JOURNAL OF CLINICAL MICROBIOLOGY, May 1990, p. 876-881 Vol. 28, No. 50095-1137/90/050876-06$02.00/0Copyright © 1990, American Society for Microbiology

Electrophoretic Karyotyping of Typical and AtypicalCandida albicans

MONA MAHROUS,' T. J. LOTT,2 S. A. MEYER,' A. D. SAWANT,' AND D. G. AHEARNl*Laboratory for Microbial and Biochemical Sciences, Georgia State University, Atlanta, Georgia 30303,

and Division of Mycotic Diseases, Centers for Disease Control, Atlanta, Georgia 303332Received 28 September 1989/Accepted 17 January 1990

Electrophoretic karyotypes of atypical isolates of Candida albicans, e.g., strains that were germ tubenegative, failed to express proteinase activity, demonstrated low virulence for mice, formed hyperchlamydo-conidia, produced hyperhyphae, or were sucrose negative (including the type strain of Candida stellatoidea),were compared with those of typical C. albicans. Karyotypes of whole-cell DNA of classical C. albicansexamined with transverse alternating-field electrophoresis under specific conditions were composed of sevenDNA bands with a specific migration pattern. Certain atypical strains and representatives of the three serotypesof C. stellatoidea produced discrete karyotypes with 5 to 10 bands. All isolates demonstrated a significant degreeofDNA relatedness, suggesting their conspecificity. Densitometric tracings of DNA bands provided an objectiveand standardized method for comparing bands within the gels.

The increased incidence of deep candidiasis, particularlyas an often fatal iatrogenic disease, has stimulated interest indeveloping more finite procedures for epidemiological stud-ies of the most common etiological agent, Candida albicans.Various methods, including differences in streak morphol-ogy (2), resistance to chemicals (31), a combination ofphysiological characteristics and resistances to chemicals(20, 21), differences in carbohydrate assimilation (33), sero-typing (5), and sensitivity to yeast toxins (25), have beenproposed for subdividing C. albicans into subgroups orbiotypes. Some success has been achieved, but in generalthe methods recognize either too many or an insufficientnumber of subgroups for practical use. In certain instancesthe procedures have lacked reproducibility in different lab-oratories (10, 22).The development of karyotyping data and endonuclease

digestion patterns of genomic DNA has recently offeredmore fundamental and possibly more reproducible biotypingcapabilities (27). Lott et al. (11) observed, by using fieldinversion gel electrophoresis, five distinct chromosomalmobility groups among isolates of C. albicans. Seven to ninebands with 14 distinct electrophoretic patterns were ob-served by Merz et al. (15), who used orthogonal-field alter-nating-gel electrophoresis in an epidemiologic study of iso-lates of C. albicans from different patients; and Magee et al.(12) resolved up to 11 DNA bands by applying contour-clamped homogeneous-field gel electrophoresis, orthogonal-field alternating-gel electrophoresis, and field inversion gelelectrophoresis. These procedures in conjunction with geneprobes led Magee et al. to postulate the presence of sevenchromosomes. Lasker et al. (9), with a similar protocol in astudy of two isolates, indicated the presence of at least eightchromosomes. Kwon-Chung et al. (7, 8) with an orthogonal-field alternating-gel electrophoresis procedure found distinctkaryotype patterns for two types of Candida stellatoidea(considered herein a variety of C. albicans). One form, typeII, had seven DNA bands in a pattern similar to that of C.albicans serotype A; the other, type t, serotype B, had eightor nine bands.

In most investigations, conditions for obtaining karyo-

* Corresponding author.

types have not been fully described, and only strains readilyidentified as C. albicans or C. stellatoidea have been inves-tigated. Atypical forms of C. albicans that lack classicalidentification characteristics have been of increasing inci-dence among yeast cultures submitted to the Division ofMycotic Diseases, Centers for Disease Control, for identifi-cation. Karyotyping of atypical C. albicans and the use of atransverse alternating-field electrophoresis (TAFE) systemhas not been reported. In this study we compare TAFEkaryotypes of whole-cell DNA of classical and atypical C.albicans.

