0095-1137/96/$04.0010
Copyrightq1996, American Society for Microbiology
Species-Specific Identification of Candida krusei by
Hybridization with the CkF1,2 DNA Probe
A. CARLOTTI,* A. COUBLE, J. DOMINGO, K. MIROY,ANDJ. VILLARDLaboratoire de Mycologie Fondamentale et Applique´e aux Biotechnologies Industrielles, Faculte´ de Pharmacie, 69373 Lyon Cedex 08, France
Received 20 February 1996/Returned for modification 16 March 1996/Accepted 20 April 1996
The species specificity of the Candida krusei DNA fingerprinting probe CkF1,2 has been investigated. A total of 149 pathogenic and nonpathogenic fungal and bacterial DNAs were screened with CkF1,2. The probe was cold labeled with peroxidase, and its specificity was assessed by using Southern blot, dot blot, and colony blot hybridization. Its sensitivity was determined by dot blot hybridization. The CkF1,2 probe proved to be species specific. It hybridized with DNA for the 112 C. krusei strains studied, whereas it failed to hybridize under low-stringency conditions to 37 DNAs from 27 different yeast species, including Candida albicans, Candida
glabrata, Candida norvegensis, Candida inconspicua, Candida tropicalis, Candida valida, Candida zeylanoides, and Yarrowia lipolytica, as well as DNAs from the filamentous fungi and bacteria tested. However, CkF1,2 hybridized
strongly with DNA of the yeast species Issatchenkia orientalis, the putative ascogenous perfect state of C. krusei. Amounts as small as 60 to 120 ng of C. krusei target DNA were detected by dot blot hybridization with CkF1,2. It permitted the direct screening of colony blots for early identification. The CkF1,2 probe has potential value as a diagnostic reagent for identifying C. krusei.
The yeast species Candida krusei (putative asexual state of
Issatchenkia orientalis) is widespread in nature (3). It now
rep-resents one of the four most common yeast pathogens in highly compromised patient populations, e.g., neutropenic patients (10, 18, 29). In such patients it causes life-threatening systemic infections (12). Moreover, most strains of C. krusei are natu-rally resistant to the antifungal agent fluconazole (8, 21), which is being used increasingly. Hence, it is crucial to reliably and rapidly identify strains of this species. Unfortunately, several problems exist in the current laboratory methodology for iden-tification of yeasts, particularly for C. krusei.
The conventional methods used for yeast identification, based on biochemical features (3, 15), are time-consuming and tedious. As C. krusei uses a limited number of substrates, these methods fail to easily differentiate it from other phenotypically similar species, such as Candida valida (3). Miniaturized iden-tification systems, involving a reduced number of tests, such as API strips (bioMerieux, Marcy l’Etoile, France) or the Vitek Yeast Biochemical Card (Vitek Systems, Hazelwood, Mo.), which are used in the routine clinical laboratory do not always provide conclusive results (6, 7, 20, 23). C. krusei can be mis-identified as Candida inconspicua, Candida lipolytica, Candida
norvegensis, Candida rugosa, C. valida, Candida zeylanoides, or
even Candida glabrata. Furthermore, physiological tests do not allow yeast strain discrimination at the infraspecific level (19). We recently developed the CkF1,2 DNA probe for finger-printing C. krusei strains (5). CkF1,2 is composed of two cloned
EcoRI restriction fragments of C. krusei K31 genomic DNA,
F1 and F2. Hybridization of Southern blots of HinfI-digested whole-cell DNA with CkF1,2 generates patterns containing a variable number of bands, depending on the strain. Band num-ber and molecular weights differ among strains, providing a means for strain discrimination (5). We recently showed, by sequence analysis, that F1 and F2 are polymorphic forms of the
nontranscribed intergenic region of rRNA genes (unpublished data) and contain a repeated sequence, which we named CKRS-1, that is made up of seven to eight shorter repeated sequences of about 165 bp. Preliminary results indicated that the probe could be species specific. Most of the fingerprinting probes described for fingerprinting Candida albicans isolates, and also made up of middle repetitive sequences, are species specific (1, 11, 16, 22).
