11231128.

55. White, T. C. 1997. Increased mRNA levels of ERG16, CDR, and MDR1correlate with the increases in azole resistance in Candida albicans isolatesfrom a patient infected with human immunodeficiency virus. Antimicrob.Agents Chemother. 41:14821487.

56. Wickes, B. L., and R. Petter. 1996. Genomic variation in C. albicans. Curr.Top. Med. Mycol. 7:7186.


0296-05.pdf

Documents