MATERIALS AND METHODSCultures. Isolates were selected from cultures submitted

for identification to the Division of Mycotic Diseases, Cen-ters for Disease Control (C isolates), from the AmericanType Culture Collection (A isolates), and from the culturecollection at Georgia State University (G isolates). The yeastisolates were screened with the API 20C (Analytab Prod-ucts, Plainville, N.Y.) yeast system and identified by stan-dard assimilation and fermentation tests and morphologicalstudies of pseudomycelium, chlamydospores, and germtubes (1, 30). The sources and identifications of the culturesare presented in Table 1. Cultures were lyophilized within 1month of their submission to Georgia State University, andworking stocks were maintained on Sabouraud dextroseagar.

Biotyping. Selected characteristics, namely, resistance toflucytosine and safranin, citrate utilization, pH tolerance,and proteinase production from the procedures of Odds andAbbott (20), were employed for biotyping. Proteinase pro-duction was detected by the bovine serum albumin-agarplate method of Ruchel et al. (26). Serotypes were deter-mined with the Candida Check RM 302 Iatron Kit (IatronLab, Inc., Tokyo, Japan). Strains previously serotyped bystandard procedures were employed as controls (29).

Virulence to mice. C. albicans strains were grown in 50 mlof Sabouraud broth on a reciprocal shaker (200 rpm) at 37°Cfor 24 to 48 h. Cells were harvested by centrifugation at 6,000x g for 10 min and washed three times with normal saline(0.9% [wt/vol] NaCl). The inoculum preparation consisted ofcells suspended in saline to an optical density of 0.12 at 600

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TABLE 1. Physiological and morphological characteristics of selected isolates of C. albicans

Virulence' API MorphologybStrain Source Yr isolated Serotype to mice Proteinase profile G C HG-30 Vagina 1977 A 0/10 + 2576170 + + +G-M-44 Clinical 1988 A 0/10 + 6572170 + + +G-9 Feces 1977 A 0/10 + 2560170C + + +G-364 Mouse feces 1987 A 10/10 - 2576170C-655 Skin lesion 1982 A 10/10 + 2572171 + + +C-402 Clinical 1987 A 0/10 + 6776171C + + +C-051 Lung 1987 A 10/10 - 2576171 + + +C-448 Blood 1987 A 6/10 + 2566170 - - +C-B-1071 Mouth Unknown B 0/6 + 2172170 + + +G-1114 Clinical Unknown A ND + 2552150c + - +A-11006d Vagina 1941 B + 2562150 + - +C-B-1034e Skin C ND + 2546050C + - +

a Number of survivors/number inoculated. ND, Not determined.b G, Germ tube; C, chlamydospores; H, hyphae.C Unlisted or uncommon digit for C. albicans.d Type culture of Candida stellatoidea ATCC 11006. See reference 8 for information on virulence in mice.e Previously described as strain J-1012 (23). See reference 23 for the year isolated.

nm (about 2 x 106 cells per ml). CFW White Swiss mice fromCharles River Laboratories (Wilmington, Mass.) were inoc-ulated with 0.1 ml of cell suspensions via the tail vein.Autopsy was performed on mice that died and mice thatwere sacrificed 14 days after inoculation.DNA characteristics. DNA was isolated by the method of

Marmur (13) as modified by Meyer and Phaff (17) and thebase compositions (moles percent guanine plus cytosine)were determined at least twice for each strain by the thermaldenaturation method (14). The DNA relatedness of selectedstrains was obtained by the procedures of Seidler andMandel (28) as modified by Kurtzman et al. (6).

