Medical Mycology 2001, 39, 185–193
Accepted 29 August 2000
PCR ngerprinting: a convenient molecular tool to distinguish between Candida dubliniensis and Candida albicans W. MEYER, K. MASZEWSKA & T. C. SORRELL Centre for Infectious Diseases and Microbiology, Molecular Mycology Laboratory, The University of Sydney at Westmead Hospital, Sydney, Australia Candida dubliniensis was recently identied as a germ-tube- and chlamydosporepositive 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 signicant problems in the correct identication of C. dubliniensis in a standard clinical mycology laboratory. Polymerase chain reaction (PCR) ngerprinting was successfully applied here to distinguish between clinical isolates of the two closely related species. Microsatellite ([GACA]4 ) and minisatellite ([50 -GAGGGTGGCGGTTCT-30 ], derived from the core-sequence of the wild-type phage M13) specic 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 identied by a distinct species-specic multilocus pattern, allowing identication to species level for all clinical isolates. In addition, the PCR ngerprinting generated strainspecic proles, making this method applicable to epidemiological investigations. PCR ngerprinting 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. Keywords printing
Candida albicans, Candida dubliniensis, identication, PCR nger-
Introduction Since its rst description in 1995 [1], infection by Candida dubliniensis has been increasingly reported in a number of human immunodeciency virus (HIV)positive individuals and acquired immune deciency syndrome (AIDS) patients from around the world. In these individuals it has been primarily isolated from the
Correspondence: Dr Wieland Meyer, Centre for Infectious Diseases and Microbiology, Molecular Mycology Laboratory, The University of Sydney at Westmead Hospital, ICPMR, Level 3, Room 3114A, Darcy Road, Westmead, NSW 2145, Australia. Tel.: ‡61 2 98456895; fax: ‡61 2 98915317; e-mail: meyer@ angis.usyd.edu.au
oral cavities [1–7], but isolation from other anatomical sites (vagina and lung) has also been reported [1,8]. It has been shown that C. dubliniensis can be a constituent of the normal human oral ora, with the potential to cause oral candidosis [1,8]. C. dubliniensis expresses the C. albicans serotype A [1,4] and is able to form germ tubes, pseudohyphae and abundant chlamydospores [1,3,8]. These features have been diagnostic for C. albicans, making it phenotypically and morphologically indistinguishable from this phylogenetically most closely related yeast species. In comparison to C. albicans, C. dubliniensis isolates are characterized by a higher resistance to the commonly used antifungal drug uconazole, and uconazole-susceptible isolates are able to develop resistance to the drug in vitro [1,9]. For this
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reason it is important to identify clinical isolates correctly at a very early stage of infection. Despite the phenotypical similarities between these two fungal pathogens, C. dubliniensis differs slightly in its carbohydrate assimilation prole, as determined with the commercially available biochemical yeast identi cation systems (API ID 32C and API 20C AUX kits from bioMe´ rieux, Marcy l’Etoile, France) [10], in particular the absence of ß-glucosidase [1,2]. However, these assays are sometimes difcult to interpret. C. dubliniensis also differs from C. albicans in its inhibited growth at elevated temperatures of 42 oC [1,3,8] or 45 oC [11], its growth on the recently developed commercial chromogenic agar medium CHROMagar Candida [12], where it forms dark green colonies, and the absence of uorescence in the colonies on methyl blue-Sabouraud agar plates [13]. However, individual strain variations have been reported for each of these characteristics [1,3,8], making these criteria unreliable for a correct identi cation. Because of the difculties in using methods based on phenotypic