Department of Molecular Microbiology, B. Rappaport Faculty of Medicine1, and Department of Clinical Microbiology2, Rambam Medical Center, Haifa 31096, Israel
Author for correspondence: Daniel Kornitzer. Tel: +972 4 829 5258. Fax: +972 4 829 5254. e-mail: danielk{at}tx.technion.ac.il
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ABSTRACT |
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Keywords: molecular typing, fungal VNTR, ploidy level, sexual versus clonal reproduction
Abbreviations: AP-PCR, arbitrarily primed PCR; VNTR, variable numbers of tandem repeats
The GenBank accession numbers for the sequences determined in this work are AF326279AF326292.
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INTRODUCTION |
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The most widely used and versatile approach for the molecular epidemiology of microbial infections is DNA fingerprinting, which encompasses a variety of methods that detect variations at the DNA sequence level (Soll, 2000 ). The amount of variation between isolates gives a measure of their relatedness. Importantly, implied in this type of analysis is that the organism reproduces clonally, or at least that sexual reproduction, and its associated recombination of markers, is infrequent within the studied population. The choice of fingerprinting method will depend on the type of polymorphism that needs to be detected, which in turn depends on the purpose of the strain comparison. For the determination of the origin of a nosocomial infection, the main question is whether a set of isolates are epidemiologically related or not. For this purpose, the markers to be used should be stable within the time frame of transmission from one patient to another, and sufficiently polymorphic to allow a reasonable amount of discriminatory power (Hunter, 1991
). An additional important consideration in the clinical setting is the ease and reproducibility of the method.
One method of choice to detect genetic polymorphisms is arbitrarily primed PCR (AP-PCR), where short oligonucleotides are used to amplify random genomic fragments (Welsh & McClelland, 1990 ). This method is technically simple, and if enough primers are used alone or in combinations, the large number of bands obtained ensures that some of the genomic polymorphisms between unrelated strains will be detected with a reasonable probability. However, AP-PCR suffers from a high sensitivity to reaction conditions (Caetano-Anolles, 1993
; Meunier & Grimont, 1993
), resulting in high centre-to-centre, and even experiment-to-experiment, variability (Becker et al., 2000
; Taylor et al., 1999
; Tyler et al., 1997
). An alternative approach is to use locus-specific primers for known highly polymorphic loci. Such loci generally consist of variable numbers of simple tandem repeats, or VNTRs (Taylor et al., 1999
; van Belkum, 1999
). Variability of VNTRs is probably due to DNA polymerase slippage during replication (Strand et al., 1993
). VNTRs often consist of microsatellites, or short (16 nucleotide) sequence repeats, but can also consist of repeats of longer sequences, or minisatellites. One such variable minisatellite is the C. krusei repeated sequence 1 (CKRS-1), which consists of a 165 bp repeat within the rDNA locus (Carlotti et al., 1997
). One drawback of VNTRs is that because of their simple structure, alleles can be identical by mutation rather than by descent, which reduces their value for phylogenetic studies (Metzgar et al., 2001
; Orti et al., 1997
). Furthermore, VNTRs of Candida albicans have been observed to vary over time even within a single patient, suggesting that the speed with which VNTRs evolve may be too high even for the relatively short time frame of epidemiological research (Metzgar et al., 1998
). To increase the confidence of VNTR analysis and to prevent misclassification of related strains as unrelated, multiple loci need to be assayed.
Potential VNTRs can be identified by computerized searches for simple sequence repeats. Beyond a certain repeat length, such regions have a high probability of being polymorphic (Field & Wills, 1998 ). This approach, which has been successfully used, for example, with C. albicans (Field et al., 1996
; Lunel et al., 1998
), requires knowledge of a substantial part of the genome sequence of the organism. However, for pathogens for which little sequence information exists, other methods are required to isolate highly variable regions. Here, we describe the identification by AP-PCR of a highly polymorphic locus in C. krusei. Surprisingly, sequence analysis indicated that the polymorphism occurred within a degenerate trinucleotide repeat. Haplotype analysis suggested that, unlike simple sequence repeats, the degenerate repeats in this locus are relatively stable. Since most haplotypes differ by length, this polymorphism allows the easy and rapid characterization of isolates with high discriminatory power. Furthermore, analysis of the haplotype distribution provides insight into the mode of reproduction of this yeast.
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METHODS |
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AP-PCR.
Reactions were performed with nine random 10-mers, including UBC143 and UBC734 (Zeng et al., 1996 ) and seven others (sequences available upon request) at annealing temperatures of 4045 °C, each yielding on average 10 bands of median size 0·51 kb, i.e. 510 kb were covered per experiment. Coverage may have been lower, as short-length polymorphisms may not have been detectable in the larger fragments. The primer that detected the CKTNR polymorphism was RAPD4 (5'-AAGAGCCCGT-3').
Amplification of CKTNR sequence.
The following primers were designed for specific amplification of the CKTNR polymorphic sequence: CKTNR5 (5'-ACAGCAGTCGCAGGCCC-3') and CKTNR3 (5'-GTCGGAGACATAACCGC-3'). All primers were produced by Sigma-Genosys, Cambridgeshire, UK. For each 20 µl reaction, 10 ng genomic DNA was used; alternatively, a small amount of material from a pure fungal colony was directly resuspended in the PCR reaction mix. Amplification conditions were: 94 °C for 4 min, then 30 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s. Reaction products were separated by electrophoresis for 60 min at 200 V on 6 cm polyacrylamide gels (40:1, 10%) supplemented with Spreadex polymer (Elchrom Scientific). For sequencing of individual alleles, the bands were cut out of the gel, eluted and reamplified, and the PCR products were subjected to direct sequencing using an ABI PRISM 310 automated sequencer. For the GENESCAN procedure, a TET-modified CKTNR3 primer was used, and the reaction products were run on an ABI PRISM 310 machine together with the MapMarker 400 TMRA-marked size standards (BioVentures).