Karyotyping by TAFE. Gel electrophoresis of whole-cellDNA was determined by a modification of the method ofLott et al. (11). Cells grown for 14 to 18 h in 20 ml of 1%(wt/vol) yeast extract-2% (wt/vol) peptone-1% (wt/vol) glu-cose at 30°C were harvested by centrifugation, washedtwice, and suspended at approximately 1:10 (vol/vol) in 0.05M EDTA (pH 7.5, adjusted with NaOH). To 1.0 ml of thiscell suspension, 100 ,ul of Zymolyase 60 T (Miles Laborato-ries, Inc., Naperville, Ill.; 2 mg/ml in Tris-EDTA buffer) and1.0 ml of 1.0% (wt/vol) LE agarose (Beckman Instruments,Inc., Palo Alto, Calif.) in 0.125 M EDTA were added. Thissuspension was vortexed and poured into molds (4 by 9 by 2mm). After the agarose in the molds solidified, the solidifiedagarose plugs were incubated overnight at 37°C in 0.5 MEDTA-0.01 M Tris (pH 7.5) with 7.5% (wt/vol) 2-mercapto-ethanol. The plugs were rinsed with sterile distilled waterand incubated for an additional 12 to 18 h at 50°C in 0.5 MEDTA-1% lauryl sarcosine-0.01 M Tris (pH 9.5) containing2 mg of proteinase K (Sigma Chemical Co., St. Louis, Mo.)per ml. The excess buffer was removed, and the plugs wererinsed with sterile distilled water and were stored at 4°C untilprocessed, which was always within 2 weeks. The agaroseplugs were cut into smaller plugs to fit the size of the wells ingel plates (1% LE agarose in TAFE buffer). The smallerplugs were held in sterile distilled water for about 1 h at roomtemperature before they were loaded into the wells of the gelplates. The wells with plugs were sealed with melted aga-rose, and the gel plates were placed in the vertically posi-tioned track in the chamber of a TAFE system (Beckman)filled with 1 x TAFE buffer. The temperature of the chamberwas held at 10 to 13°C, and the voltage, switch time, andrunning time were varied for the optimal resolution of DNA

bands. Gels were stained with ethidium bromide, destainedin distilled water, and photographed under UV light.The lanes on the negatives (a 1.6-mm width in the center of

the lane, every 20 ,um of migration distance) were scannedwith an Ultrascan XL enhanced laser densitometer (LKBProdukter AB, Bromma, Sweden). The data obtained wereanalyzed with the computer software Gelscan XL (LKB)under the following conditions: peak width for peak search,8; area rejected, 1.0; integration method, signal; and baseline option, common.

RESULTS

Phenotypes. The characterization of the isolates is pre-sented in Table 1. Sucrose-negative C. albicans strains wereconsidered C. albicans var. stellatoidea. This variety isindicated by the API 20C profile that terminates in the digits50. Other variations in profile numbers that indicated differ-ences in assimilation of glycine, xylose, arabinose, xylitol,and methyl-d-glucoside fell within the profile codes forpossible C. albicans. The assimilation and fermentationpatterns (data not shown) of all strains by standard tests (30)were in general agreement with those established for C.albicans. Some reactions that were negative with the API20C system (e.g., xylose assimilation) were positive in theliquid assimilation media. Isolates that readily producedgerm tubes and chlamydospores and expressed virulence formice, in conjunction with compatible physiological charac-teristics (e.g., G-30, G-M-44), were considered classical ortypical isolates of the species. These typical isolates wereproteinase positive, showed tolerance to pH 1.4, were citratepositive, and were resistant to safranin and flucytosine.These properties grouped the typical isolates with two of themore common biotypes in the scheme of Odds and Abbott(20). These characteristics for distinguishing the commonbiotypes were not always reproducible; e.g., distinction of a

positive or negative reaction was arbitrary for the atypicalstrains of C. albicans.

All strains readily expressing proteolysis, except for strainC-655, a hyphal form, were virulent for mice. A repre-sentative of classical C. albicans (G-30) produced in miceintensive tissue invasion and necrosis. The yeast was recov-

ered from the kidney, heart, and spleen and occurred intissues in hyphal and yeast forms. Details of the virulence of

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TABLE 2. G+C content and DNA relatedness of selectedisolates of C. albicans

Paired DNA (molto G+C) % DNArelatedness

G-364 (36) + G-30 (35) .......................................... 100G-655 (36) + G-364 (36) .......................................... 100C-448 (36) + G-30 (35).......................................... 95A-11006 (34) + G-1114 (36) ....................................... 98G-1114 (36) + G-30 (35) .......................................... 91

this strain (G-30, also known as CA 30) for mice werepresented by Cole et al. (3). Isolate G-364 (germ tubenegative), even in conjunction with an adjuvant (gastricmucin), was recoverable from the kidney of only 1 sacrificedmouse out of the 10 examined. Microscopic examination oftissues from this mouse revealed negligible tissue invasion.The recovered yeast isolate showed the same characteristics(i.e., no germ tubes, proteinase negative, etc.) as the inocu-lum.The moles percent G+C of the DNA and DNA reassoci-

ations of selected isolates are given in Table 2. Classical C.albicans G-30 showed significant (>90%) DNA relatednesswith the representative morphologically and physiologicallyatypical strains, including C. albicans var. stellatoidea (G-1114).

