Fingerprinting reveals gamma-ray induced mutations in fungal DNA: implications for identification of patent strains of Trichoderma harzianum

We have analyzed different patent strains and gamma-ray induced mutants of Trichoderma harzianum by DNA fingerprinting and PCR fingerprinting (RAPD). Applying wild-type phage M13 DNA, with the oligonucleotides (CT)₈ and (GTG)₅ as probes for hybridization, as well as the oligonucleotides GGCATCGGCC, (GTG)₅, (CAC)₅ and the M13 sequence GAGGGTGGCGGTTCT as primers in PCR, we were able to obtain different and discriminative fingerprint patterns for all strains and mutants investigated. Irradiation of fungi led to mutations which resulted in new fingerprint patterns. Consequently, irradiation-induced mutants can be clearly distinguished from the original wild-type isolates by genomic fingerprinting which is of importance for the patent protection of fungal strains. Sequencing of the ITS-1 and ITS-2 regions of the rDNA gene complex revealed the same sequence for all mutant strains and the original wild-type strain.     

Differentiation of species and strains among filamentous fungi by DNA fingerprinting

Summary We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)₈, (GTG)₅ and (GACA)₄, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)₄ produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.     

PCR fingerprinting: a convenient molecular tool to distinguish between Candida dubliniensis and Candida albicans.

Candida dubliniensis was recently identified as a germ-tube- and chlamydospore-positive yeast, phenotypically and morphologically indistinguishable from the phylogenetically closely related yeast species C. albicans and its synonymized variant C. stellatoidea. The high similarity between these yeast species causes significant problems in the correct identification of C. dubliniensis in a standard clinical mycology laboratory. Polymerase chain reaction (PCR) fingerprinting was successfully applied here to distinguish between clinical isolates of the two closely related species. Microsatellite ([GACA]4) and minisatellite ([5'-GAGGGTGGCGGTTCT-3'], derived from the core-sequence of the wild-type phage M13) specific oligonucleotides were used as single primers in PCR to amplify hypervariable inter-repeat DNA sequences from 16 C. dubliniensis strains and 11 C. albicans strains. Each species, represented by its ex-type strain, could be identified by a distinct species-specific multilocus pattern, allowing identification to species level for all clinical isolates. In addition, the PCR fingerprinting generated strain-specific profiles, making this method applicable to epidemiological investigations. PCR fingerprinting using the primer M13 is proposed here as a simple, reliable and highly reproducible molecular tool to differentiate between strains of C. albicans and C. dubliniensis.     

Incidence of SUC-RTM telomeric repeated genes in brewing and wild wine strains of Saccharomyces

When over-expressed, RTM yeast genes confer resistance to the toxicity of molasses. They are found in distiller's and baker's industrial yeasts in multiple copies, scattered on the telomeres and physically linked to the telomeric SUC genes. Because these genes are absent from some laboratory strains, we explored the genomes of other industrial yeasts (brewing strains) and wine wild strains. A collection of 47 wine yeast strains (S. cerevisiae and S. bayanus) and 15 brewing strains, lager, ale and possible ancestors (S. monacensis, S. paradoxus and S. carlsbergensis) were screened for the presence of RTM genes. Only three wine strains and all brewing strains proved to contain RTM sequences in different copy numbers. PCR and chromosome blotting confirm the presence of SUC sequences in tandem with RTM. Moreover, analysis of the entire S. cerevisiae genome sequence shows that three other, non-telomeric, genes related to RTM are scattered on different chromosomes. Received: 4 December 1996     

Species and genotypic diversities and similarities of pathogenic yeasts colonizing women

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 19 women with AIDS, (ii) 11 pregnant women without human immunodeficiency virus (HIV) infection, and (iii) 19 women who were neither pregnant nor infected with HIV. Seven yeast species were recovered, including 59 isolates of Candida albicans, 9 isolates of Candida parapsilosis, 5 isolates of Candida krusei, 3 isolates of Candida glabrata, 2 isolates of Saccharomyces cerevisiae, and 1 isolate each of Candida tropicalis and Candida lusitaniae. Seventy unique genotypes were identified by PCR fingerprinting with the M13 core sequence and by random amplified polymorphic DNA analysis. Of the nine shared genotypes, isolates from three different hosts were of one genotype and pairs of strains from different body sites of the same host shared each of the other eight genotypes. Genetic similarities among groups of strains were calculated and compared. We found no significant difference in the patterns of relatedness of strains from the three body sites (oral cavity, vagina, and rectum), regardless of host conditions. The yeast microflora of all three host groups had similar species and genotypic diversities. Furthermore, a single host can be colonized with multiple species or multiple genotypes of the same species at the same or different body sites, indicating dynamic processes of yeast colonization on women.     