To determine whether CkF1,2 might be species specific and may therefore function as a diagnostic probe, we screened a panel of pathogenic and nonpathogenic fungal DNAs with CkF1,2. The species specificity was assessed by Southern blot, dot blot, and colony blot hybridizations. We report here that the CkF1,2 probe may be a valuable diagnostic reagent, since it fails to hybridize to other fungal or bacterial DNAs under low-stringency conditions but hybridizes with DNAs of all the
C. krusei strains tested.
MATERIALS AND METHODS
Strains.The 149 strains of yeasts, filamentous fungi, and bacteria examined in this study are listed in Table 1. The 38 yeast reference strains belonged to 28 different species according to Kreger-van Rij (15). The type strain of the species C. krusei, CBS 573T(Centraal Bureau voor Schimmelcultures, Delft, The Neth-erlands), was included, as well as C. krusei LMCK31, from which the CkF1,2 DNA probe was originally selected (5). I. orientalis CBS 5147T, the putative perfect state of C. krusei, was also included in the study. A total of 112 strains of C. krusei from clinical (n5110) or environmental (n52) sources, labeled K1 to K128, were also studied. Species phenotypically related to C. krusei (i.e., C. valida) or difficult to distinguish from C. krusei by means of miniaturized iden-tification systems (e.g., C. glabrata, C. inconspicua, Candida lambica, C. norveg-ensis, C. rugosa, C. zeylanoides, Yarrowia lipolytica [formerly C. lipolytica], and Zygosaccharomyces rouxii) were also included. Representative pathogenic fila-mentous fungi and bacterial strains were included too (Table 1). Fungal strains were grown in YM medium (glucose, 1%; yeast extract, 0.3%; malt extract, 0.3%; Bacto Peptone, 0.5%) at 308C. When the strains were not purchased from international collections, their identifications were made according to conven-tional methods (3, 15) and were further verified with the API 32C system (bioMerieux). Yeasts were maintained at 48C on YM agar slants. Bacterial strains were grown as described previously (4).
DNA extraction.Yeast whole-cell DNA was extracted by the procedure de-scribed by Scherer and Stevens (25), with slight modifications. Driselase (Fluka Chemie AG, Buchs, Switzerland) was used instead of Zymolyase for spheroplast production. DNAs from filamentous fungi were extracted as described previously (2). Bacterial DNAs were extracted as described elsewhere (9). DNA
concen-* Corresponding author. Mailing address: Laboratoire de Mycologie Fondamentale et Applique´e aux Biotechnologies Industrielles, Faculte´ de Pharmacie, 8, Avenue Rockefeller, 69373 Lyon Cedex 08, France. Phone: (33) 78-77-70-20. Fax: (33) 78-77-72-12.
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trations and A260/A280ratios were determined spectrophotometrically with an Ultrospec UV monitor (LKB, Cambridge, England).
Development of the CkF1,2 DNA probe.The CkF1,2 DNA probe is composed of two cloned EcoRI restriction fragments of C. krusei K31 genomic DNA, F1 and F2 (5), with estimated molecular sizes of approximately 5.2 and 5.4 kb, respectively. These two fragments were selected among others because (i) they corresponded to repeated sequences and (ii) in a preliminary study we deter-mined that they differ from the two main EcoRI restriction fragments of
tran-scribed rRNA genes of, respectively, 2.9 and 3.55 kb (5). Each fragment was ligated into pBluescript II KS1(Stratagene, La Jolla, Calif.) opened at the EcoRI site. Epicurian Coli Sure competent cells (Stratagene) were used for transformation. Bacterial and plasmid genetic manipulations were performed as outlined by Sambrook et al. (24). After plasmid digestion with EcoRI, the inserts were purified by using the Geneclean II kit (BIO 101) and combined to make the CkF1,2 DNA probe, which contained equal proportions (wt/wt) of the two cloned EcoRI fragments.