Karyotypes. In preliminary studies with C. albicans G-30,the karyotype patterns obtained with pulsed-field electro-phoresis showed differences in the number of DNA bands(and their resolution), varying mainly with alterations in therunning time, voltage, and switching time. Maximal andusually comparable resolution of bands occurred at 72 to 96h with test conditions of 50 mA and a 10-min switch time.Under the conditions of 50 mA and a 10-min switch time for96 h, isolates G-30, G-9, G-M-44, and C-B-1071 demon-strated nearly identical karyotype patterns consisting of fourobvious electrophoretic mobility groups or clusters, indi-cated as A 1,2, B 3, C 4,5, and D 6,7. Two of these strains areshown in Fig. 1, lanes 1 and 3. With conditions recom-mended by the manufacturer (Beckman) of 140 mA and a60-s switch time for 18 h for DNA from Saccharomycescerevisiae (334 pulsed-field electrophoresis certified) and 38mA and a 60-min switch time for 140 h for Schizosaccharo-myces pombe (Y-12796), 13 and 3 bands, respectively, wereproduced. Only 11 and 2 bands, respectively, of DNA fromthese species were obtained with the standard conditions forC. albicans (data not shown).The resolution of DNA bands and comparisons of karyo-

types were facilitated by densitometric tracings. Computeranalysis of scan data, under conditions that recognizedseven bands for G-30, indicated that the broad and diffuse Acluster of C-402 and the D cluster of C-B-1071 were com-posed of two bands (Fig. 1, cluster D of lane 3 and cluster Aof lane 4; Fig. 2, C-B-1071 and C-402). Karyotypes anddensitometer scans of the same strain which were preparedseparately and from different-age cultures were nearly iden-tical.Some atypical strains had distinct karyotypes from those

of the classical group. This distinct karyotype was reproduc-ible. Only six definite bands were resolved for isolate G-364lacking one band in cluster A; probably this is a result ofcomigration of bands A 1,2 (Fig 1, lane 2). Isolate C-448 hadadditional bands in groups B and D (Fig. 3). A small shouldercorresponding in location to band 10 of C-448 was observedin G-30. This slight shoulder was observed for a few otherstrains at the same location, but our standard densitometric

1 2 3 4___-_!l

A t.B

c

FIG. 1. Ethidium bromide-stained gel showing chromosomesizes of C. albicans DNA separated by TAFE at 50 mA with a10-min switch time for 96 h. Lanes: 1, strain G-30; 2, G-364; 3,C-B-1071; 4, C-402.

conditions did not recognize it as a peak except for C-448,which was the only strain where band 10 was detectable onphotographs of gels. The karyotype of C. albicans C-655(hyphal form) was unique under a variety of test conditionsbecause it gave a maximum of only five bands (Fig. 4).Three serotypes of C. albicans var. stellatoidea (G-1114,

serotype A; A-11006, serotype B; and C-B-1034, serotype C)produced three different electrophoretic karyotype patterns(Fig. 5). Photographs of the karyotyping gel of strain G-1114appeared to have only a single band at cluster A. However,with densitometric analysis the karyotype of G-1114 wassimilar to that of classical C. albicans (G-30) (Fig. 5).

0 10 20 30 40 50 60 70MIGRATION DISTANCE (mm)

FIG. 2. Densitometric tracings of chromosome-size DNA bandsseparated by TAFE at 50 mA with a 10-min switch time for 96 h.

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0 10 20 30 40 50MIGRATION DISTANCE (mm)

60 70

FIG. 3. Densitometric tracings of chromosome-size DNA bandsseparated by TAFE at 50 mA with 10-min switch time for 96 h.