Statistical genetics.
For the calculation of deviations from the HardyWeinberg equilibrium, Guo and Thompsons Markov chain approach was used, because of its appropriateness for large numbers of alleles and small sample sizes (Guo & Thompson, 1992 ). It was implemented using the GENEPOP software package (Raymond & Rousset, 1995
). Where alleles characterized by sequencing were considered together with alleles characterized only by length, the ambiguous alleles were grouped together. This is a conservative assumption with regard to rejection of the null hypothesis of panmixia. When the whole sample was considered, the null hypothesis could be rejected with P<0·0001. When only the sequenced subset of isolates was considered, the same result was obtained, with P=0·0001. Finally, due to the fact that our sample contained isolates from different geographical locations, which may distort the result due to a populations admixture effect, we performed the test with only the Haifa subset of the sample; here, the null hypothesis was still rejected with P=0·0193.
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RESULTS |
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To obtain these data, twelve PCR reactions were performed on four clinical isolates and one reference laboratory stock strain with nine different AP-PCR primers, alone or in combinations (see Methods). A single primer identified a variable band of about 500 bp (Fig. 1). This fragment was cloned from three strains. Sequencing of one clone from each strain revealed a length polymorphism within a degenerate trinucleotide repeat. We designated this locus CKTNR. The sequence information was then used to design locus-specific primers CKTNR5 and CKTNR3. The CKTNR locus was isolated and subjected to sequence analysis in additional clinical isolates. Most of the clinical isolates yielded two different alleles (Fig. 2
and Tables 1
and 3
). This observation supports the suggestion, based on segregation of a uracil auxotrophy following UV irradiation, that C. krusei is diploid (Whelan & Kwon-Chung, 1988
). The CKTNR primers did not react with C. albicans DNA (Fig. 2
). One or two slower-migrating bands were detectable in most lanes (indicated with an asterisk in Fig. 2
), the exception being the homozygous reference strain. Upon extraction from the gel and reamplification, each of these bands yielded the original gel pattern again, whereas reamplification of the faster-migrating bands yielded the single amplified band only (not shown). We concluded that these slower-migrating species represent heteroduplexes of the two alleles.
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The mode of reproduction of a micro-organism clonal versus sexual greatly influences its epidemiological tractability by molecular fingerprinting. When a single locus is considered, as is the case here, the predictions for a primarily clonal mode of reproduction are fixed heterozygosity and absence of segregation genotypes, or, more generally, deviations from the HardyWeinberg equilibrium (Tibayrenc et al., 1991 ). Given the nature of our sample, deviations from the HardyWeinberg equilibrium could be most rigorously tested. In the case of panmixia (frequent exchange of alleles by random mating between all members of the population), the alleles are predicted to be at equilibrium. We found that the null hypothesis of panmixia could be rejected with a very high probability (see Methods), suggesting that C. krusei/I. orientalis reproduction is primarily clonal.
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DISCUSSION |
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An epidemiological typing system should ideally be (1) easy to apply, (2) stable enough over time to prevent misclassification of related strains as unrelated, and (3) polymorphic enough to prevent misclassification of unrelated strains as related (Hunter, 1991 ; Soll, 2000
). The CKTNR locus conforms to these three requirements: (1) locus-specific primers allow rapid and unequivocal genotyping of any given strain, without the need to test the whole set of strains together; (2) the CKTNR haplotypes appear to be extremely stable over time, which should prevent misclassification of related strains as being unrelated; and (3) the considerable length polymorphism of the CKTNR locus and the occurrence of two alleles results in a high discriminatory power, i.e. a low probability that two epidemiologically unrelated strains will exhibit the same profile. For these reasons, the CKTNR locus could be used to test a suspicion that a cluster of isolates are related. If the CKTNR profiles support the suspicion of relatedness, additional assays such as AP-PCR or CKRS-1 profile determination could be used for confirmation.
We found that both original C. krusei strains and I. orientalis strains, including the I. orientalis type strain CBS 5147, react with the CKTNR PCR primers and carry many alleles identical to those found in our clinical C. krusei isolates (Table 3). Thus, our data support the reclassification of both sets of strains as a single species (Barnett et al., 2000
). Some strains of I. orientalis have been shown to be able to sporulate, mate and exchange markers (Kurtzman & Smiley, 1976
; Kurtzman et al., 1980
). The ability to epidemiologically track a micro-organism by molecular fingerprinting presupposes a primarily clonal, rather than sexual, mode of reproduction. The question of whether C. krusei reproduces sexually has additional implications, most notably with regard to resistance to antifungals. Even if an organism exhibits the ability to reproduce sexually in the laboratory, whether it does so in nature can only be addressed by a population genetics approach (Tibayrenc et al., 1991
). The diplotype distribution is not consistent with panmixia, suggesting that the reproductive mode of C. krusei is primarily clonal. However, a low level of sexual reproduction cannot be ruled out. It actually may explain the observation that identical alleles are found in association with various different partners.
Concluding remarks
The large-scale AP-PCR methodology we used to identify the CKTNR locus may be applied to isolate additional polymorphic microsatellites, whether the sequence of the organism in question is known or not. Computer-based searches aimed at the detection of simple microsatellites depend on extensive sequence information and may often miss degenerate microsatellites (depending on the allele that is present in the sequenced isolate: note that many of the CKTNR alleles have no more than four contiguous CAA repeats). The combination of length polymorphism, stability and ease of detection potentially provided by degenerate microsatellite loci such as CKTNR makes them useful molecular epidemiology tools.
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ACKNOWLEDGEMENTS |
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Received 27 March 2001;
accepted 8 April 2001.