Genomic complexity and chromosomal rearrangements in wine-laboratory yeast hybrids

In this report we describe the genomic complexity of a number of Saccharomyces yeast strains isolated from sherry wine (flor yeasts), and the genomic stability of a yeast hybrid derived from one of these and a laboratory strain. Flor yeast strains largely differed in their DNA content, but showed very few variations their molecular karyotype. These strains contained a large number of Ty2 sequences, but lacking the Ty1 elements commonly found in laboratory strains. The genetic analysis of a flor-laboratory hybrid indicated that flor yeasts were aneuploid. Hybridization patterns obtained with Ty1 and Ty2 probes in the meiotic progeny of this hybrid suggested that recombination may occur not only among homologous chromosomes of similar length, but also among polymorphic partners with different sizes. New chromosomal variants were frequently observed in the meiotic products, suggesting that polymorphism in chromosome length may itself be a major source of karyotypic variation. The genetic analysis of such variants indicated that recombinational events leading to new chromosomal forms may occur both mitotically and meiotically. Received: 2 April / 26 May 1996     

Differentiation of species and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD)

Polymerase chain reaction (PCR)-based technology, involving random amplification of polymorphic DNA (RAPD), was used to assess the genomic variability between 24 isolates of deuteromycetous fungi (Metarhizium anisopliae, Metarhizium flavoviride, unidentified strains of Metarhizium and Beauveria bassiana) which were found to infect grasshoppers or locusts. M. flavoviride showed little intraspecific variability in PCR-amplified fragments when compared to M. anisopliae. The high level of variability in PCR-amplified fragments contained within M. anisopliae was similar to the total variability between B. bassiana, M. anisopliae and M. flavoviride, and suggests that M. anisopliae may include a number of cryptic species. Four polymorphic RAPD fragments were used to probe the genomic DNA of the various species and strains. On the basis of these probes the fungi can be grouped into M. flavoviride, M. anisopliae, or B. bassiana. According to PCR-amplified fragments, previously-unidentified Metarhizium strains were characterized as M. flavoviride. There was little evidence that these fungi, all isolated from, or virulent towards, grasshoppers or locusts, showed host-selection in PCR-amplified fragments. Nor was geographical origin a criterion for commonalty based on PCR-amplified fragments. PCR-fragment-pattern polymorphisms and the construction of probes from one or more of these fragments may provide a useful and rapid tool for identifying species and strains of entomopathogenic fungi.     

An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera

Summary Sequences hybridzing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana. Sequence order for the 34.5 kbp mtDNA of E. nana is identical to that for mtDNAs from B. custersianus (28.5 kbp) and B. naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species. A closer relationship is suggested for D. intermedia and D. bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites. Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S. cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species. An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence. Likewise mtDNA from B. anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence. By contrast the largest mtDNA (101.1 kbp) from B. custersd has the cytochrome b and LrRNA sequences in the same orientation. In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites. This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues.     

Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.     

Specific primers for rapid detection of Microsporum audouinii by PCR in clinical samples

This report describes application of PCR fingerprinting to identify common species of dermatophytes using the microsatellite primers M13, (GACA)4, and (GTG)5. The initial PCR analysis rendered a specific DNA fragment for Microsporum audouinii, which was cloned and sequenced. Based on the sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers were designed and verified by PCR to establish their reliability in the diagnosis of M. audouinii. These primers produced a singular PCR band of 431 bp specific only to strains and isolates of M. audouinii, based on a global test of 182 strains/isolates belonging to 11 species of dermatophytes. These findings indicate these primers are reliable for diagnostic purposes, and we recommend their use in laboratory analysis.     