We have recently established by using sequencing, restriction mapping, and cross-hybridization that F1 and F2 are two polymorphic forms of the nontran-scribed intergenic region of rRNA genes. They both contain a repeated se-quence, which we named CKRS-1, that is made up of eight and seven shorter repeated sequences of about 165 bp in F1 and F2, respectively (unpublished data).
Southern blot hybridization.Whole-cell DNA samples (5 to 8mg) were di-gested with EcoRI (Appligene) (5 U/mg of DNA) as recommended by the supplier. After 5 h of incubation at 378C, the reaction was stopped by heating the reaction vials to 708C for 10 min. DNA fragments were separated electrophoreti-cally on 0.8% (wt/vol) agarose (type II medium EEO; Sigma Chemical Co., St. Louis, Mo.) gels in Tris-borate-EDTA buffer (pH 8.1). Electrophoresis was performed at 2 V/cm for 18 h. The gels were stained with ethidium bromide (0.5 mg/ml) and photographed with 300-nm transillumination through an orange filter and with Polaroid 55 film, and DNA fragments were transferred to a positively charged nylon membrane (Appligene) by Southern blotting (26) under a vacuum and then baked at 808C for 15 min. The phagelDNA digested with HindIII was used as a standard. The CkF1,2 DNA probe (100 ng) and standard (100 ng) were labeled directly with horseradish peroxidase by using the ECL kit (Amersham, Les Ulis, France) and detected by enhanced chemiluminescence as described previously (5). Prehybridization (2 h), hybridization (overnight), and washings of Southern blots were performed at 428C under nonstringent condi-tions as described previously (5). Chemiluminescence detection was performed as described previously (5), by using Hyperfilm-ECL (Amersham) with exposure times of 10 to 15 min. Total RNA, extracted from Saccharomyces cerevisiae CBS 1369 (14), labeled with peroxidase was used as a positive control for hybridiza-tion.
Dot blot hybridization.For sensitivity assays we used genomic DNA of C. krusei CBS 573Twhich was serially diluted in 40ml of bidistilled water from 2 to 0.001mg, subsequently denatured by the addition of 20ml of 0.2 N NaOH, and then dot blotted onto positively charged nylon membranes (Appligene) by using the Milliblot apparatus (Millipore Corporation, Bedford, Mass.). Prehybridiza-tion and hybridizaPrehybridiza-tion condiPrehybridiza-tions were the same as described above.
For specificity assays we used (i) C. krusei CBS 573Twhole-cell DNA serially diluted to 2, 1, 0.10, 0.05, and 0.01mg in a final volume of 40ml of bidistilled water and (ii) equivalent amounts of DNA (at least 10mg) of each isolate of the non-C. krusei species in 40ml of bidistilled water. That amount of DNA repre-sented 100-fold the detection limit of CkF1,2, as determined by a dot blot hybridization sensitivity assay. DNAs were denatured by the addition of 20ml of 0.2 N NaOH and dot blotted onto positively charged nylon membranes (Appli-gene) by using the Milliblot apparatus (Millipore Corporation). Prehybridization and hybridization conditions were the same as described above.
Yeast colony hybridization.Colony screening with the CkF1,2 DNA probe was done by the protocol described in the instruction booklet for the ECL kit (Amersham), with slight modifications. Fresh cells were plated on YM agar and incubated overnight at 308C. Colony lifts were done on positively charged nylon membranes (Appligene) which had been first saturated with a Driselase (Fluka) solution (10 mg/ml in 0.4 M sorbitol, 50 mM phosphate buffer [pH 7.5], 0.1% [vol/vol] 2-mercaptoethanol). The lifted colonies were incubated at 378C for 30 min. They were lysed by placing the membranes (colony side up) for 15 min on two sheets of Whatman 3MM paper (Whatman International Ltd., Maidstone, England) which had been saturated with 0.5 N NaOH. The blots were then rinsed in 400 ml of 53SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate). DNA released from the yeast lysates was bound to the membranes by subsequent baking at 808C for 15 min. Prehybridization and hybridization procedures were carried out as described above, but for 1 and 6 h, respectively, at 428C in order to do the experiment within 1 working day.
