Multilocus Sequence Typing for Differentiation of Strains of Candida tropicalis

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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.11.5593–5600.2005

Multilocus Sequence Typing for Differentiation of

Strains of

Candida tropicalis

Arianna Tavanti,

1

Amanda D. Davidson,

1

Elizabeth M. Johnson,

2

Martin C. J. Maiden,

3

Duncan J. Shaw,

1

Neil A. R. Gow,

1

and Frank C. Odds

1

*

Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom1_{; HPA Mycology Reference Laboratory, Myrtle Road, Kingsdown, Bristol BS2 8EL,}

United Kingdom2_{; and The Peter Medawar Building for Pathogen Research and Department of Zoology,}

University of Oxford, OX1 3SY, United Kingdom3

Received 8 June 2005/Returned for modification 20 July 2005/Accepted 27 August 2005

A system is described for typing isolates of the pathogenic fungus Candida tropicalis, based on sequence polymorphisms in fragments of six genes: ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1a. The system differentiated 87 diploid sequence types (DSTs) among a total of 106 isolates tested or 80 DSTs among 88 isolates from unique sources. Replicate isolates from the same source clustered together with high statistical similarity, with the exception of one isolate. However, a clade of very closely related isolates included replicate isolates from three different patients, as well as single isolates from eight other patients. This clade, provi-sionally designated clade 1, was one of three clusters of isolates with high statistical similarity. Five of six isolates in one cluster that may acquire clade status were resistant to flucytosine. This study addsC. tropicalis

toCandida albicansandCandida glabrataasCandidaspecies for which a multilocus sequence typing (MLST) system has been set up. TheC. tropicalisMLST database can be accessed at http://pubmlst.org/ctropicalis/.

Strain typing by sequencing of several housekeeping genes in a microbial species (multilocus sequence typing; MLST) has rapidly developed as a reliable technology for epidemiological studies of infectious disease (5, 23). The method involves de-termination of DNA sequence polymorphisms between iso-lates with a set of fragments of five to seven genes, which are ideally under neutral selective pressure and chromosomally dispersed. The data obtained are highly reproducible, amena-ble to statistical analyses to quantify similarities and putative genetic relationships between isolates, and able to be stored in a single central database for global internet access.

Among fungal diseases, deep-seatedCandidainfections are the most commonly encountered opportunistic problems that affect seriously immunocompromised or debilitated hosts. Su-perficialCandida infections are responsible for considerable morbidity among neonates, the elderly, and patients with AIDS (oral infections) and among women of childbearing age (vulvovaginal infections). Candida albicans accounts for the majority of these infections, but otherCandidaspecies, partic-ularlyC. glabrata, C. parapsilosis, andC. tropicalis, are by no means uncommon causes, with some authorities documenting a rise in prevalence of the latter at the expense ofC. albicans. There is therefore a clear need for strain typing of these spe-cies for epidemiological purposes. MLST technology has been developed forCandida albicans(3, 4, 22) andC. glabrata(7). MLST could not be used for C. parapsilosis because of the paucity of allelic polymorphisms in this species (20). The MLST approach provides for definition of population struc-tures within a species and can reveal differences in

geograph-ical origins, anatomgeograph-ical sources, and other properties between clades of closely related isolates (2, 3, 7, 19, 21).

We have now developed MLST for the speciesC. tropicalis, a species that has accounted for 5 to 30% ofCandida blood-stream infections during the past 15 years (17). C. tropicalis

probably has an entirely diploid genome (1, 8, 13) and, as with

C. albicans, MLST can take advantage of the additional poly-morphisms that are due to allelic heterozygosities.

MATERIALS AND METHODS

Isolates.All but 2 of the 106C. tropicalisisolates (Table 1) were originally cultured from clinical material; the exceptions were two isolates from the stom-ach contents of a chameleon. All isolates were reidentified by standard morpho-logical and physiomorpho-logical criteria. Historical isolates came from our collection of pathogenic fungi and fresh isolates beyond those we received for routine test purposes came from the Mycology Reference Laboratory in Bristol, United Kingdom, and the Women’s and Children’s Hospital, Adelaide, Australia. The sources for 30 isolates were 10 patients and 1 animal from whom two or more cultures were obtained from different anatomical sites or at different times. A set of 88 single-source isolates was therefore available, including one chosen ran-domly from each of the sets of multiple isolates. The isolates represented prob-able genetic diversity based on their date, anatomical site, and geographical source of isolation. The yeasts were maintained on Sabouraud agar (Oxoid, Basingstoke, United Kingdom).