Use of polymerase chain reaction and specific monoclonal antibodies as rapid method to recognize Borrelia burgdorferi sensu stricto,B. garinii and B. afzelii among Italian isolates of B. burgdorferi

We previously classified locally isolated strains of Borrelia burgdorferi by a restriction fragment length polymorphism analysis of total DNA, by DNA/DNA Southern Blot hybridization and by a hybridization with rRNA 16 + 23 S from Escherichia coli [Cinco et al. (1993) Microbiologica 16:323–332] into three genetic groups which, according to the reference strains used, should correspond to the three species so far described as B. burgdorferi sensu stricto, B. garinii and B. afzelii. To find a simpler method for strain identification, in this study we analyzed the Italian strains and some strains identification, in this study we analyzed the Italian strains and some strains originating from other European countries, employing the species-specific 16S rRNA primers in the polymerase chain reaction technique (PCR) and some phenotypic markers like the B. afzelii-specific monoclonal antibodies and the battery of OspA-specific monoclonal antibodies which were reported to give a reactivity pattern correlated to the species [Wilske et al. (1993) J Clin Microbiol 31:340–350]. The PCR results confirmed those obtained previously by identifying the three groups as B. burgdorferi sensu stricto, B. garinii and B. afzelii; the reactivity patterns obtained with the monoclonal antibodies (mAb) also corresponded to those described as typical of the three species. We standardized the PCR technique to amplify a sample of crude template DNA obtained from a culture of 10⁵ spirochetes.     

Reconstruction of the genome origins and evolution of the hybrid lager yeast Saccharomyces pastorianus

Inter-specific hybridization leading to abrupt speciation is a well-known, common mechanism in angiosperm evolution; only recently, however, have similar hybridization and speciation mechanisms been documented to occur frequently among the closely related group of sensu stricto Saccharomyces yeasts. The economically important lager beer yeast Saccharomyces pastorianus is such a hybrid, formed by the union of Saccharomyces cerevisiae and Saccharomyces bayanus-related yeasts; efforts to understand its complex genome, searching for both biological and brewing-related insights, have been underway since its hybrid nature was first discovered. It had been generally thought that a single hybridization event resulted in a unique S. pastorianus species, but it has been recently postulated that there have been two or more hybridization events. Here, we show that there may have been two independent origins of S. pastorianus strains, and that each independent group—defined by characteristic genome rearrangements, copy number variations, ploidy differences, and DNA sequence polymorphisms—is correlated with specific breweries and/or geographic locations. Finally, by reconstructing common ancestral genomes via array-CGH data analysis and by comparing representative DNA sequences of the S. pastorianus strains with those of many different S. cerevisiae isolates, we have determined that the most likely S. cerevisiae ancestral parent for each of the independent S. pastorianus groups was an ale yeast, with different, but closely related ale strains contributing to each group’s parentage.     

Molecular characterization and comparison of Clostridium botulinum type C avian strains

Type C botulinum neurotoxin (BoNT/C)-producing Clostridium botulinum causes animal botulism worldwide and has become a serious problem in poultry flocks and waterfowl in Sweden. The objectives of the present study were to isolate, characterize and subtype C. botulinum type C avian isolates in order to increase the knowledge of the genetic diversity. Isolates from 13 birds were identified by 16S rRNA sequencing and BoNT/C gene detection by real-time polymerase chain reaction (PCR). Conventional PCR was used to distinguish a chimeric BoNTC/D gene, often associated with avian botulism, from the BoNT/C gene. The isolates analysed all contained the gene coding for a chimeric toxin type C/D. Two fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD), were optimized and used to investigate the epidemiological relatedness among the strains. The isolates were divided into three different pulsotypes based upon their restriction profiles for SmaI and SalI. The RAPD system proved to be as discriminative as PFGE. This study reveals a small genetic diversity among Swedish type C strains, with a high similarity between strains from broilers and herring gulls.     

