Abstract: Six strains of Candida albicans were subjected to pulsed-field gel electrophoresis (PFGE) using the CHEF-DRIII system (BioRad). Hansenula mingei YB-4662-VIA and Saccharomyces cerevisiae YNN 295 (BioRad) were used as size markers (1.05-3.13 and 0.22-2.2 megabase pairs [Mbp] respectively) for comparison of DNA molecules. The DNAs were resolved bv a three-block protocol with pulse times of 120 s for 24 h, 240 s for 36 h and 300 s for 17 h. The voltage was set at 4.; V/cm for the first two blocks and 4.0 V/cm for the final block. PFGE was carried out under these conditions using different agarose concentrations, types and concentrations of buffer, temperatures, and sizes of agarose gel plug. The resolution and mobility of DNAs were affected by some of these variables. Separation of C. albicans by PFGE was optimal at 12 deg C with 1.0 x Tris-borate-EDTA (TBE) buffer using 1.2% agarose. Resolution of banding patterns was dependent on size of DNA plug used.
Key words: Candid albicans. Electrophoresis, gel, pulsed-field. Mycological typing techniques.
Introduction
There has been increasing interest in the epidemiological study of Candida albicans in human immunodeficiency virus (HIV)-infected individuals1,2 and in bone-marrow transplant recipients3 5 in whom extensive use of prophylactic antibiotics has resulted in the increased incidence of yeast infections. In spite of its importance as an infectious agent in progressive immunodeficiency and HIV infections, very little is known of the epidemiology of C. albicans in this context.
In recent years, molecular typing systems, such as pulsed-field gel electrophoresis (PFGE), have been used successfully to evaluate strain relatedness by analysing the very large chromosomal DNA found in yeasts.6-8 Contour-clamped homogeneous electric field (CHEF) electrophoresis is a modified version of PFGE, and is reported to be more sensitive than restriction enzyme analysis for typing of Candida species.9,10 Several studies have demonstrated the advantage of CHEF in typing C. albicans.1,9-12 DNA molecules resolved by PFGE range in size from <10 kilobase pairs (Kbp) to intact chromosomal DNAs of a few megabase pairs (Mbp).13,14 The delineation of these large DNA molecules is often constricted by a number of experimental variables. These variables are critical to the consistency and reproducibility of CHEF banding patterns, and have not been adequately studied.
The aim of this study is to assess the effects of different experimental conditions on electrophoretic patterns. The variables studied are: electrophoretic parameters; buffer types and concentrations; agarose concentrations; temperature; proteinase K digestion time of spheroplasts; and sizes of DNA plugs.
Materials and methods
Yeast strains
Six strains of C. albicans (I-VI) were isolated from patients who had received a bone marrow transplant. These strains were identified as C. albicans by germ tube formation in human serum after 3 h of incubation at 37 deg C and confirmed by the yeast API-20 cAux (bioMerieux, Basingstoke, UK). Isolates were stored in Sabouraud's dextrose agar slopes (Oxoid, Basingstoke, UK) at 4 deg C until studies were performed.
Yeast DNA insert preparation
A single colony of C. albicans in Sabouraud's dextrose agar (Oxoid) was inoculated into 25 mL YEPD broth (1% [w/v] yeast extract [Oxoid], 1% [w/v] peptone bacteriological [Oxoid] and 2% [w/v] dextrose) and incubated overnight at 37 deg C in a water bath with agitation. Yeast cells were then harvested, washed (x3) with 50 mmol/L EDTA (pH 7.5) and finally suspended in 5 mL 50 mmol/L EDTA (pH 7.5). The concentration of yeast cells was determined by using a haemocytometer, and 6 x 10^sup 8^ cells were resuspended in 315 mu L 50 mmol/L EDTA (pH 7.5). Prior to mixing the cells with agarose, 15 tL lyticase (2.5 mg/ mL) (Sigma, Poole, UK) was added and the mixture incubated at 37 deg C for 1 h. After equilibrating at 50 deg C, 0.63 mL of the cell suspension was mixed with 0.37 mL 2% [w/v] InCert agarose (FMC Bioproducts, Rockland, USA) to give an agarose plug with a final concentration of 0.75% [w/v]. The mixture was transferred into a disposable plug mould (10.0 x 5.0 x 1.5 mm) (BioRad, Hercules, USA) and allowed to solidify at 4 deg C for 30 min. The agarose plugs were transferred to a polystyrene container and incubated overnight at 30 deg C in 2.5 mL 50 mmol/L EDTA, 10 mmol/L Tris-HCI (pH 7.5). The plugs were then washed (x3) in 0.5 mol/L EDTA (pH 9.5). Cell lysis was completed by adding 2 mL buffer (10 mmol/L Tris-HCI [pH 7.5], 0.5 mol/L EDTA [pH 8.0], 1% lauryl sarcosine) containing 30 U proteinase K (Sigma). Digestion was carried out at 50 deg C for either 24 h or 48 h. The plugs were then rinsed (x3) with 50 mmol/L EDTA (pH 7.5).