characteristics to distinguish between C. dubliniensis and C. albicans, the focus has shifted in the past few years to molecular methods, which exploit genetic differences between the two fungal pathogens to develop more efcient differentiation techniques. These techniques range from restriction fragment length polymorphism (RFLP) generated using the enzymes HaeIII and HinfI [1,14], to DNA-hybridization ngerprinting using the C. albicans-specic repeat sequence 27A [1,15] and synthetic oligonucleotide probes homologous to eukaryotic microsatellite sequences [1]. Other techniques used are: random amplied polymorphic DNA (RAPD) analysis [1], sequence analysis of the V3 region of the large ribosomal subunit gene [1,16], electrophoretic karyotyping [1], and specic amplications of certain genes by polymerase chain reaction (PCR) [17]. The most convincing evidence that C. dubliniensis is a distinct taxon from C. albicans has been the number of nucleotide differences (13–15 nucleotides) in the ribosomal RNA gene sequence, when compared with C. albicans serotype A and B strains as well as strains representing the synonym C. stellatoidea [1,16,18] . However, most of these techniques are labour intensive, cumbersome and time-consuming. In spite of the identi ed differences, a rapid and accurate discrimination between C. albicans and C. dubliniensis remains problematic in the average clinical mycology laboratory. In the original studies on C. dubliniensis by Sullivan et al. [1], a number of microsatellite-speci c oligonucleotides (e.g. [GTG]5, [GACA]4 and [GATA]4) were used as hybridization probes in DNA-ngerprinting. Over the
past 6 years, our group has successfully shown that the same oligonucleotides can be used as single primers in PCR-ngerprinting experiments to obtain highly informative multilocus proles from a number of fungal genera [19–24]. In these studies a number of synthetic oligonucleotides have been tested as single primers: the core sequence of the wild-type phage M13 (50 -GAGGGTGGCGGTTCT-30 ), which is specic to minisatellite DNA sequences, and (CT)8, (GTG)5, (GACA)4 and (GATA)4, which are specic to simple repetitive DNA (microsatellite) sequences. It was found that the primers M13 and (GACA)4 generate the most discriminatory and informative ngerprint proles. In earlier studies, our group established the potential of this PCR based technique to be used as a method for a fast and reliable differentiation of several anamorphic species of the yeast genus Candida and its associated teleomorphic species. All investigated yeast species could be clearly differentiated based on the unique species-specic banding pattern when these two primers were used as single primers in the PCR [19,21]. In addition the generated PCR ngerprinting proles displayed variation at the strain level, allowing the separation of individual strains within each species [19,21–23]. It was also demonstrated that the PCR ngerprints are stable in vitro and in vivo [22]. All of the ndings mentioned above make this technique an ideal tool for a fast and reliable identi cation of clinical fungal specimens. Based on the these ndings, PCR ngerprinting was applied with the objective of differentiating the phylogenetically closely related yeast species C. albicans and its variant C. stellatoidea from the recently described species C. dubliniensis.
Materials and methods Fungal isolates Sixteen C. dubliniensis isolates, including the ex-type culture (CBS 7987 = NCPF 3949), 10 C. albicans isolates, including the ex-neotype culture, which is serotype A (CBS 562) and a representative serotype B isolate (CBS 5983), as well as the ex-neotype culture for the conspecic variant C. stellatoidea (CBS 1905) (Table 1) were examined in this study. The isolates were obtained from the Centraalbureau voor Schimmelcultures (CBS, Delft, The Netherlands), the Clinical Mycology Laboratory at Westmead Hospital, Westmead, Australia, Sullivan & Nicolaides Pathology, Brisbane, Australia and the Mycology Unit, Microbiology Department, Auckland Hospital, Auckland, New Zealand. Cultures were grown on yeast peptone glucose (YPD) agar plates at 27 oC prior to DNA isolation. All isolates were