Choice of loci for MLST.Initially, 13 gene fragments were chosen for a pilot MLST study of 20C. tropicalisstrains. We reduced the set to the minimum of six gene fragments (Table 2) which, in combination, yielded the highest interstrain discrimination (largest number of different strain types). One fragment was obtained with primers described for the C. albicans ZWF1agene encoding glucose-6-phosphate dehydrogenase (22), which amplified products from several otherCandidaspecies; the remainder wereC. tropicalisgenes whose sequences were obtained from the GenBank database. Primers were designed to amplify gene fragments of 500 to 750 bp and are also described in Table 2, with the corresponding PCR product sizes given.

DNA extraction.Genomic DNA was extracted from yeasts grown in YPD broth comprising 2% glucose, 2% mycological peptone (Oxoid), and 1% yeast extract (Difco, Detroit, MI). Briefly, cells were harvested in the stationary phase and lysed by vortexing the pellet for 5 min with 0.3 g of glass beads (0.45 to 0.52 mm in diameter; Sigma, St. Louis, MO) in 200␮l of buffer (100 mM Tris HCl,

* Corresponding author. Mailing address: Institute of Medical Sci-ences, Aberdeen, AB25 2ZD, United Kingdom. Phone and fax: 44 1224 555828. E-mail: [email protected].

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TABLE 1. Details ofC. tropicalisisolates tested by MLST, listed in order of their DST showing genotypes for seven DNA fragments sequenced

Strain reference no.

Date

isolated Country

a _Source Description or patient

origin (reference)

No. of isolates of DST or genotype: DST ICL1 MDR1 SAPT2 SAPT4 XYR1 ZWF1␣

J942105 1994 US Blood Original no. 90B-4 (16) 1 1 1 1 1 1 1

J930222 1993 Belgium Nail 2 1 2 2 2 1 2

J930582/3 1993 Belgium Chameleon intestine 3 1 3 1 3 2 3

J930581 1993 Belgium Chameleon intestine 3 1 3 1 3 2 3

J930949 1993 Germany Oral (AIDS patient) 4 2 4 3 3 3 3

L601 1986 UK Pus Patient 1 (15) 5 1 5 4 4 4 1

J981212 1998 US Vagina 6 1 6 3 5 5 3

J981248 1998 US Vagina 6 1 6 3 5 5 3

J980156 1997 US Blood Same patient origin as

J980160, J980162

7 3 7 3 6 6 4

J980160 1997 US Bronchial aspirate Same patient origin as J980156, J980162

7 3 7 3 6 6 4

J941019 1994 Belgium Oral 8 2 8 5 7 5 5

75/036 1975 UK Oral 9 4 9 3 8 4 6

BSS601952 2001 UK Gastric contents Same patient origin as BUC808390

10 3 10 1 7 5 4

BUC808390 2001 UK Urine Same patient origin as

BSS601952 11 3 11 1 7 5 4 CDC451 US 12 1 1 1 1 5 1 J930575/2 1993 Belgium Vagina 12 1 1 1 1 5 1 L823 1986 UK Sputum Patient 1 (15) 13 1 12 4 4 4 1 J950787/T 1995 Greece 13 1 12 4 4 4 1 L644 1986 UK Feces Patient 2 (15) 13 1 12 4 4 4 1 L711 1986 UK Oral Patient 2 (15) 13 1 12 4 4 4 1 L712 1986 UK Feces Patient 2 (15) 13 1 12 4 4 4 1 J942213 1994 13 1 12 4 4 4 1 A427748 2001 UK Blood 14 3 7 1 6 6 7