Determination of electrophoretic parameters
Electrophoresis was conducted in a CHEF-DRIII system. Portions of the prepared agarose plugs containing whole yeast cell DNA were loaded onto a 15lane, 140 x 200 mm horizontal agarose gel in 1xTBE buffer (89 mmol/L Tris, 10 mmol/L boric acid, 2.5 mmol/L sodium EDTA [pH 8.0]). The DNA samples were then resolved by CHEF-DRIII, using different combinations of voltage, pulse time and running time (Table 1). The combination which gave the best resolution of DNA bands was used to assess the remaining variables. Gels were stained with ethidium bromide (1 mu g/mL) for 30 min and were then destained in distilled water for 30 min. DNA bands were visualised by ultraviolet (UV) transillumination.
Effect of various experimental conditions on DNA resolution
To study different experimental conditions, including buffer types and concentrations, agarose concentration, temperature, proteinase K digestion time and DNA plug size, PFGE was performed using the experimental variables listed in Table 2. However, only electrophoresis protocol B was used in this part of the study (Table 1).
DNA size marker
Chromosomes of Hansenula mingei YB-4662-VIA and Saccharomyces cerevisiae YNN 295 were used as size markers for comparison with C. albicans.
Results
Electrophoretic parameters
Various parameters were modified continually to achieve optimal resolution and sharpness of DNA bands. Two sets of parameters (protocols A and B) were found to give the best resolution and sharpness (Table 1).
The resolution and sharpness of DNA bands achieved with the two protocols were very similar (Fig. lA and 1B), and no extra bands were seen among the six isolates. Protocol B was used throughout the remaining experiments under different conditions because the extended time allowed easy identification of DNA bands that were close to each other.
Different buffers and concentrations
The PFGE patterns of C. albicans using TBE and TAE (40 mmol/L Tris-acetate, 1 mmol/L EDTA pH 8.0) buffers at 0.5 x and 1 x concentration are shown in Fig. 2. Comparison of the two buffers at 0.5 x concentration (Fig. 2B and 2C) showed better separation of the DNA bands in TAE than in TBE. In addition, the high molecular weight DNA bands (>2.2 Mbp) were more prominent using TBE than TAE. Comparison of the two TBE concentrations (Fig. 2A and 2B) showed that 1 x TBE produced sharper DNA bands, although the migration rate was much slower. We were unable to compare TAE concentrations because the CHEF-DRIII failed to operate when 1 x TAE buffer was used. This was probably due to fluctuation in current flow caused by the high concentration of buffer.
Effect of agarose concentration
The PFGE patterns of C. albicans DNA molecules subjected to agarose concentrations of 1.0, 1.2 and 1.5% (w/v) are shown in Fig. 3. In 1% agarose (Fig. 3A), larger DNA molecules (>2.2 Mbp) were diffused and smeared, however, smaller DNA molecules (1.01.81 Mbp) migrated further than was seen in 1.2% or 1.5% agarose. In 1.2% agarose (Fig. 3B), both large and small DNA molecules gave better resolution than was seen in 1.5% agarose (Fig. 3C), and some distinct separation of the larger molecules (>2.2 Mbp) seen in 1.2% agarose appeared to be compressed or smudgy in 1% or 1.5% agarose.