Computerized analysis.The computer program NCSA Gel Reader (NCSA, Champaign, Ill.) was used to read the migration distances of the fragments on numerized (300 dots per in. [1 in.52.54 cm]) images of the gels on a Macintosh Centris 660 AV computer (Apple Computer Inc., Cupertino, Calif.) and to calculate their molecular sizes by extrapolation in comparison with the standard phagelDNA HindIII digest (Appligene) run in a reference lane.
RESULTS
The CkF1,2 DNA probe is species specific for C. krusei in Southern hybridization.To determine the species specificity of the CkF1,2 DNA probe, DNAs of the medically most impor-tant yeast species were first screened by Southern hybridiza-tion. Genomic DNAs from reference strains of C. albicans,
[image:2.612.59.296.102.599.2]Candida guilliermondii, Candida lusitaniae, Candida kefyr, C.
TABLE 1. Yeast, filamentous fungus, and bacterial strains screened with CkF1,2 and coordinates of the corresponding DNA samples in
the dot blot experiments shown in Fig. 3B
Species Strain designation a
(no. of isolates) Coordinates
Yeasts
Candida albicans ATCC 2091 B7
Serotype A LML 1a B8
Serotype B LML 1b B9
Candida boidinii 34 F 1 D7
Candida famata CBS 1795T C5 Candida glabrata CBS 138T C4 Candida guilliermondii CBS 6021T D9 Candida humicola CBS 2839T D5 Candida inconspicua CBS 180T C3 Candida kefyr CBS 607T B10 Candida krusei CBS 573T, K1-128 (112)b A1–A5 Candida lambica CBS 1876T D6 Candida lusitaniae CBS 6936T B11 Candida norvegensis LML Q243 E5
Candida parakrusei LML 5201 B3
Candida parapsilosis CBS 604T C1 Candida rugosa SIPHV 823 C7
Candida tropicalis CBS 94T C2 Candida valida CBS 638T, CBS 635 (2) B5, B6 Candida zeylanoides IPP 207 C6
Cryptococcus neoformans
CBS 132T E7
Geotrichum candidum CBS 109.12 D1
Issatchenkia orientalis CBS 5147T B1 Kluyveromyces
marxianus
CBS 712T E6
var. bulgaricus CBS 2762T E3
var. dobzanskii CBS 2104T E4
var. drosophilarum LML Kly69 D10
var. lactis CBS 683T D11
var. vanudenii CBS 4372T E2
var. wickerhamii LML Kp E1
Rhodotorula glutinis CBS 20T C9 Rhodotorula rubra CBS 17T C10 Saccharomyces
cerevisiae
CBS 1171T E8
Trichosporon cutaneum IPP 654 C8
Yarrowia lipolytica CBS 6124T, LML 1051 (2) B2, B4 Zygosaccharomyces
rouxii
LML Z1 D8
Filamentous fungi
Aspergillus fumigatus LML AR27 D2
Aspergillus flavus LML AF7 B12
Mucor mucedo LML M4 C12
Bacteria
Escherichia coli JM109 D12
Nocardia asteroides IPP 1750-88 D3
Rhodococcus sp. LML R1 D4
Staphylococcus aureus LML S1 C11
aAbbreviations: ATCC, American Type Culture Collection, Rockville, Md.;
CBS, Centraal Bureau Voor Schimmelcultures, Delft, The Netherlands; IPP, Institut Pasteur, Paris, France; LML, Laboratoire de Mycologie de la Faculte´ de Pharmacie, Lyon, France.
bThe number in parentheses is the total number of C. krusei strains studied.