DISCUSSION

The karyotypes of strains of C. albicans determined byTAFE varied with changes in electrophoretic conditionsand, in some instances, between runs with the same condi-tions (gel-to-gel variations). A control DNA of a classical C.albicans (G-30) therefore was included in each gel. Densi-tometric tracings of gels faciitated the detection of bands notreadily observed on photographs of gels, the discernment ofbroad or diffuse bands in the gels, and the comparison ofkaryotypes obtained in different runs. Classical isolates of C.albicans produced a distinctive karyotype of 7 DNA bandsin four mobility groups, whereas some atypical strainsproduced different patterns of 5 bands (C-655, hyphal form),9 or 10 bands (C-448), or 6 bands (G-364, avirulent form).Though the karyotypes of each of the three representativesof C. albicans var. stellatoidea were distinct, C. albicans

A

BG-30 1

12 34

C-655 2

0 10 20 30 40 0 60 70

20 30 40 50MIGRATION DISTANCE (mm)

FIG. 5. Densitometric tracings of chromosome-size DNA bandsseparated by TAFE at 50 mA with a 10-min switch time for 96 h.

and C. albicans var. stellatoidea demonstrated significantDNA relatedness, supporting their conspecificity as notedby Meyer (16). Kwon-Chung et al. (7) suggested that type IIC. stellatoidea was a sucrose-negative mutant of C. albicansserotype A. Probably, type I C. stellatoidea strains ofKwon-Chung et al. (7) (represented herein by A-11006) arederived from C. albicans serotype B.

In comparison of DNA of S. cerevisiae with moleculesthat may be within a range of 260 to 2,200 kilobases (18, 24)

c

0 10 20 30 40 50 60 70

DG-30 2345

10 20 30 40 50 60 70

MIGRATION DISTANCE (mm)

FIG. 4. Densitometric tracings of chromosome-size DNA bands separated by TAFE as follows: (A) 60 mA, 5-min switch time, 72 h; (B)50 mA, 5-min switch time, 96 h; (C) 50 mA, 10-min switch time, 96 h; (D) 38 mA, 60-min switch time, 140 h.

G-30 3

C-655 2

. ..34

C-655 4i 3

2

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880 MAHROUS ET AL.

and Schizosaccharomyces pombe with molecules of 3,000 to9,000 kilobases (4), we estimated that chromosome-sizeDNA molecules of classical C. albicans range from about1,000 to 5,000 kilobases. Perfect et al. (24) with a contour-clamped homogeneous-field gel electrophoresis procedureestimated that chromosome-size DNA molecules of C. albi-cans range from 900 to greater than 2,200 kilobases. How-ever, for reasons cited in the paragraph below, particularlyconformational influences, direct size comparisons of DNAbands from different species may be somewhat inaccurate.

C. albicans, which has not demonstrated a sexual cycle,has been reported to be diploid with strains showing possibleaneuploidy (2n + n) (32). Magee et al. (12) reported thathomologous chromosomes may migrate separately, possiblybecause of deletions, translocations, or differences in theamount or distribution of repetitive DNA. Chromosomes ofnearly the same molecular weight may comigrate as oneband in a gel (15). Conversely, similarly sized chromosomescould differ in configuration and the stereochemical state oftheir DNA and therefore migrate differently (19). Some ofour data suggest that large DNA molecules or complexedDNA molecules in the wells may fail to enter the gel. Thiswas possibly the case with C. albicans C-655. Also, theDNA bands in our system did not migrate at the same ratethroughout the entire gel distance. For example, the firstgroup of bands entering the gel (the D cluster) in classical C.albicans contained three closely packed bands at 150 mAwith a 90-s switch time for 24 h; these bands converged in thegel to two bands by 96 h. Therefore the number of DNAbands in our TAFE system (as noted by others with differentsystems) does not necessarily represent the number ofchromosomes.The current status of gel electrophoretic karyotyping of C.

albicans involves varied procedures that provide DNA bandpatterns of apparent value in biotyping, but the patterns are

not comparable between laboratories. To our knowledge thisis the first presentation of karyotypes for C. albicans with a

TAFE procedure. It permitted distinction of strain typesamong phenotypically diverse isolates of C. albicans whoseidentification had been verified by molecular procedures.Further studies with establishment of karyotype data banksand refinement of procedures for additional species are

required before karyotyping can be employed in practicalepidemiological studies of unknown yeast species.

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