Microsatellite-primed PCR and random primer amplification polymorphic DNA for the identification and epidemiology of dermatophytes

This study demonstrates the capacity of the one-step polymerase chain reaction (PCR) fingerprinting method using the microsatellite primers (GACA)₄ or (GTG)₅ (MSP-PCR) to identify six of the most frequent dermatophyte species causing cutaneous mycosis. PCR with (GACA)₄ was a suitable method to recognise Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale among 82 Argentinian clinical isolates, producing the most simple and reproducible band profiles. In contrast, the identification of Trichophyton mentagrophytes and Trichophyton tonsurans was achieved using PCR with (GTG)₅. In this way, the sequential application of PCR using (GACA)₄ and (GTG)₅ allowed the successful typification of clinical isolates which had not been determined by mycological standard techniques. In this work, the intraspecies variability among 33 clinical isolates of M. canis was detected using random amplification of polymorphic DNA (RAPD-PCR) with the primers OPI-07 and OPK-20. The genetic variations in the isolates of M. canis were not associated with clinical features of lesions or pet ownership, but a geographical restriction of one genotype was determined with OPK-20, suggesting a clonal diversity related to different ecological niches in certain geographical areas. The results of this work demonstrate that the detection of intraspecies polymorphisms in M. canis by RAPD-PCR may be applied in future molecular epidemiological studies to identify endemic strains, the route of infection in an outbreak or the coexistence of different strains in a single infection.     

Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting.

The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.     

Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species.

A total of 49 type and neotype isolates and 32 clinical isolates of the anamorph genus Candida and related teleomorph genera were obtained from different culture collections and clinical laboratories. Isolates were subjected to two phenotypic methods of identification, Vitek yeast biochemical card (YBC) and API ID 32C, both based on carbohydrate assimilation, and one genotypic method, PCR fingerprinting, based on the detection of DNA polymorphisms between minisatellite-specific sequences with the primer M13 (5' GAGGGTGGCGGTTCT 3'). The correct identification of a strain at the Centraalbureau voor Schimmelcultures was used as the gold standard for the identification of an isolate. When the study was restricted to species included in the respective biochemical databases, the Vitek YBC and API ID 32C systems performed adequately with positive identification rates of 87.3 and 76.8%, respectively. When uncommon species were added to the study, several of which are not included in the databases, the identification efficiencies were 76.5 and 77.5%, respectively. By comparison, all isolates were correctly identified by PCR fingerprinting, with 63 reference species profiles in the databank. Sufficient polymorphisms among the total set of banding patterns were observed, with adequate similarity in the major patterns obtained from a given species, to allow each isolate to be assigned unambiguously to a particular species. In addition, variations in minor bands allowed for differentiation to the strain level. PCR fingerprinting was found to be rapid, reproducible, and more cost-effective than either biochemical approach. Our results provide reference laboratories with an improved identification method for yeasts based on genotypic rather than phenotypic markers.     

Physiological and molecular characterization of atypical isolates of Malassezia furfur

The species constituting the genus Malassezia are considered to be emergent opportunistic yeasts of great importance. Characterized as lipophilic yeasts, they are found in normal human skin flora and sometimes are associated with different dermatological pathologies. We have isolated seven Malassezia species strains that have a different Tween assimilation pattern from the one typically used to differentiate M. furfur, M. sympodialis, and M. slooffiae from other Malassezia species. In order to characterize these isolates of Malassezia spp., we studied their physiological features and conducted morphological and molecular characterization by PCR-restriction fragment length polymorphism and sequencing of the 26S and 5.8S ribosomal DNA-internal transcribed spacer 2 regions in three strains from healthy individuals, four clinical strains, and eight reference strains. The sequence analysis of the ribosomal region was based on the Blastn algorithm and revealed that the sequences of our isolates were homologous to M. furfur sequences. To support these findings, we carried out phylogenetic analyses to establish the relationship of the isolates to M. furfur and other reported species. All of our results confirm that all seven strains are M. furfur; the atypical assimilation of Tween 80 was found to be a new physiological pattern characteristic of some strains isolated in Colombia.     