AM2002/0110 2002 Germany Oral Same patient origin as AM2002/0111

15 1 7 1 9 5 1

AM2002/0111 2002 Germany Oral Same patient origin as AM2002/0110 15 1 7 1 9 5 1 J940670B 1994 Belgium Oral 16 2 12 6 10 7 5 J932716 1993 Belgium Vagina 17 5 7 1 11 4 7 L474 1986 UK Vagina Patient 1 (15) 18 1 12 4 12 4 1 L501 1986 UK Feces Patient 1 (15) 18 1 12 4 12 4 1 L590 1986 UK Vagina Patient 1 (15) 18 1 12 4 12 4 1 L831 1986 UK Oral Patient 1 (15) 18 1 12 4 12 4 1 J951358 1995 UK 19 3 13 4 7 1 1 J920533 1992 Sweden Vagina 20 1 14 4 4 4 1 J930561/2 1993 Belgium Vagina 21 3 9 7 8 4 6 NCCLS39 US 22 1 15 1 13 8 1

J941836 1994 The Netherlands Blood 23 1 16 3 7 9 3

J950226 1995 US 24 3 7 4 10 1 1

NCCLS71 US 27 3 7 1 6 6 4

AM2005/2002 2005 UK 28 2 18 5 10 10 5

81/056 1981 UK Nail 29 1 19 3 7 3 1

J980157 1998 US Abdominal biopsy 30 3 7 3 15 6 4

75/035 1975 UK Oral 31 1 20 4 4 4 1

A700246 1999 UK Tracheal aspirate 31 1 20 4 4 4 1

b30343/7/04 2004 UK Blood 31 1 20 4 4 4 1

b31429/7/04 2004 UK Blood Same patient origin as

b31252/7/04

31 1 20 4 4 4 1

A700639 1999 UK Tracheal aspirate 32 1 2 3 3 2 1

b30431/5/04 2004 UK 32 1 2 3 3 2 1

81/031 UK 33 3 7 3 13 4 3

81/022 UK 34 1 21 4 5 6 1

J942104 US Original no. MY-1012

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35 1 7 8 6 2 4

83/002 1983 UK 36 1 21 9 5 6 1

J980162 1998 US Bronchoalveolar

lavage

Same patient origin as J980156, J980160

37 3 7 3 6 11 4

L470 1986 UK Sputum Patient 1 (15) 38 1 20 4 12 4 1

J990332/2 1999 US Vagina 39 1 22 3 10 9 3

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pH 8.0, containing 2% Triton X-100, 1% sodium dodecyl sulfate, 1 mM EDTA) and 200␮l of 1:1 (vol/vol) phenol-chloroform solution. After vortexing, 200␮l of TE (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) was added to the lysate; the mixture was microcentrifuged at full speed for 5 min, and the aqueous phase was

trans-ferred to a new tube. DNA was precipitated by addition of 1 ml of ethanol to the supernatant. Samples were centrifuged for 2 min, and the pellet was resuspended in 400␮l of TE containing 100␮g of RNase (10␮l of a 10-mg/ml solution; Sigma). The mixture was incubated for 1 h at 37°C, and then DNA was

precip-TABLE 1—Continued Strain reference no. Date isolated Country a

Source Description or patient origin (reference)

No. of isolates of DST or genotype: DST ICL1 MDR1 SAPT2 SAPT4 XYR1 ZWF1␣

J980184 1998 US Tracheal aspirate 40 1 23 3 16 12 8 L391 1986 UK Vagina 41 1 7 4 12 13 1 J981379 1998 US Vagina 42 1 24 3 7 14 6 81/024 UK 43 7 25 3 7 5 1 AM2005/0003 2003 UK 44 3 26 3 7 15 7 b30980/5/04 2004 UK Blood 45 1 3 3 17 16 3 AM2003/0078 2003 UK Blood 45 1 3 3 17 16 3 b30167/5/04 2004 UK Blood 46 1 27 1 11 4 3

b30642/4/04 2004 UK CAPD fluidb _{Same patient origin as}

b30451/4/04 47 3 4 3 3 17 3 b30524/4/04 2004 UK Urine 48 1 20 1 4 13 1 b31389/3/04 2004 UK Blood 49 8 28 3 18 17 7 b30931/5/04 2004 UK Blood 50 1 1 10 1 1 1 b30277/4/04 2004 UK Urine 51 1 29 3 19 13 1