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Table 1
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List of type cultures and clinical isolates used in this study
Species/isolate
Alternative number*
Geographical origin
Year of isolation
Candida albicans (Robin) Berkhout (1923) WM 83 Sydney/Australia 1995 WM 204 Sydney/Australia Unknown WM 205 WH 96-250-1065 Sydney/Australia 1996 WM 211 WH 95-201-1014 Sydney/Australia 1995 WM 212 WH 96-256-0566 Sydney/Australia 1996 WM 229 CBS 562 Uruguay 1935 WM 231 CBS 5983 Probably USA Unknown WM 622 ACC3243 Auckland/New Zealand 1998 WM 623 MA9026 Auckland/New Zealand 1992 WM 772 WH 99-105-2447 Sydney/Australia 1999 Candida albicans synonym Candida stellatoidea (Jones & Martin) Langeron & Guerra (1939) WM 230 CBS 1905 Unknown Unknown Candida dubliniensis Sullivan (1995) WM 206 WH 96-241-1055 Sydney/Australia 1996 WM 602 CBS 7987 Dublin/Ireland NCPF 3949 WM 606 CBS 7988 Melbourne/Australia 1995 WM 607 CBS 8500 Nijmegen, Netherlands 1998 WM 608 CBS 8501 Nijmegen, Netherlands 1998 WM 609 WH 98-079-1183 New York, USA 1998 WM 611 WH 96-212-2001 Sydney/Australia 1998 WM 615 MD8127 Auckland/New Zealand 1998 WM 616 SH3240 Auckland/New Zealand 1998 WM 617 BS5206 Auckland/New Zealand 1997 WM 618 BY9374 Auckland/New Zealand 1998 WM 619 BI3140 Auckland/New Zealand 1996 WM 620 SG8798 Auckland/New Zealand 1997 WM 621 CB1830 Auckland/New Zealand 1998 WM 624 WH 99-025-1889 Sydney/Australia 1999 WM 771 BC901 Brisbane/ Australia 1999
Comments
HIV¡, from oral cavity From sputum From necrotic tissue NT, Serotype A, from skin Serotype B HIV¡, from a mouth swab HIV¡, from sputum NT From sputum 1995 HIV¡, from oral cavity From blood HIV‡, from oral cavity From throat HIV¡, from sputum HIV¡, from sputum HIV¡, from sputum HIV¡, from sputum HIV¡, from abdominal uid HIV¡, from throat swab HIV¡, from lip swab From sputum HIV‡, from blood
CBS, Centraalbureau voor Schimmelcultures, Baarn-Delft, The Netherlands; WH, Westmead Hospital, The University of Sydney, Sydney, Australia; T, ex-type culture; NT, ex-neotype culture. *, Numbers dening the same strain in a different culture collection are given under alternative number column.
maintained on Sabouraud agar slants at 4 oC, as water cultures at room temperature and as glycerol stocks at ¡70 oC.
PCR ngerprinting
Genomic DNA was isolated as described previously [21]. PCR ngerprinting was carried out using oligonucleotides of the minisatellite specic core sequence of the wild-type phage M13 (50 -GAGGGTGGCGGTTCT-30 ) [25] and of the microsatellite (simple repetitive DNA sequence) (GACA)4 [26] as single primers. Amplication reactions were performed in 50 m l volumes containing 25 ng of genomic DNA template, 10 mM Tris-HCl, pH 8¢3, 50 mM KCl, 1¢5 mM MgCl, 0¢2 mM each of dATP, dCTP, dGTP and dTTP (Boehringer Mannheim, Mannheim, Germany), 3 mM magnesium acetate, 30 ng primer and 2¢5 units of AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT, USA). The PCR reactions were performed in a Perkin Elmer thermal cycler (model
480) with 35 cycles of 20 s denaturation at 94 oC, 1 min annealing at 50 oC for the primers M13 and (GTG)5, and at 43 oC for the primer (GACA)4, and 20 s extension at 72 oC, followed by a nal extension cycle for 6 min at 72 oC. The PCR products were removed, concentrated to approximately 20 m l (Eppendorf Concentrator, model 5301; Eppendorf-Netheler GmbH, Hamburg, Germany), and separated by electrophoresis in 1¢4% agarose gels in Tris-borate-EDTA buffer for 10 h at 3 V cm¡1. Amplication products were detected by staining with ethidium bromide (0¢5 m g ml¡1) and visualized under UV light. The 1 kb DNA ladder from GIBCO-BRL was used as molecular size standard.