Effect of temperature
PFGE was carried out at either 12 deg C or 14 deg C, and the mobility of the C. albicans DNA molecules was similar at both temperatures. However, the smaller DNA molecules appeared to migrate at a slightly faster pace at 14 deg C, and thicker bands and sharper resolution were observed at 12 deg C (Fig. 4A). Proteinase K digestion
Preparation of DNA plug inserts is critical to the success of electrophoretic karyotyping of C. albicans, and most preparation is based on a procedure described by Schwartz and Cantor.6 Incomplete digestion of spheroplasts failed to produce acceptable DNA bands in the PFGE. We digested the spheroplasts of C. albicans for 24 h (Fig. 5A) and 48 h (Fig. 5B); the results did not show any distinct differences.
Different C. albicans DNA plug size
No apparent difference in DNA-molecule mobility was seen in the three different plug sizes (0.5 x 5.0 x 1.5 mm, 1.0 x 5.0 xl.5 mm) cut from the same piece of DNA insert preparation (Fig. 6). However, the resolution of smaller molecules (<1.02 Mbp), as seen with S. cerevisiae YNN 295, appeared to be more distinct and brighter as plug size increased ( x 2, x 4). In the case of larger DNA molecules (>1.05 Mbp), as seen in samples I and II (Fig. 6), intensity of DNA bands increased as plug sizes increased. Sharpness of bands decreased when the plug size was increased to 2.0 x 5.0 x 1.5 mm and smearing of the DNA bands appeared.
Discussion
Optimisation of methods for karotyping C. albicans DNA is a complex and time-consuming procedure, and a variety of parameters can influence PFGE performance. We examined a number of these parameters systematically, in order to understand the separation process and to reduce the number of variables so that optimum running conditions could be achieved. Comparison of the selected variables (Table 2) was performed at least twice on separate occasions, and the results were found to be consistent and reproducible.
We developed a 68-h two-block protocol and a 77-h three-block protocol for karotyping C. albicans DNA using CHEF-DRIII. Both provided excellent resolution and discrete DNA bands; however, the threeblock protocol was preferred for separation of DNA bands in close proximity. Variation in the number of DNA bands has been reported widely in C. albicans, and may be due to the use of different typing systems by various investigators.7,11,15-19 Technically, it is not feasible to delineate DNA molecules >2.2 Mbp and <1.6 Mbp simultaneously, without running separate systems using different parameters to suit the movement of individual DNA molecules.'" However, using the methods reported here, we observed good separation between DNA molecules >2.2 Mbp, and those <1.02 Mbp.
Different investigators have used different types of buffer of different concentration. Under normal electrophoretic conditions, ionic concentrations in the buffer are closely related to electric field strength and pulse time, and the mobility of DNA molecules is directly related to both of these parameters.13 In our study, the mobility of DNA molecules increased, with decreased resolution, at lower buffer concentrations. The reduction in ionic concentration of TBE buffer could have affected the electric field strength, which in turn affected the speed of DNA re-orientation and caused the DNA molecules to migrate faster. Comparison of the two TBE concentrations (Fig. 2A and 2B) showed that Ix TBE produced sharper bands, although the migration rate was much slower. At the lower concentration (x0.5), TAE produced better resolution and less hazy DNA bands than did TBE. Overall, this demonstrated that different buffers at different concentrations do affect the migration and resolution of DNA bands. Generally, a faster migration rate was accompanied by a decrease in band sharpness, and lx TBE offered better resolution than that obtained with 0.5x TBE and 0.5x TAE.
Mobility of DNA molecules is dependent on the maximum matrix pore size of agarose, and this decreases progressively with increasing agarose concentration." In our study, this effect was observed when the agarose concentration was increased from 1% to 1.5% (Fig. 3), and a 1.2% concentration proved optimal for PFGE separation. The mobility of DNA molecules in PFGE is also dependent on buffer temperature.'2 As investigation into the effect of temperature on mobility was carried out over a very narrow range, the reported observation that DNA mobility increases with increasing temperature could not be demonstrated.12 Regarding spheroplast preparation, sharpness of DNA bands was not improved when proteinase K digestion time was increased from 24 h to 48 h; however, the effect of different lytic enzymes and solubilising agents needs further investigation.
Considerable emphasis has been placed on the standardisation of cell count,1,8,20 but little attention paid to the size of DNA plug inserted into the well of the agarose gel. We found that small plug size (0.5 x 5.0 x 1.5 mm) gave better resolution of larger DNA molecules (>1.05 Mbp), and larger plug size (2.0 x 5.0 x 1.5 mm) gave better resolution of smaller DNA molecules (<1.02 Mbp).