b30451/4/04 2004 UK CAPD fluid Same patient origin as b30642/4/04 52 3 4 3 3 3 3 b31245/5/04 2004 UK Urine 53 5 16 3 20 9 1 b30274/5/04 2004 UK Blood 54 3 30 3 7 18 7 b30096/6/04 2004 UK Urine 55 1 31 3 21 3 1 b30441/6/04 2004 UK Blood 56 1 7 11 21 19 1 b30588/6/04 2004 UK Catheter 57 9 3 3 10 19 9 b31083/6/04 2004 UK 58 10 32 4 22 18 7 b31084/6/04 2004 UK Sputum 59 1 7 4 4 20 1 b31514/6/04 2004 UK 60 10 7 3 8 11 10 b31595/6/04 2004 UK Sputum 61 1 33 12 23 21 3 b31597/6/04 2004 UK 62 10 7 3 6 11 4 b30349/7/04 2004 UK Blood 63 3 9 13 24 4 3 b30488/7/04 2004 UK Bronchoalveolar lavage 64 1 7 1 6 22 4 b30604/7/04 2004 UK Sputum 65 1 20 4 4 4 11 b30613/7/04 2004 UK Blood 66 1 34 2 13 23 2 b31200/7/04 2004 UK Urine 67 1 3 4 11 24 1

b31252/7/04 2004 UK Urine Same patient origin as

b31429/7/04

68 1 20 4 10 4 1

b31581/7/04

69 2 20 4 10 25 5

b31580/7/04

69 2 20 4 10 25 5

b31586/7/04 2004 UK Catheter 70 2 20 6 10 26 5

WC02-203295 2000 Australia Blood Same patient origin as WC02-203295

71 1 7 1 7 28 9

72 1 7 1 7 28 13

73 11 37 14 25 29 14

WC04-200749 2000 Australia Venous catheter tip Same patient origin as WC04-200748 74 12 37 15 25 29 15 AM2003/0076 2003 UK Blood 75 13 38 16 26 30 16 AM2005/0005 2004 UK 76 14 39 17 27 30 17 J981352 1998 USA Vagina 77 1 24 3 7 33 4 73/108 1973 UK Oral 78 1 1 18 1 5 3 J940788 1994 Belgium Oral 79 2 20 5 10 26 5 CDC44 US 80 1 1 3 1 5 1 81/020 1970s UK 81 1 7 18 21 1 1 L634 1986 UK Vagina Patient 1 (15) 82 15 38 19 28 28 13 AM2005/0004 2004 UK Blood 83 1 39 3 7 31 17 b30357/6/04 2004 UK Vascular catheter 84 9 40 3 4 19 9 AM2005/0006 2004 UK Blood 85 9 26 3 11 24 4 AM2005/0007 2005 UK Blood 86 9 26 1 11 32 4 AM2005/0008 2005 UK 87 9 26 1 11 33 10 a

US, United States; UK, United Kingdom. b

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itated with 1 ml of isopropanol and 10␮l of 3 M sodium acetate, dried, and redissolved in 50␮l of TE, pH 8.0.

Amplification and nucleotide sequence determination.PCRs were used to amplify the gene fragments listed in Table 2. Reaction volumes of 50␮l con-tained 100 ng of genomic DNA, 2.5 UPfuDNA polymerase (Promega, Madison, WI), 5␮l of 10⫻buffer (supplied with the enzyme), 200␮M deoxynucleoside triphosphates (dNTPs) (Promega), and 10␮M forward and reverse primers. A Flexigene thermocycler (Techne, Cambridge, United Kingdom) was set up with a first cycle of denaturation for 7 min at 94°C followed by 30 cycles of denatur-ation at 94°C for 1 min, annealing at 52°C for 1 min, and elongdenatur-ation at 74°C for 1 min 5 s, with a final extension step of 10 min at 74°C. The amplified products were precipitated in microdilution plates. Briefly, 60␮l of a 20% polyethylene glycol (Sigma)–2.5 M NaCl solution was added to each well containing 40␮l of PCR product. The microdilution plate was then sealed, vortexed, incubated at room temperature for 30 min, and centrifuged for 1 h at 2,250⫻g(4°C). The supernatant was discarded, and the plate was inverted onto a piece of 3-mm chromatography paper and centrifuged again at 500⫻gfor 1 min to remove any residual polyethylene glycol from the wells. Pellets were washed with 150␮l of 70% ethanol, precipitated as described above, and resuspended in 60␮l of sterile water. Both strands of purified gene fragments were sequenced on an ABI 3700 DNA analyzer (Foster City, Iowa) with a 2.5␮M concentration of the same primers that were used in the PCR step. The sequence data were coupled with DNASTAR software. Heterozygosities were defined by the presence of two coincident peaks in the forward and reverse sequence chromatograms. The one-letter code for nucleotides from the International Union of Pure and Ap-plied Chemistry (IUPAC) nomenclature was used to define results.

