Vol. 29, No. 7

JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1364-1367

0095-1137/91/071364-04$02.00/0 Copyright © 1991, American Society for Microbiology

Restriction Fragment Length Polymorphism Analysis of AzoleResistant and Azole-Susceptible Candida albicans Strains MICHELLE A. PEARCE AND SUSAN A. HOWELL* Institute of Dermatology, St. Thomas' Hospital, Lambeth Palace Road, London SE] 7EH, United Kingdom Received 1 October 1990/Accepted 9 April 1991

Restriction fragment length polymorphism analysis was performed with the endonucleases EcoRI, Bglll, and HinfI on a collection of Candida albicans strains comprising eight strains randomly selected from clinical microbiology laboratory specimens, three reported azole-resistant strains from treatment failures, and several subcultures of the azole-resistant strain NCPF 3310 (also known as the Darlington strain) received from different laboratories. The results demonstrated a diversity of the restriction fragment length polymorphism patterns that were obtained and revealed that two of the proposed Darlington subcultures had patterns distinct from each other and from those of the other Darlington isolates; both were also found to have lost their azole resistance. on Sabouraud dextrose agar (Oxoid, Basingstoke, United Kingdom). Yeast cells were harvested by centrifugation at 2,000 x g for 5 min from an overnight suspension of cells grown in YEPD broth (yeast extract [0.3%], mycological peptone [1%], and glucose [2%]; Oxoid) at 37°C on an orbital incubator. The DNA was extracted by the method of Durkacz et al. (2), with modifications to the procedure for the removal of proteins. Briefly, this involved the production of spheroplasts by incubation with Zymolyase 20T (ICN Biomedicals, High Wycombe, United Kingdom) followed by lysis with 10% sodium dodecyl sulfate. After the addition of potassium acetate and centrifugation, DNA was precipitated with ice-cold isopropanol. The DNA was collected by centrifugation at 8,000 x g for 10 min, dried, dissolved in 1 ml of 5 x TE buffer (50 mM Tris, 5 mM EDTA), and incubated at 37°C for 60 min with 100 ,ul of RNase A (5 mg/ml; Sigma, Poole, United Kingdom) and then for 120 min with proteinase K (2 mg/ml; Sigma). The DNA was precipitated overnight at -20°C with 2 volumes of 95% ethanol, collected by centrifugation at 1,000 x g for 10 min, redissolved in 1 x TE buffer (10 mM Tris, 1 mM EDTA), and frozen until required. Cesium chloride density gradient centrifugation. The DNA extract was purified by CsCl density gradient centrifugation on an MSE Europa 65M ultracentrifuge at 120,000 x g for 44 h at 20°C. The gradient contained a 1:1 (gram per milliliter) ratio of DNA solution to cesium chloride and 2 mg of ethidium bromide per ml. The ethidium bromide was removed from the purified DNA by partitioning with n-butanol and dialysis overnight against 1 x TE buffer. The DNA was precipitated overnight with absolute ethanol, pelleted by centrifugation in an MSE Microcentaur at high speed for 10 min, and dried by desiccation. Endonuclease restriction and electrophoresis. The purified DNA was dissolved in distilled water, and a 25-,il aliquot was digested with 4 ,l of restriction endonuclease (GIBCOBRL, Uxbridge, United Kingdom) and 3 ,ul of core buffer for 3 h at 37°C. The reaction was stopped by heating the solution to 65°C for 10 min and the subsequent addition of 10 RI of glycerol blue. Electrophoresis was performed on a 0.8% agarose gel and with TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 30 V for 18 h. The gel was stained with ethidium bromide, and the DNA bands were visualized under long-wave UV light.

Previous epidemiological study of Candida albicans has relied upon various phenotypic techniques such as morphotyping (10), resistotyping (13), and biotyping (9). More recently, genetic analyses by restriction fragment length polymorphisms (RFLPs) have been applied (8), and several DNA probes have been developed for Southern blotting to endonuclease-digested DNA (7, 14). C. albicans NCPF 3310 (also known as the Darlington strain), which was isolated from a patient with chronic mucocutaneous candidiasis, was the first clinically resistant isolate reported in the United Kingdom (12). Since then, subcultures of this strain have been passed between several laboratories in attempts to identify its mechanism of azole resistance, which remains to be fully understood (3, 4). An azole-susceptible variant of this strain (3310S) was found following repeated subculture (6), and subsequently, its sterol composition was shown to be different from those of several of the other Darlington strain subcultures (5). The identity of this susceptible variant as a Darlington subculture was determined by RFLP analyses, and the results were compared with those for other clinically resistant and susceptible C. albicans organisms isolated from patients with candidiasis. MATERIALS AND METHODS Strains. The sources of the strains used in this study have been described in detail elsewhere (5). They consist of a collection of azole-resistant strains (strains NCPF 3303, NCPF 3310, NCPF 3342, and NCPF 3363, isolated from patients with chronic mucocutaneous candidiasis who failed treatment) that have been deposited at the Mycological Reference Laboratory, Colindale, London, United Kingdom; NCPF 3153, which is a standard reference strain; randomly selected strains from clinical microbiology laboratory specimens, strains Cl through C10; and a collection of seven isolates (isolates B34226, B35972, B44726, Darlington, NCPF 3310, 3310R, and 3310S) representing subcultures of the Darlington strain, all of which had been passed to various laboratories over a period of 8 years. Growth and DNA extraction. All cultures were maintained *