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krusei, Candida parapsilosis, Candida tropicalis, and C. valida,
as well as Y. lipolytica, were digested with EcoRI, and the restriction products were separated by agarose gel electro-phoresis. Complex patterns of bands were observed on ethidium bromide-stained gels (Fig. 1A). Brightly stained bands were clearly distinguished from the rest of the digestion products, which varied in both number and size, depending on the species. C. krusei CBS 573Tand C. valida CBS 638T re-striction patterns were not easily differentiated. Two promi-nent bands of approximately 2.9 and 3.55 kb were easily dis-tinguishable in the C. krusei restriction pattern. Other brightly stained bands were observed in both the 4.4- to 6.6-kb and 7.0-to 9.4-kb size ranges. As shown in Fig. 1B, the use of CkF1,2 7.0-to probe the Southern blot of the gel resulted in strong
hybrid-ization to C. krusei CBS 573TDNA in the 4.4- to 6.6-kb size range, but not to other yeast species DNAs. Two distinct hy-bridizing fragments with estimated molecular sizes of 5.0 and 5.2 kb were observed.
To obtain an appropriate positive control, the blot was stripped and rehybridized with peroxidase-labeled 18S and 25S rRNA from S. cerevisiae. Because of the high degree of evo-lutionary conservation of transcribed ribosomal DNA se-quences, these ribosomal probes hybridized to each yeast DNA assayed under identical low-stringency hybridization and wash-ing conditions (data not shown). Two distinct strongly hybrid-izing fragments of 2.9 and 3.55 kb, which corresponded to the main rRNA gene EcoRI restriction fragments, were observed with C. krusei CBS 573T.
The same species specificity of CkF1,2 was observed by Southern hybridization analyses when Candida famata, C.
gla-brata, C. humicola, C. inconspicua, C. rugosa, C. zeylanoides,
and S. cerevisiae DNAs were screened (data not shown). No significant cross-hybridization signal was observed.
The CkF1,2 probe hybridized strongly to each of the 112 C.
krusei DNAs screened by Southern hybridization and detected
restriction fragment length polymorphisms as previously re-ported (5). An example of hybridization patterns obtained for 10 C. krusei strains is given in Fig. 2. Nine isolates were of clinical origin either from eight distinct patients (isolates K1, K17, K18, K27, K35, K60, and K65) or from the same patient (K62 and K64, isolated 15 days apart). The type strain was also included. The CkF1,2 probe hybridized with one to three frag-ments of various sizes in the 4.4- to 6.6-kb range, depending on the isolate. Nine distinct hybridization patterns were observed for the 10 isolates. Isolates K62 and K64, originating from the same patient, were characterized by the same pattern. We had already shown that CkF1,2 detected restriction fragment length polymorphisms in C. krusei strains and proved useful for fingerprinting 58 C. krusei strains (5).
Sensitivity of the CkF1,2 DNA probe in dot blot hybridiza-tion.To determine the sensitivity of the CkF1,2 DNA probe, whole-cell DNA from C. krusei CBS 573Twas serially diluted from 2 to 0.001mg, denatured, and dot blotted on positively charged nylon membrane. CkF1,2 gave strong hybridization signals when the amounts of target DNA were in the range of 2 to 0.125 mg (Fig. 3A). Hybridization signals with lower in-tensities were recorded for 0.06- and 0.03-mg amounts of target DNA. The results suggested that as little as 60 to 120 ng of C.
krusei DNA was detectable. No hybridization signal was
ob-served when the amount of DNA was,30 ng. The same results were obtained when DNAs from five randomly chosen C.
kru-sei strains were used as targets, with detection limits in the
range of 30 to 120 ng (data not shown). The limit of detection was arbitrarily set at 100 ng on the basis of these results.
Species specificity of the CkF1,2 DNA probe in dot blot hybridization assays using large amounts of target DNA.To determine the species specificity of the CkF1,2 probe with a large number of either yeasts, filamentous fungi, or bacterial species, dot blot hybridization was carried out. Thirty-nine DNAs from 28 yeast species, including the putative sexual state of C. krusei, I. orientalis, and those species which resemble C.
krusei, as well as both filamentous fungi and bacteria, were
screened. At least 10mg of DNA of the investigated non-C.