Statistical analysis of MLST data.Phylogenetic analyses by unweighted pair-group method with arithmetic average (UPGMA) and neighbor-joining algo-rithms were conducted with MEGA version 2.1 (14) applied to modified se-quence data. The analyses were based only on the results for variable loci to maximize their power to discriminate between isolates; the data set of only the variable bases was suitable for pairwise difference analysis, which has been used previously withC. albicansMLST (3, 22). To obtain sequences that could be handled by the MEGA software, which is not programmed to analyze heterozy-gous code data, the following procedure was used. The results for the variable loci from the six gene fragments sequenced were concatenated into a single sequence. For any pair of isolates, each with a diploid genome, the base at each variable locus could be homozygous and identical between the isolates, heterozy-gous and identical, homozyheterozy-gous and different, or heterozyheterozy-gous in one isolate and homozygous in another. For example, the sequencing result (in the IUPAC single-letter code) for a given locus across a set of strains might appear as A, T, or W (⫽A⫹T). Data from the variable loci from the sixC. tropicalisalleles were therefore conjoined into a single sequence, and then each base in the sequence was rewritten twice for a homozygous (A, C, G, or T) datum or as the two component bases for a heterozygous (K, M, R, S, W, Y) datum. These revised sequences could then be used to generate an unrooted UPGMA dendrogram

based on pair differences in MEGA 2.1. In this form, they were the functional equivalent of scoring a pair of results as 1 for homozygous or heterozygous identical data, 0 for homozygous different data, and 0.5 when one allele had a heterozygous result and the other a homozygous result and then creating a difference matrix. The significance of the cluster nodes was determined by boot-strapping with 1,000 randomizations. The same data were also used to generate unrooted neighbor-joining trees based onp-distance, also with 1,000 random bootstrapping operations to determine significance of nodes.

The eBURST package (http://eburst.mlst.net/) (9) was used to determine putative relationships between isolates. This software scans pairs of alleles and records isolates as related when five of the six alleles are identical between a pair. The eBURST algorithm places all related isolates into clonal complexes and, where possible, predicts the founding, or ancestral diploid sequence type (DST) of each complex. The output is a display of the most parsimonious patterns of descent of each DST from the ancestral type.

Discriminatory power was calculated according to Hunter (11).

RESULTS

C. tropicalis strain differentiation by MLST. The six frag-ments sequenced allowed for differentiation of 87 DSTs among 106 isolates (Table 1), indicating a discriminatory power of 0.994. For the set of 88 isolates from unique sources used to determine population structure (below), 80 DSTs were found. Of the six gene fragments used for MLST,XYR1showed the highest typing efficiency, distinguishing 3 genotypes per poly-morphism for only 11 polymorphic sites (Table 3), andSAPT2 TABLE 2. List of gene fragments and primer details forC. tropicalisMLST

Gene

Fragment Gene product accession no.GenBank Primer

c Amplicon

size (bp)

Sequence startb Sequenced fragment

(bp)

5⬘ 3⬘

ICL1 Isocitrate lyase D00703 Fwd, 5⬘-CAACAGATTGGTTGCCATCAGAGC-3⬘ 737 CGAAGCTG TGGCAATT 447