Corresponding author. 1364

VOL. 29, 1991

RFLPs OF AZOLE-RESISTANT AND -SUSCEPTIBLE C. ALBICANS 1

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RESULTS AND DISCUSSION Endonuclease digestion with EcoRI of DNA from all of the C. albicans strains (Fig. 1) produced an identical and characteristic brightly stained banding pattern with molecular masses of 11.2, 9.4, 7.7, 5.6, and 4.4 kb. This closely corresponds to the molecular mass of the circular mitochondrial genome of 41 kb described by Wills et al. (14). Because of the heavy background of digested nuclear DNA, few differences between the strains could be readily detected. Table 1 shows some of the similarities and differences in the fragment sizes of the RFLP patterns produced by restriction endonucleases.

FIG. 2. (A) BgIII-digested whole-cell DNA of some clinical strains. Lanes: 1, HindIII-digested lambda DNA; 2, Cl; 3, C4; 4, C7; 5, C9; 6, C8. -(B) BgII digestion of azole-resistant strains. Lanes: 1, HindIII-digested lambda DNA; 2, Darlington; 3, NCPF 3303; 4, NCPF 3346; 5 and 6; NCPF 3363.

Clearer and less ambiguous RFLPs were observed on restriction with the endonuclease BglII, which made the differentiation of some of the clinical strains easier (Fig. 2A), but it failed to distinguish strain NCPF 3303 (Fig. 2B, lane 3) from strain NCPF 3346 (Fig. 2B, lane 4) or the Darlington strain from strain C3, C5, or C10 (data not shown). In Fig. 3 a common BglII banding pattern was obtained for the Darlington isolates B34226, B35972, Darlington, NCPF 3310, and 3310R, hence, differentiating these Darlington strain subcultures from strain 3310S (Fig. 3, lane 7), which

TABLE 1. Major differentiating DNA restriction fragments following digestion with EcoRI, BglIl, or Hinfl Position (kb) of the following differentiating DNA restriction fragments':

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4.1, 5.6, 4.1, 4.3, 5.0,

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2.7, 2.0, 2.0, 1.9, 2.0, 2.6, 2.6, 1.9, 2.0, 2.0, 3.0, 2.0,

1.7, 1.4 (19) 1.9, 1.7 (4) 1.9, 1.8, 1.7, 1.4 (3) 1.7, 1.4, 1.3 (1) 1.4 (1) ND 2.3, 1.4, 1.3 (1) 1.9, 1.4 (3) 1.9, 1.4 (4) 1.7, 1.4 (3) 1.9, 1.7, 1.4 (1) 2.0, 1.9, 1.7, 1.4 (4) 1.7, 1.4 (1) 1.7 (3) 1.7, 1.4 (4) 1.9, 1.4 (3) 2.0, 1.4 (5) 1.9, 1.7, 1.4 (1)

1.9, 2.0, 2.0, 2.0, 1.7,

a The number of repeated analyses for each strain is indicated in parentheses. ND, not done. b All strains tested had bands at 11.2, 9.4, 7.7, 5.8, 5.6, and 4.4 kb. C All strains tested had bands at 12.1, 6.2, 2.5, and 1.2 kb and, with the exception of NCPF 3346, bands at 2.4 and 1.6 kb. d All strains tested had bands at 1.9 and 1.7 kb. ' The Darlington isolates Darlington, 3310R, NCPF 3310, B34226, and B35972 had the same patterns. f-, no additional band seen.

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_ _ FIG. 3. BglII digests of the seven isolates representing subcultures of the Darlington strain. Lanes: 1, HindIll-digested lambda DNA; 2, B34226; 3, B35972; 4, B44726; 5, Darlington; 6, NCPF 3310; 7, 3310S; 8, 3310R; 9 (unnumbered), HindIll-digested lambda DNA.

had a band at 5.4 kb, and strain B44726 (Fig. 3, lane 4), which had two bands of 2.3 and 1.8 kb. The strain 3310S subculture was isolated as an azolesusceptible variant of strain NCPF 3310 and in this study was found to have remained azole susceptible (1), whereas the other Darlington isolates, with the exception of B44726, remained azole resistant. The BglII restriction pattern of strain 3310S contained an additional band of 5.6 kb but lacked the bands of 4.1 and 1.4 kb present in the other Darlington isolates (Fig. 3), suggesting an altered base pair composition that resulted in the loss of a BgIlI restriction site. However, restriction with Hinfl, an enzyme shown recently by Smith et al. (11) to be useful for the RFLP analysis of C. albicans, demonstrated an extra bright band of 2.4 kb in the RFLP pattern of strain 3310S (Fig. 4, lane 7) that was faint or absent from the digests of the other Darlington isolates and faint bands at 2.3 and 2.2 kb that were very bright in the other RFLP patterns. There were several differences in the restriction patterns obtained with the different endonucleases between strain 3310S and the other Darlington isolates that could not be explained by a simple mutation event. Strain B44726 was thought to be an authentic subculture of the Darlington strain, and by using EcoRI it had an RFLP pattern identical to those of the other Darlington isolates. However, digestion of B44726 with BglII produced two extra bands of 2.3 and 1.8 kb that were absent from the RFLPs of the other subcultures (Fig. 3, lane 4), and digestion with Hinfl (Fig. 4, lane 4) showed a difference in the 4-kb region. Strain B44726 was found to be susceptible to ketoconazole (1). Although B44726 had a sterol composition similar to that of 3310S (5) and both were sensitive to ketoconazole, results of the RFLP analysis suggest that B44726 mutates in a manner different from that of 3310S. The RFLP patterns produced by EcoRI digestion were difficult to interpret because of the high background of nuclear DNA. The endonucleases BglII and Hinfl produced less background and made easier the differentiation of