Analysis of genetic relatedness PCR ngerprinting proles were analysed using the GelComparII version 1.01 software (Applied Maths, Kortrijk, Belgium) [27]. All visible bands were included
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in the analysis regardless of the band intensity. The bands for each ngerprinting pattern were dened using the manual option in the GelComparII software with a band tolerance of 1%. This was the minimum position tolerance that was required to normalize the molecular size markers to 100% identity. Similarity coefcients were calculated using the dice algorithm, and cluster analysis performed by the unweighted pair group method for arithmetic averages (UPGMA). For epidemiological purposes the ngerprinting data obtained using the primers M13 or (GACA)4 were combined as a composite data set and corrected for internal weights, to produce cluster analysis based upon the combined ngerprint data.
Results and discussion The current study includes the ex-type or ex-neotype cultures of C. albicans, C. stellatoidea and C. dubliniensis, as well as several clinical isolates. All the clinical isolates studied were tentatively identied as C. dubliniensis, on the basis of giving an atypical C. albicans identication prole using commercially available biochemical identication systems (API ID32C; bioMe´ rieux) and failing to grow at elevated temperatures but producing germ tubes and chlamydospores. PCR ngerprinting was carried out using the primers M13 or (GACA)4 as single primers to amplify hypervariable inter-repeat DNA fragments from all investigated yeast strains. The PCR proles obtained were highly informative and generated clearly distinct banding patterns for the C. albicans and C. dubliniensis isolates (Figs. 1a and 2a). The same PCR based techniques were used previously in our laboratory to distinguish other medically important yeasts [19,21–23]. The primer M13 generated DNA fragments ranging in sizes of 500–1900 bp for C. albicans and 350–2500 bp
for C. dubliniensis (Fig. 1a). The bands obtained with the primer (GACA)4 ranged 600–2040 bp for C. albicans and 400–4000 bp for C. dubliniensis (Fig. 2a). All clinical isolates could be clearly assigned to one or the other species based on the species-specic banding patterns obtained. C. albicans serotypes A and B, and the C. stellatoidea showed similar PCR ngerprinting proles, with a band sharing similarity of 70% for both primers used (Figs. 1a and 2a). These band sharing values are within the intraspecies range of variation (70–85%) observed previously for other Candida species [19,21], conrming that the species are conspecic in spite of differing in one or two nucleotides in the V3 region of the ribosomal RNA gene [18,28,29]. The differences observed between the ngerprinting patterns (Figs. 1a and 2a) are due to strainspecic variation within a single species [19,21]. Major discriminatory or diagnostic ngerprinting bands for each species were identied and their molecular weights determined using the computer program GelComparII version 1.01, based upon ngerprint patterns representing each of the isolates in this study. Most of these major bands were present in all isolates studied (Figs. 1a and 2a). The major discriminatory bands are presented in Table 2 and arrow heads in Figures 1a and 2a indicate their locations within the multilocus proles, obtained with the two primers respectively. Similarity coefcients for all possible pairs of isolates were calculated using the Dice algorithm with a band position tolerance of 1% and dendrograms were generated by the UPGMA analysis. These dendrograms split the investigated yeast isolates into two groups. Isolates representing C. albicans and its conspecic variant formerly known as C. stellatoidea form one cluster clearly separated from the isolates clustered with the ex-type culture of C. dubliniensis, which form a second distinct cluster. All of the clinical isolates are
Table 2 Molecular sizes of major discriminatory/diagnostic PCR ngerprinting bands of C. albicans and C. dubliniensis, for the primers M13 and (GACA)4 Primer
Species
No. of major diagnostic bands
Molecular sizes of major bands (bp)*
C. albicans C. dubliniensis
9 11
1800y, 1670, 1420, 1170, 1120, 1050y, 750, 710, 605 2330, 1960, 1825, 1410, 1120, 1090, 907y, 765, 654y, 535, 375