Rev, 5⬘-CGAAGTCATCAACAGCCAAAGCAG-3⬘

MDR1 Multidrug resistance protein

AF194419 Fwd, 5⬘-TGTTGGCATTCACCCTTCCT-3⬘ 663 TGATGGTG GCCYTTAT 425

Rev, 5⬘-TGGAGCACCAAACAATGGGA-3⬘

SAPT2 Secreted aspartic protease 2

AF115320 Fwd, 5⬘-CAACGATCGTGGTGCTG-3⬘ 658 CTGGTGTC TXTTCCAA 525

Rev, 5⬘-CACTGGTAGCTGAAGGAG-3⬘

SAPT4 Secreted aspartic protease 4

AF115322 Fwd, 5⬘-TGCTTCTCCTACAACTTCACCTCC-3⬘ 483 CATXATTA CAACAATT 390

Rev, 5⬘-ATTCCCATGACTCCCTGAGCAACA-3⬘

XYR1 D-Xylose reductase I or II

AB002105 Fwd, 5⬘-AGTTGGTTTCGGATGTTG-3⬘ 479 TCTACAAT AAATTGGT 370

Rev, 5⬘-TCGTAAATCAAAGCACCAGT-3⬘

ZWF1a Putative glucose-6-phosphate dehydrogenase

Fwd, 5⬘-GGTGCTTCAGGAGATTTAGC-3⬘a ₆₄₇ _TGCCTTGTTT _ATTGTTCAGT ₅₂₀

Rev, 5⬘-ACCTTCAGTACCAAAAGCTTC-3⬘a a

Primers described forC. albicans CaZWF1agene, encoding glucose-6-phosphate dehydrogenase (21). b

X signifies a polymorphic base. c

Fwd, forward; Rev, reverse.

TABLE 3. Properties of sixC. tropicalisfragments used for MLST of 106 isolates Gene Fragment No. of polymorphic sites No. of genotypes No. of genotypes/ polymorphism Ratio of nonsynonymous to synonymous changes ICL1 21 15 0.71 0.15 MDR1 24 40 1.67 0.14 SAPT2 37 19 0.51 0.23 SAPT4 31 28 0.90 0.41 XYR1 11 33 3.00 0.03 ZWF1a 18 17 0.94 0.06

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was the least efficient fragment in terms of genotypes per polymorphism (Table 3). For all of the fragments, the ratios of nonsynonymous to synonymous amino acid changes resulting from the sequence polymorphisms were less than 1, indicating the genes were under neutralizing selective pressure (Table 3). Only four of the C. tropicalis isolates, WC04-200748 and WC04-200749 (both from the same patient), L634, and AM2005/0005 showed variations at large numbers of the poly-morphic sites identified. These variations were mostly seen as heterozygosities at multiple loci rather than as homozygous differences from other isolates, and they were particularly abundant in theSAPT2andSAPT4fragments. The majority of the isolates were discriminated on the basis of a smaller num-ber of polymorphisms in each fragment than is implied by the data in Table 3.

Reproducibility of MLST data. A total of 10 different C. tropicalisisolates were submitted for full or partial MLST in duplicate on different occasions and blinded to the person conducting the sequencing. The duplicate tests involved a total of 1,286 fragment comparisons, of which 10 (0.8%) gave ferent results between duplicate tests. In eight cases, the dif-ference resulted from a single-locus homozygous/heterozygous

difference, and in two cases there were homozygous/heterozy-gous discrepancies at two sites in a single fragment. The se-quence reproducibility was therefore better than 99%.

Nucleotide polymorphisms and amino acid changes. The 142 nucleotide polymorphisms among the six sequenced frag-ments resulted in 24 nonsynonymous changes in amino acids encoded by sequence-variable triplets. Of these, 18 changes were nontrivial (e.g., acidic to basic side chains, aliphatic to aromatic side chains). In ZWF1a, the only nonsynonymous amino acid change was between a tyrosine residue and a stop codon. However, all isolates with theZWF1astop codon poly-morphism were heterozygous at the site. In the SAPT2 se-quence, a switch between tyrosine and a stop codon also re-sulted from the polymorphism at position 125 in the sequence. Only 2 of the 99 isolates sequenced encoded the stop codon, but in both cases, the alleles were homozygous for the stop codon.