FIG. 4. Hinfl digests of the seven Darlington isolates. Lanes: 1, HindlIl-digested lambda DNA; 2, B34226; 3, B35972; 4, B44726; 5, Darlington; 6, NCPF 3310; 7, 3310S; 8, 3310R.

strains. The Darlington isolates were easily distinguished from the other azole-resistant strains when the endonuclease BglII was used. The identities of the two strains 3310S and B44726 as subcultures of the Darlington strain were shown to be in doubt, because their RFLP patterns produced by BgIII and Hinfl differed from those of the subcultures of the five other Darlington isolates. The RFLP patterns of these two isolates did not correspond to any of the azole-resistant treatment failure strains deposited at the Mycological Reference Laboratory, and so the origins of these isolates are uncertain. ACKNOWLEDGMENTS We are indebted to all those who donated strains. We thank Y. M. Clayton for performing the MIC determinations, and we are grateful for the financial support for this project provided by the Dunhill Medical Trust. REFERENCES 1. Clayton, Y. M. Personal communication. 2. Durkacz, B., D. Beach, D. Hayles, and P. Nurse. 1985. The fission yeast cell cycle gene cdc2: structure of the cdc2 region. Mol. Gen. Genet. 201:543-545. 3. Hitchcock, C. A., K. J. Barrett-Bee, and N. J. Russell. 1986. The lipid composition of azole-sensitive and azole-resistant strains of Candida albicans. J. Gen. Microbiol. 132:2421-2431. 4. Hitchcock, C. A., K. J. Barrett-Bee, and N. J. Russell. 1987. Inhibition of 14a-sterol demethylase activity does not correlate with resistance to azoles. J. Med. Vet. Mycol. 25:329-333. 5. Howell, S. A., A. I. Mallet, and W. C. Noble. 1990. A comparison of the sterol content of the Candida albicans Darlington strain with other clinically azole resistant and azole sensitive strains. J. Appl. Bacteriol. 69:692-696. 6. Johnson, E. M. 1986. Ph.D. thesis. University of Bristol, Bristol, United Kingdom. 7. Magee, B. B., T. M. D'Souza, and P. T. Magee. 1987. Strain and species identification by restriction fragment length polymorphisms in the ribosomal repeat of Candida species. J. Bacteriol.

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169:1639-1643. 8. Matthews, R., and J. Burnie. 1989. Assessment of DNA fingerprinting for rapid identification of outbreaks of systemic candidiasis. Br. Med. J. 298:354-357. 9. Odds, F. C., C. E. Webster, P. G. Fisk, V. C. Riley, P. Mayuranathan, and P. D. Simmons. 1989. Candida species and C. albicans biotypes in women attending clinics in genitourinary medicine. J. Med. Microbiol. 29:51-54. 10. Phongpaichit, S., D. W. R. Mackenzie, and C. Fraser. 1987. Strain differentiation of Candida albicans by morphotyping. Epidemiol. Infect. 99:421-428. 11. Smith, R. A., C. A. Hitchcock, E. G. V. Evans, C. J. N. Lacey, and D. J. Adams. 1989. The identification of Candida albicans

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strains by restriction fragment length polymorphism analysis of DNA. J. Med. Vet. Mycol. 27:431-434. 12. Warnock, D. W., E. M. Johnson, M. D. Richardson, and C. F. H. Vickers. 1983. Modified response to ketoconazole of Candida albicans from a treatment failure. Lancet i:642-643. 13. Warnock, D. W., D. C. E. Speller, J. D. Milne, A. L. Hilton, and P. I. Kershaw. 1979. Epidemiological investigation of patients with vulvovaginal candidosis. Application of a resistogram method for strain differentiation of Candida albicans. Br. J. Vener. Dis. 55:357-361. 14. Wills, J. W., B. A. Lasker, K. Sirotkin, and W. S. Riggsby. 1984. Repetitive DNA of Candida albicans: nuclear and mitochondrial components. J. Bacteriol. 157:918-924.

Restriction fragment length polymorphism analysis of azole-resistant and azole-susceptible Candida albicans strains.

Restriction fragment length polymorphism analysis was performed with the endonucleases EcoRI, BglII, and HinfI on a collection of Candida albicans str...
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