C. albicans C. dubliniensis
7 13
1975, 1890, 1255, 1160, 1065, 865, 835 4150, 3620, 3270, 2290, 1600y, 1450y, 1400y, 1150, 1010, 860, 815, 730, 540y
M13
(GACA)4
*, Molecular sizes of the PCR ngerprint bands were determined automatically from computer-scanned photographs of agarose gels by comparison with the molecular size standard (1 kb marker) using the computer program GelComparII version 1.01; y, major diagnostic PCR-ngerprinting bands which may not be present in all investigated strains. Downloaded from https://academic.oup.com/mmy/article-abstract/39/2/185/986647 by guest on 05 June 2018
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Fig. 1 (a) Electrophoretic separation of PCR ngerprints obtained by amplifying genomic DNA from a number of type, neotype and clinical isolates of C. albicans and C. dubliniensis using the M13 core sequence (50 -GAGGGTGGCCGGTTCT-30 ) as a single primer in the PCR. For full description of the investigated isolates, see Table 1. Major discriminatory/diagnostic PCR ngerprinting bands are indicated by arrows and values given in Table 2. (b) Dendrogram derived from the similarity matrix obtained from the PCR-ngerprint pattern generated with the primer M13 after analysis with the program GelComparII version 1.01 using Dice coefcient with a band tolerance of 1% and UPGMA analysis. ã 2001 Medical Mycology, 39, 185–193 Downloaded fromISHAM, https://academic.oup.com/mmy/article-abstract/39/2/185/986647 by guest on 05 June 2018
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Fig. 2 (a) Electrophoretic separation of PCR ngerprints obtained by amplifying genomic DNA from a number of type, neotype and clinical isolates of C. albicans and C. dubliniensis using (GACA)4 as a single primer in the PCR. For full description of the investigated isolates, see Table 1. Major discriminatory/diagnostic PCR ngerprinting bands are indicated by arrows and values given in Table 2. (b) Dendrogram derived from the similarity matrix obtained from the PCR ngerprint pattern generated with the primer (GACA)4 after analysis with the program GelComparII version 1.01 using Dice coefcient with a band tolerance of 1% and UPGMA analysis. Downloaded from https://academic.oup.com/mmy/article-abstract/39/2/185/986647 by guest on 05 June 2018
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grouped correctly with their respective standard ex-type or ex-neotype strains (Figs. 1b and 2b). The computer scanned PCR proles generated in this study and from other Candida species in previous studies [19,21] have formed the basis of a computer database, which can be used for future identi cations of atypical or unidentiable Candida isolates. The band tolerance of 1% used in the present analysis was determined during computer analysis of the molecular weight standard (1 kb ladder; GIBCO-BRL), which was included at three intervals on each gel. It represents the tolerance required for corresponding bands in each molecular weight standard to be interpreted as identical in the dendrogram, compensating for small, unavoidable differences in the running conditions of each gel. No further computer-enhanced optimizations were made. The results presented in this study demonstrate again that PCR ngerprinting data do not reect denitive phylogenetic relationships between a given set of isolates [21]. This technique provides an indication of the relationships of isolates according to the primer/PCR system utilized (Figs. 1b and 2b). To overcome the fact that each primer amplies only a small independent
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subset of the genome which is not representative of the true phylogenic relationships between the investigated isolates, the obtained data sets have to be combined for epidemiological purposes to obtain accurate genetic relationships (Fig. 3). In addition to correctly identifying each of the clinical isolates to the species level, the primers used were also able to distinguish all investigated isolates. The average intra-species variation of the PCR ngerprints obtained with the primer M13 was 18¢3% (standard deviation of 7¢7) for C. albicans/C. stellatoidea and 10¢4% (standard deviation of 6¢8) for C. dubliniensis. With primer (GACA)4, there was an average intra-species variation of 18¢3% (standard deviation of 7¢4) for C. albicans/ C. stellatoidea and 15¢9% (standard deviation of 12¢7) for C. dubliniensis. The current analysis revealed that the minisatellite specic primer M13 is more discriminatory than the simple repetitive DNA sequence (microsatellite) specic primer (GACA)4. The primer M13 could distinguish all isolates investigated. With primer (GACA)4, three isolates appeared to be identical even though there was no apparent correlation between the patients they came from. One such isolate was from the USA (WM609)