Genomic and phenotypic evaluation of Salmonella typhimurium and Salmonella enteritidis in Iran

Genomic and phenotypic evaluation of Salmonella typhimurium and Salmonella enteritidis by two simple, fast, and applicable methods of random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and antibiotic susceptibility for employment in epidemiologic surveillance screening systems in Iran was the objective in this study. We selected 60 (30 S. typhimurium and 30 S. enteritidis) isolates from different animal sources and different times and places among 1975–2006 in Iran. Serotyping with traditional method and reliable antisera was done and then confirmed with multiplex PCR (with four and three pair of primers, respectively). All of S. typhimurium and S. enteritidis isolates have virulence genes of invA and spv, respectively. Then, from nine primers, two primers that have enough discriminatory power were selected for RAPD analysis. We set up the quality and quantity of DNA template, primers, MgCl₂, Taq DNA polymerase, and deoxyribonucleotide triphosphates concentration for RAPD analysis. We also examined the strains with disk diffusion standard antibiotic susceptibility test. With the application of primer P1254; we saw four and seven and primer 23L, six and three profiles in RAPD analysis and 13 and six profiles of R-type in susceptibility test of S. typhimurium and S. enteritidis, respectively. Combination of two methods differentiated these 30 strains to 20 and 16 different profiles, respectively. The finding of this study indicated that clonality of S. enteritidis is more than S. typhimurium in Iran and RAPD-PCR in combination with antibiotic susceptibility test were fast, easy, relatively reliable, and discriminatory molecular and phenotypic methods in the differentiation of S. typhimurium and S. enteritidis for epidemiologic purposes in Iran.     

Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae

A loop-mediated isothermal amplification (LAMP) system, the illumigene Mycoplasma DNA amplification assay (Meridian Bioscience, Inc., Cincinnati, OH) was evaluated to determine its analytical sensitivity, specificity, and clinical application in comparison to historic culture in a collection of archived respiratory specimens. The illumigene limit of detection was ≤88 CFU/reaction for 10 Mycoplasma pneumoniae reference strains. This assay correctly identified 36 M. pneumoniae reference strains and clinical isolates from various geographic origins, including both of the main subtypes. No cross-reactions were detected with other mycoplasmas, ureaplasmas, other bacterial species, viruses, yeasts, or human DNA. Among 214 respiratory specimens previously cultured for M. pneumoniae, when real-time PCR with bidirectional sequencing of the PCR products was used to resolve discrepancies, the sensitivity was 22 of 22 (100%) and the specificity was 190 of 192 (99%). This commercial LAMP assay is a useful rapid method for detecting M. pneumoniae in clinical specimens. Additional prospective clinical trials with direct comparison to culture and PCR are warranted.     

Rapid identification and fingerprinting of Candida krusei by PCR-based amplification of the species-specific repetitive polymorphic sequence CKRS-1.

A PCR method was developed to identify and fingerprint Candida krusei isolates simply and rapidly. The primer pair Arno1 and Arno2 was designed to amplify the polymorphic species-specific repetitive sequence CKRS-1 (C. krusei repeated sequence 1) that we identified in the nontranscribed intergenic regions (IGRs) of rRNA genes in C. krusei LMCK31. The specificity, sensitivity, reproducibility, and fingerprinting ability of the PCR assay were evaluated. Amplification products were obtained from all 131 C. krusei isolates studied. No other yeast species of medical importance (n = 26), including species similar to C. krusei, species of pathogenic filamentous fungi, or a variety of pathogenic bacteria, yielded a PCR product with these primers. This PCR assay allowed for the identification of C. krusei in less than 6 h. The PCR assay was sensitive enough to detect as little as 10 to 100 fg of C. krusei-purified DNA and proved to be reproducible. Since amplification products varied both in number and in molecular weight according to the strains, PCR patterns allowed strains to be distinguished. To ascertain the epidemiological usefulness of this PCR fingerprinting, the patterns of the 131 isolates were compared. A total of 95 types which corresponded to 95 independent strains were delineated (discriminatory power = 1 with n = 95). Comparison of the results of PCR fingerprinting and those of fingerprinting with the CkF1,2 probe showed that they concurred. In addition, this work yields insights into the mechanisms involved in generating polymorphisms in the IGRs of C. krusei. Since this method is simpler and faster than established identification and genotyping methods of this important pathogenic species, it is a critical improvement for clinical microbiology laboratories relevant not only to diagnosis but also to epidemiology.