Similarity of isolates from the same source. A neighbor-joining dendrogram for all 30 duplicate and multiple isolates from single sources is shown in Fig. 1. Twelve isolates from three different patients coclustered with very high similarity (topmost cluster in Fig. 1). Ten isolates from sources referred to as patients 1 and 2 in Table 1 were all superficial surveillance isolates from individuals undergoing chemotherapy for hema-tological malignancy in the same hospital in the 1980s (15); the other two were blood and urine isolates from a patient in a different hospital in 2004. Isolate L634 was a further isolate from patient 1, but it did not cluster anywhere close to the remainder of patient 1 isolates. The considerable difference of L634 from the other patient 1 isolates may have resulted from an error in maintenance of the isolate in our collection. Be-cause of its obvious difference, L634 was treated as an isolate from a separate source for subsequent analysis. All other sets of duplicate and triplicate isolates from individual patients formed separate clusters according to their source, with high isolate similarity confirmed by the high bootstrap values at the dendrogram nodes. The ability of MLST to distinguish possible errors in labeling or storage of isolates in the way we recog-nized a problem with L634 is a further advantage of the system: we have noted similar “maverick” strain types by MLST in groups of isolates from single sources with our C. albicans

system.

Population structure of 88 isolates from separate sources.

eBURST analysis of the genotypes and DSTs for 88C. tropi-calisisolates from separate sources revealed one cluster of 8 DSTs and another of 6 DSTs as the only related sets of any size to emerge from this analysis. There were clusters with three and four DSTs and four pairs of related DSTs, but most of these comprised multiple isolates from the same patients; the majority of isolates appeared as unrelated singletons.

A UPGMA dendrogram based on pairwise differences be-tween MLST sequences (Fig. 2) indicated a population struc-ture for C. tropicalis isolates that included three clades of

FIG. 1. Unrooted neighbor-joining tree showingp-distance for 30

C. tropicalisisolates in pairs or multiples from 10 patients and one animal. Numbers within the tree indicate the bootstrap values for the cluster nodes.

FIG. 2. Unrooted UPGMA dendrogram for pairwise differences between 88C. tropicalisisolates from separate sources. Numbers within the dendrogram indicate bootstrap values for cluster nodes. Numbers immediately to the right of the dendrogram show the eBURST clonal clusters to which the isolates indicated belong. Three proposed clades of closely related isolates are indicated, as well as a loose cluster of isolates with resistance to the antifungal agent flucytosine.

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isolates that were reasonably robust (bootstrap values of at least 68%) and closely related (within ap-distance of 0.02 or less). These clusters were provisionally designatedC. tropicalis

clades 1 through 3. All but two of the isolates in the group designated clade 1 were in the largest eBURST clonal cluster. The other two UPGMA clusters also correlated with eBURST data. Among all 106 isolates typed in this study, a total of 20 isolates from 11 patients belonged to provisional clade 1. Six isolates, loosely clustered on the UPGMA dendrogram, were all resistant to flucytosine (Fig. 2), but the low bootstrap value for the node of this cluster precludes its designation as a putative clade.

Properties of isolates in different clades. Numbers of iso-lates in the three definable clades were too small to permit statistical analysis of isolate properties by clade. Fifty-six (59.7%) of the 106 isolates shown in Fig. 2 came from the United Kingdom. Clade 1 was dominated by isolates from the United Kingdom (8 of 11 isolates), and there were no clade 1 isolates from North America, whereas clades 2 and 3 showed a small majority (4/6 and 5/9, respectively) of isolates from other countries. The distribution of isolates from blood or other sterile source, among all isolates where the source was known, was uneven: 1/9 in clade 1, 2/4 in clade 2, and 3/5 in clade 3. The set of loosely clustered flucytosine-resistant isolates (Fig. 2) was the only group in the set tested that showed a possible cluster-related property. There was no conspicuous relation-ship between individual alleles and flucytosine resistance.