Fig. 3 Dendrogram generated form the composite data set after combining the PCR ngerprinting data obtained with the primers M13 and (GACA)4 , using internal weights dened in the individual GelComparII analysis for each primer, showing the genetic relationships between the individual isolates. ã 2001 Medical Mycology, 39, 185–193 Downloaded fromISHAM, https://academic.oup.com/mmy/article-abstract/39/2/185/986647 by guest on 05 June 2018
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while two were from New Zealand (WM618 and WM619), isolated from different patients several years apart. These ndings again emphasize that it is important to use at least two independent markers for the identication of any given clinical isolate, in order to overcome the bias caused by differences in distribution and rates of evolution of certain loci. The fact that the presented identication system allows both species and strain identi cation differentiates it from a number of previously suggested identi cation systems that only identify isolates to the species level [17,29]. The main difference of the proposed PCR ngerprinting technique is that the primers used are specic to mini- and microsatellites, genetic elements that are found in a wide range of eukaryotic organisms. That is why the same primers and PCR conditions can be used for the identi cation of a wide range of fungal pathogens. The species-specic major banding patterns generate a unique species identi cation pattern. The identication power is only limited by the size of the available databases (meaning the number of included species). Previously described molecular methods were only applicable to single species or to a small range of species they had been specically developed for. In addition to circumventing this limitation, the PCR ngerprinting technique is also a very useful method for epidemiological investigations, particularly for the identication of nosocomial outbreaks and of drug resistant strains, if the independent ngerprinting data sets are analysed in a combined fashion (Fig. 3). The proposed identication technique is simple, reliable and does not require radioactively labelled probes, such as those recently developed for C. dubliniensis [30], making it an ideal identi cation system for routine clinical mycology laboratories. The close phylogenetic relationship between the two yeast species is demonstrated in the relatively high interspecies homology of their ngerprinting proles. The observed inter-species homology was 57¢9% for the primer M13 and 58¢6% for the primer (GACA)4, compared with previously obtained data from other yeast species, which were characterized by 15–30% interspecies homology [14,21,22]. We propose PCR-ngerprinting using especially the primer M13 as a simple, rapid, stable, sensitive, highly reproducible and cost effective molecular tool to distinguish C. albicans and C. dubliniensis.
Acknowledgements We thank Ok Cha Lee, Clinical Mycology Laboratory, Westmead Hospital, Westmead, Australia, for the biochemical yeast identi cation, Heide-Marie Daniel
for maintenance of cultures and preparation of photographs and Sarah Kidd, both Molecular Mycology Laboratory, Westmead Hospital, for proofreading the manuscript. We appreciate the gifts of cultures from: David Yarrow, CBS; Ok Cha Lee, Jenny Robson and Susan Benson, Sullivan & Nicolaides Pathology, Brisbane, Australia, and Dinah Parr and Karen Rogers, Mycology Unit, Microbiology Department, Auckland Hospital, Auckland, New Zealand. This investigation was supported by an NH&MRC CARG grant (#960792) from the National Health & Medical Research Council, Canberra, Australia to W. Meyer, and two grants-in-aid from the Millennium Foundation of the Westmead Institute of Health Research, Westmead, Australia to W. Meyer.
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