krusei species was dot blotted on positively charged membrane;
this amount corresponded to 100-fold the limit of detection of
C. krusei DNAs (Fig. 3A). Under low-stringency hybridization
[image:3.612.59.297.72.496.2]and washing conditions, the CkF1,2 DNA probe hybridized very strongly (Fig. 3B) with both control C. krusei DNAs (from 2 to 0.100 mg) (coordinates A1, A2, and A3 in Fig. 3B) and DNA of its putative sexual state, I. orientalis (coordinates B1),
FIG. 1. Southern blot analysis of Candida species done with the CkF1,2 probe. Whole-cell DNA samples (5 mg) from reference strains of different species, including C. krusei CBS 573T
, were digested with EcoRI, electropho-resed on a 0.8% agarose gel at 40 V for 18 h, stained with ethidium bromide, and photographed (A) prior to being blotted on positively charged nylon membrane. The blot was probed with about 100 ng of peroxidase-labeled CkF1,2 probe and revealed by chemiluminescence (B). AlDNA HindIII digest was used as the standard.
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while it failed to hybridize significantly to all other fungal or bacterial DNAs. Under these conditions, with very large amounts of non-C. krusei target DNA some very weak hybrid-ization signals were observed with DNAs of C. inconspicua (coordinates C3), a Rhodococcus sp. (coordinates D4), and
Kluyveromyces marxianus var. vanudenii (coordinates E2), but
they were not strong enough to interfere with detection of C.
krusei isolates. Effectively, they were always of intensities lower
than the intensity of the control, 0.100mg of C. krusei DNA. This is particularly clear when the weak hybridization signals are compared with the signal obtained with I. orientalis DNA, which corresponded to similar amounts of target DNA. The weak hybridization signals were therefore considered nonsig-nificative.
Screening of colony blots with CkF1,2.In an effort to use the CkF1,2 probe for direct detection of C. krusei colonies on agar-plated samples, 6-h hybridization assays were performed with colony replicas containing C. krusei CBS 573T, C. krusei LMCK31, I. orientalis CBS 5147T, and 12 species which resem-bled C. krusei (Fig. 4). CkF1,2 hybridized to released DNA
from C. krusei and I. orientalis colonies, while it failed to hy-bridize to released DNA from colonies of the other yeast species (Fig. 4). To ensure that DNA was effectively released from all the colonies, the membrane was further hybridized with 18S and 25S rRNA from S. cerevisiae. Hybridization sig-nals were obtained for the 15 corresponding colony locations (data not shown). CkF1,2 permitted the detection of C. krusei colonies within 1 working day.
DISCUSSION
The two main characteristics to consider when assessing the value of any diagnostic probe are specificity and sensitivity. The secondary factors to consider are the cost, ease of use, and rapidity.
We have demonstrated here that the DNA fingerprinting probe CkF1,2 from C. krusei, which originates from the inter-genic region of rRNA genes, was specific for C. krusei isolates and C. krusei’s putative perfect state, I. orientalis. It enabled the detection and reliable identification of this species by hybrid-ization on Southern blots and dot blots, as well as colony blots. Notably, it permitted unequivocal distinguishing of C. krusei from C. valida, C. glabrata, C. inconspicua, C. norvegensis, C.
rugosa, C. zeylanoides, and Y. lipolytica, whereas these species
are known to be difficult to separate by current laboratory methodology for identification of yeasts (6, 7, 20, 23). Wickes et al. (28) have reported the isolation of a C. krusei species-specific probe, which they named CK13, in a study on the relationships among organisms of different groups of medically important yeasts. Notably, they compared three C. krusei strains—including the ATCC 6258 type strain (equivalent of CBS 573T)—with the type strain of I. orientalis. The CK13 DNA probe was made of a 0.9-kb-long restriction fragment of
C. krusei genomic DNA obtained by random cloning. The
hybridization of this probe to Southern blots of
[image:4.612.60.296.73.455.2]EcoRI-di-FIG. 2. Examples of patterns obtained for EcoRI-digested CkF1,2 whole-cell DNA of C. krusei strains. CBS 573T, K65, and K60 to K1 were unrelated strains. K64 and K62 were related strains isolated from the same patient 15 days apart. AlDNA HindIII digest was used as the standard. Conditions were as described in the legend to Fig. 1.