DISCUSSION

Our data show it is possible to differentiate isolates of C. tropicaliswith a high degree of reproducibility by multilocus sequence typing with the set of six gene fragments described in this study. The six genes chosen meet the requirement for MLST fragments to encode a low ratio of nonsynonymous to synonymous amino acid changes, suggesting they are not under selective pressure (12). Only two of the fragments yielded more than one genotype per polymorphic locus (Table 3). However, these data are biased by the unusual occurrence of a small subset of isolates showing polymorphisms at many more sites in some of the gene fragments than the rest of the isolates. If the five isolates AM2003/0076, AM2005/0005, L634, WC04-200748, and WC04-200749 are excluded from the analysis in Table 3, the genotype/polymorphic site ratio rises to 1.7 for

ICL1, 2.2 forMDR1, 2.3 forSAPT2, 1.4 forSAPT4, and 2.1 for

ZWF1a, with the ratio forXYR1only slightly increased to 3.7. These revised ratios approach those seen with C. albicans

MLST (4). The unusually polymorphic isolates may represent a subtype within the species C. tropicalisbut are unlikely to constitute a possible separate species, since they form PCR products of the same size as all the isolates with all six primers. A totally different situation was encountered with subtypes of

C. parapsilosiswhen we attempted to devise an MLST system for strains within the species and found two stable subtypes that formed products with only a minority of primers and were confirmed as separate species (20). The overall discriminatory power of theC. tropicalisMLST system is very high: at⬎99% probably greater than that of other fungal strain typing systems (10, 18).

As withC. albicans(19), replicateC. tropicalisisolates show

high levels of similarity on an individual patient basis and high levels of difference between patients. The exception concerns isolates in the highly similar cluster here provisionally desig-nated clade 1 (Fig. 2), where isolates from three patients could not be separated by analysis of MLST data. WithinC. albicans

MLST clade 1, a similar subset of indistinguishable isolates was found which also came from several patients. For both species, there are therefore a particularly common DST and a set of very closely related DSTs that constitute a clonal cluster by eBURST analysis and may represent the strain type best adapted to coexistence with humans. Subsequent clonal repro-duction of the species leads to generation of mutants with close affinities to this “optimized” ancestral type.

The major exception to the normal tendency of isolates from the same patient to emerge as highly similar by MLST was L634, a vaginal surveillance isolate from patient 1 in a study published 20 years ago (15). The isolates from this patient have been stored in a collection that has been transported from England to Belgium and thence to Scotland, thus increasing the possibility that cross-contamination or other error means this isolate is not the original one. However, L634 was the first example we encountered of the small subset of isolates with high numbers of (usually heterozygous) polymorphisms in the six gene sequences used for MLST, and the other four exam-ples of such isolates were received subsequent to the first sequencing of L634. The isolate typed as L634 must therefore have already existed in our collection, whether or not its true origin was the same patient as for the other isolates shown as originating from “patient 1” in Fig. 1. Isolates with such un-usually high multiple sequence heterozygosities have not been encountered in our extensive experience ofC. albicansMLST. We shall prospectively investigate the possibility that such iso-lates can be generated from less heterozygous strains by ap-plication of stresses such as antifungal exposures.

The sizes of the three definable clades in the present study were too small to afford insights into possible associations with geographical areas or sites of infection; indeed, the diversity of DSTs as seen by eBURST analysis is greater than was encoun-tered in MLST surveys of smaller isolate sets ofC. albicans(3, 21) andC. glabrata(7). Nevertheless, the observation of a loose cluster of flucytosine-resistant isolates is intriguing, and we are now investigating the possibility that a common mutation un-derlies flucytosine resistance in this set in the same way as was found forC. albicansclade 1 isolates (6, 19).

The relationships between C. tropicalis strain population structure and clinically relevant properties of clades will inev-itably become clearer as the database of typed isolates (http: //pubmlst.org/ctropicalis/) expands. Meanwhile we have added

C. tropicalisto the growing list ofCandidaspecies for which MLST, with its advantages of reproducibility and portability, is now available.

ACKNOWLEDGMENTS

This study was supported by grants from the Wellcome Trust (069615, 074898).

We are grateful to the many colleagues who have supplied us withC. tropicalisisolates, in particular C. C. Kibbler and D. H. Ellis for several recent clinical isolates.

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