In summary, based on the three-block protocol, this study showed that the performance of CHEF-DRIII for C. albicans karotyping was both consistent and reproducible when operated at 12C using l.Ox TBE buffer and 1.2% agarose, with the choice of DNA agarose plug size dependent on DNA molecule size.
This study was supported by a departmental research grant from the Department of Nursing and Health Sciences, The Hong Kong Polytechnic University.
[Reference]
References
[Reference]
Lupetti A, Guzzi G, Paladini A, Swart K, Campa M, Senesi S. Molecular typing of Candida albicans in oral candidiasis: karotype epidemiology with human immunodeficiency virus
[Reference]
seropositive patients in comparison with that with healthy carriers. J Clin Microbiol 1995; 33:1238-2. Powderly WG, Robinson K, Keath EJ. Molecular typing of Candida albicans isolated from oral lesions of HIV-infected individuals. AIDS 1992; 6:814.
Barnes RA, Rogers TR. Response rates to a staged antibiotic regimen in febrile neutropenic patients. J Antimicrob Chemother 1988; 22:759-63.
[Reference]
Hoppe JE, Klausner lM, Klingebiel T, Niethammer D. Retrospective analysis of yeast colonization and infections in paediatric bone marrow transplant recipients. lycoses 1997; 40:47-54. van Belkum A, Mol W, van Saene R, Ball LM, van Velzen D, Quint W. PCR-mediated genotyping of Candida albicans strains from bone marrow transplant patients. Bone Marrow Transplant 1994; 13:811-15.
Schwartz DC, Cantor CR. Separation of yeast chromosomesized DNAs by pulsed-field gradient gel electrophoresis. Cell 1984: 37:67-75.
[Reference]
7 Magee BB, Magee PT. Electrophoretic karyotypes and chromosome numbers in Candida species. J Gen Microbiol 1987; 133:425-30.
8 Vazquez JA, Beckley A, Donabedian S, Sobel JD, Zervos M. Comparison of restriction enzyme analysis versus pulsed-field gradient gel electrophoresis as a typing system for Torulopsis glabrata and Candida species other than C. albicans. J Clin AMicrobiol 1993; 31:2021-30.
9 Sangeorzan JA, Zervos MJ, Donabedian S, Kauffman CA. Validity of contour-clamped homogeneous electric field electrophoresis as a typing system for Candida albicans. Mycoses 1995; 38:29-36.
10 Khattak MN, Burnie JP, Matthews RC, Oppenheim BA. Clamped homogeneous electric field gel electrophoresis typing
[Reference]
of Torulopsis glabrata isolates causing nosocomial infections. J Clin Microbiol 1992; 30:2211-15.
12 Mathew MK, Smith CL, Cantor CR. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature. Biochemistry 1988; 27:9204-10. 13 Mathew MK, Smith CL, Cantor CR. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 2. Effect of pulse time and electric field strength and implications for models of the separation process. Biochemistry 1988; 27:921016.
[Reference]
14 Lott TJ, Boiron P, Reiss E. An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples. Mol Gen Genet 1987; 209:1704.
16 Mahrous M, Lott TJ, Meyer SA, Sawant A, Ahearn DG. Electrophoretic karyotyping of typical and atypical Candida albicans. a Clin Microbiol 1990; 28:87681. 17 Merz WG, Connelly C, Hieter P. Variation of electrophoretic karyotypes among clinical isolates of Candida albicans. J Clin Microbiol 1988: 26:842-R
[Reference]
18 Wickes B, Staudinger J, Magee BB, Kwon-Chung KJ, Magee PT, Scherer S. Physical and genetic mapping of Candida albicans: several genes previously assigned to chromosome 1 map to chromosome R, the rDNA-containing linkage group. Infect Immun 1991; 59:24804.
19 Odds FC, Brawner DL, Staudinger J, Magee PT, Soil DR. Typing of Candida albicans strains. (Review). Z Med Met Mycol 1992; 30 Suppl 1:87-94.
20 Serwer P, Hayes SJ. Exclusion of spheres by agarose gels during agarose gel electrophoresis: dependence on the sphere's radius and the gel's concentration. Anal Biochem 1986; 158:72-8.
[Author Affiliation]
EDWIN HONG* and POLLY LEUNG
Department of Nursing and Health Sciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, People's Republic of China
[Author Affiliation]
(Accepted 28 July 1998)