FIG. 3. Dot blot hybridizations. (A) Sensitivity assay. DNA of C. krusei CBS 573T
was serially diluted in water, denatured, and dot blotted onto positively charged nylon membrane. The blot was hybridized with about 100 ng of perox-idase-labeled CkF1,2 probe to determine the limits of detection. (B) Specificity assay. Yeast, filamentous fungus, and bacterial DNAs were screened with the CkF1,2 probe. Row A, C. krusei CBS 573T
DNA serially diluted in water to 2 (A1), 1 (A2), 0.1 (A3), 0.05 (A4), and 0.01 (A5)mg; rows B to E, DNA samples, at least 10mg, from yeasts of different species (including I. orientalis [B1]), filamentous fungi, and bacteria. DNAs were denatured and dot blotted onto positively charged nylon membrane. The samples are identified by their coordi-nates in Table 1. The blot was hybridized with about 100 ng of peroxidase-labeled CkF1,2 probe.
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[image:4.612.317.555.444.629.2]gested genomic DNA from multiple Candida isolates resulted in strong hybridization to the C. krusei type strain and I.
ori-entalis DNAs in the 8- to 10-kb size range, but not to DNAs of
the C. albicans, Candida stellatoidea, C. tropicalis, Candida
paratropicalis, C. kefyr, C. parapsilosis, and S. cerevisiae strains
studied. The specificity against other yeast species, notably C.
valida; filamentous fungi; or bacteria was not reported.
Fur-thermore, Wickes et al. (28) showed that CK13 was not dis-criminatory enough for infraspecies fingerprinting, and the or-igin of the probe was not known. Finally, Wickes et al. (28) concluded that C. krusei and I. orientalis cannot be distin-guished by the methods they had used and could be considered synonyms. It is obvious from our results that the CkF1,2 probe is different from the CK13 probe of Wickes et al. (28). First, the probes differed in size: F1 and F2 are both longer than 5 kb, while CK13 is 0.9 kb long. Second, there are grounds to assert that they did not have homology, since CkF1,2 and CK13 did not hybridize with the same EcoRI restriction fragments of DNA of the type strain of C. krusei. Effectively, when used to probe Southern blots of genomic DNA of the type strain C.
krusei CBS 573T digested with EcoRI, CkF1,2 hybridized
strongly in the 4.4- to 6.6-kb size range, while CK13 hybridized in the 8- to 10-kb size range. This is also true for the other C.
krusei strains investigated. Third, we have demonstrated that
unlike CK13, CkF1,2 can be used to distinguish among C.
krusei isolates since it revealed restriction fragment
polymor-phisms (5). We had already reported that fingerprinting with CkF1,2 showed a 100% typeability, 100% reproducibility, and a discriminatory power of 1 (n 5 58 strains); thus, CkF1,2 constituted an invaluable epidemiological marker for tracing strains (5). This is further illustrated by the example shown herein.
An important point is that two different C. krusei
species-specific DNA probes, namely, CkF1,2 and CK13, both hybrid-ized to I. orientalis DNA. This further confirms the conclusions of yeast taxonomists who placed the two species in the relation of synonymy, even though interfertility between them was not established (3, 28).
We have established recently both by sequencing F1 and cross-hybridizing F1 and F2 that CkF1,2 is made up of two polymorphic forms of the nontranscribed intergenic region of rRNA genes, between the 25S and 18S RNA genes (unpub-lished data). They both contain a repeated sequence, which we named CKRS-1, that is made up of shorter repeated elements (C. krusei repeated element) of 165 bp repeated eight times in F1 and seven times in F2. Hence, the CkF1,2 probe is made up of middle repetitive sequences that are repeated as the rRNA gene clusters. Species-specific middle repetitive sequences have been described for C. albicans (1, 11, 16, 17), and
Cryp-tococcus neoformans (27). Middle repetitive genomic DNA as
presented in multiple copies per cell gave a sensitivity compa-rable to that of transcribed ribosomal or mitochondrial DNA but without the potential cross-reactivity expected from probe produced on the basis of the highly conserved sequences of these DNAs, such as those described by Sadhu et al. (22). Both the length of CkF1,2 and the repetitive nature of the sequences that it is made up of no doubt account for its high degree of sensitivity recorded herein. Effectively, CkF1,2 enabled the detection of amounts of DNA as small as 60 to 120 ng, under conditions that did not permit the significative detection of 10
mg of other fungal DNAs. The large size of the probe allows the easy incorporation of a large amount of label (peroxidase) and thus increases the DNA detection threshold. The sensitiv-ity of the CkF1,2 probe is identical to that reported by Keath et al. (13) for the detection of Histoplasma capsulatum with a 1.85-kb HindIII restriction fragment labeled with 32P, which enabled detection of 60 to 120 ng of DNA under conditions that did not permit the detection of 2mg of other fungal DNAs (13).
Because of its high degree of sensitivity, the CkF1,2 probe can be used nonradioactively labeled. The advantages of non-radioactive labeling are obvious and make molecular methods available to smaller laboratories. This eliminates the problems posed by handling and disposal of radioactivity. We have shown that excellent results can be obtained with a chemilu-minescent probe, with hybridization signals observed after 15 min of exposure. The ease of probe synthesis and the speed with which the results are available may render investigation of suspected infection with C. krusei more feasible for the clinical laboratory. The use of CkF1,2 in dot blot assays allows the screening of a large number of DNA samples (more than 90) in a few steps, in less than 36 h by the rapid DNA extraction procedure of Scherer and Stevens (25). Moreover, several blots can be screened at the same time. We recommend using 0.250
mg of C. krusei DNA as a low-range positive control and 0.5 to 1mg of target DNA for the dot blot formatted assay. Under these conditions, all hybridization signals with intensities lower than those of the low-range positive control must be consid-ered negative. The use of CkF1,2 in Southern hybridization analysis combines two main advantages: specificity (for diag-nosis) and fingerprinting ability (for typing). Southern hybrid-ization analysis with CkF1,2 is in routine use in our laboratory and has replaced the conventional identification method for C.
krusei. CkF1,2 can be gainfully used for the direct screening of
[image:5.612.61.297.72.312.2]colonies on agar plates, which is a convenient, rapid—results can be obtained within 1 working day—and simple method for testing multiple colonies in a few steps. These results may prove invaluable in medical mycology. For instance, the reli-able early detection of C. krusei among isolates of other yeast
FIG. 4. Colony blot screening with the CkF1.2 probe. Yeast lifted colonies on positively charged nylon membrane were lysed, and released DNA was bound by baking. The lift was probed with about 100 ng of CkF1,2 probe. Colonies: 1, C. inconspicua; 2, C. albicans; 3, C. krusei CBS 573T
; 4, C. krusei LMCK31; 5, Y. lipolytica; 6, C. zeylanoides; 7, C. glabrata; 8, I. orientalis; 9, C. famata; 10, C. parapsilosis; 11, C. lusitaniae; 12, C. tropicalis; 13, C. rugosa; 14, C. kefyr; 15, C. guilliermondii; 16, no colony.
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species in immunosuppressed patients will prevent the use of the antifungal agent fluconazole for treatment, since it has been suggested that its utilization may lead to the emergence of C. krusei as a pathogen in such patients (29, 30). The avail-ability of probes such as CkF1,2 is also important as an inde-pendent reference for validation of other simple methods such as the CHROMagar method. Finally, the nucleotide sequence of the species-specific DNA probe CkF1,2 is a potential target for detection of C. krusei by PCR assays.
In conclusion, the CkF1,2 probe proved to be a reliable, rapid, and efficient diagnostic tool for identifying C. krusei by Southern blot, dot blot, and colony blot hybridization, in ad-dition to having potential as a fingerprinting probe. These are classical advantages of middle repetitive DNA sequences that can be used as probes for both diagnostic and epidemiological studies.
ACKNOWLEDGMENT
This research was supported in part by a grant to A.C. from the “Poˆle Ge´nie Biologique et Me´dicale” Rhoˆne-Alpes.
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