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Journal of Clinical Microbiology, November 2001, p. 4042-4051, Vol. 39, No. 11
Departments of Laboratory
Medicine,1 Infectious
Diseases,2 and
Microbiology,3 University of
Washington, Seattle, Washington 98195
Received 11 June 2001/Returned for modification 15 August
2001/Accepted 28 August 2001
Species-specific polymorphisms in the noncoding internal
transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we
tested the hypothesis that ITS1 noncoding regions contain
diagnostically useful alleles. The length of ITS1 region PCR products
amplified from 40 species (106 clinical strains, 5 reference strains,
and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone,
compared with 16 species distinguished by using only ITS2. However,
combination of both ITS alleles permitted identification of 30 species
(98% of clinical isolates). The remaining 10 species with PCR products
of similar sizes contained unique ITS alleles distinguishable by
restriction enzyme analysis. DNA sequence analysis of amplified ITS1
region DNA from 79 isolates revealed species-specific ITS1 alleles for
each of the 40 pathogenic species examined. This provided
identification of unusual clinical isolates, and 53 diagnostic ITS1
sequences were deposited in GenBank. Phylogenetic analyses based on ITS
sequences showed a similar overall topology to 26S rRNA gene-based
trees. However, different species with identical 26S sequences
contained distinct ITS alleles that provided species identification
with strong statistical support. Together, these data indicate that the
analysis of ITS polymorphisms can reliably identify 40 species of
clinically significant yeasts and that the capacity for identifying
potentially new pathogenic species by using this database holds
significant promise.
The complexity of opportunistic
fungal infections is increasing as more patients are adversely affected
by an expanding diversity of yeasts. Invasive procedures or
immunosuppression increases the risk of fungemia (24, 34),
and drug-resistant strains have emerged (9, 21). Although
Candida albicans is still the predominate agent of
nosocomial infection, serious infections caused by other yeasts have
increased in frequency (8, 11, 29, 34). For example,
species of Cryptococcus other than Cryptococcus neoformans, previously considered to be nonpathogenic saprophytes, have been reported to cause cryptococcosis (28). Adequate
treatment of these infections depends on early detection and accurate
identification of the pathogens. However, conventional identification
by evaluation of morphological and physiological characteristics can be
laborious, sometimes leads to incorrect classification and
identification (2, 3, 13), and can be impeded by database
limitations (4, 6).
To develop a rapid molecular method for identifying yeasts, we recently
analyzed the length and sequence polymorphisms in the DNA of noncoding
internal transcribed spacer region 2 (ITS2) of the rRNA operon
(2). ITS2 region polymorphisms permitted accurate
identification of over 400 clinical strains representing 34 species of
pathogenic yeasts. ITS2 region PCR product length determined by
automated capillary electrophoresis permitted single-base-pair precision, with between-run standard deviations (SD) of In this study, we investigated the length and sequence polymorphisms in
the ITS1 region and determined their diagnostic and phylogenetic
utility for identification of medically important yeasts. These DNA
sequences contained unique alleles for all 40 pathogenic species
examined, and ITS1 alleles displayed greater interspecies variability
than ITS2 region sequences. Thirty species, comprising 98% of the
clinical isolates, were easily distinguished by simply determining the
length of the ITS1 and ITS2 region PCR products. The remaining 10 species could be differentiated by either restriction endonuclease or
DNA sequence analysis of ITS PCR products. The sequence diversity among
ITS1 alleles was useful for distinguishing closely related species,
particularly those that contained identical DNA sequences in the D1/D2
variable domain of the 26S rRNA gene. Thus, our ITS sequence data
provide a reliable means with which to rapidly identify known yeast
pathogens, will facilitate the discovery of potentially novel
pathogens, and establish a foundation for further expansion of an ITS
sequence database of medically important fungi.
Yeast isolates.
We examined 106 clinical strains and five
reference strains (Tables 1 and 2)
previously characterized genetically, morphologically, and
biochemically in detail (2). These strains were from our clinical laboratory at the University of Washington Medical Center. Twenty-three type strains from the American Type Culture Collection (ATCC) and 7 type strains from the Centraalbureau voor Schimmelcultures (CBS) were also included in this study (Table
2). These 141 strains represented 40 different species of pathogenic yeasts. Nitrate assimilation, which distinguishes Pichia fabianii from
Pichia veronae, was performed on a slant of nitrate agar
(1.4 g of potassium nitrate, 1.6 g of yeast carbon base from
Difco, 0.12 g of bromthymol blue, 16 g of Noble agar, 1 liter
of distilled water [pH 5.9 to 6.0]) incubated at 30°C for about 1 week until the agar turned from yellow or yellow-green (negative) to
blue (positive). Cryptococcus albidus and Candida
albicans were used as positive and negative controls, respectively
(17).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4042-4051.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences
Identify Medically Important Yeasts
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.5 base, and
92% of the clinical strains examined were rapidly and correctly identified by using only the unique length of their PCR products. The
remaining 8% could be identified by either restriction endonuclease or
DNA sequence analysis of their ITS2 region PCR products.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Lengths of ITS1 and ITS2 region PCR products from
clinical isolates and type strains
TABLE 2.
Length and sequence polymorphisms of ITS1 region DNAs
from clinical and type strains
0.5 base between
runs; and (iii) the apparent PCR product length strongly correlates
(R2 = 0.9992) with the actual number
of nucleotides enumerated by direct sequencing (2). Type
and reference strains were characterized by directly sequencing ITS1
region DNA. ITS1 PCR product length polymorphisms were determined for
randomly selected representatives of each of 40 species (Table 1): for
18 species, multiple isolates were characterized, and for 22 species,
only single isolates were characterized for PCR product length. All
length polymorphisms (Table 1) were subsequently confirmed by direct
sequence analysis of, in most cases, more than one representative
isolate of each species (Table 2). Among the 22 singly listed isolates
in Table 1, 7 isolates represent type strains added to this study for completeness and for which no clinical isolates were available, 7 isolates showed >99% sequence similarity with their respective type
strains, 7 isolates showed >99% sequence similarity with another
sequenced clinical isolate, and 1 clinical isolate, Cryptococcus liquefaciens, was the only representative of that species
characterized by analyzing DNA sequences from ITS1 (Table 2) and ITS2
(Table 3) and the D1/D2 variable
domain of the 26S rRNA gene (Table 4).
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PCR and DNA sequencing. DNA was extracted from yeasts after 48 h of growth on Sabouraud's agar and PCR amplified as previously described (2). ITS1 region DNA was amplified with 900 nM primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'; GIBCO BRL, Grand Island, N.Y.), 300 nM primer ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') (18), and the following thermocycler parameters: 95°C for 6 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by one final extension at 72°C for 10 min. The same parameters were used to amplify the ITS1 and ITS2 regions simultaneously as a single product by using 300 nM ITS1 and ITS4 primers (5'-TCCTCCGCTTATTGATATGC-3') (18). Similarly, 600 nM (each) NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL-4 (5'-GGTCCGTGTTTCAAGACGG-3') (14) was used to amplify the D1/D2 variable domain of the 26S ribosomal DNA (rDNA) gene: denaturation at 94°C for 5 min was followed by 30 cycles of amplification at 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min, followed by one final extension at 72°C for 7 min. Determination of the length of ITS1 PCR products by automated capillary electrophoresis and DNA sequence analysis of both the forward and reverse strands of PCR products (listed in Table 2) were performed as previously described (2).
Sequence similarity and phylogenetic analyses.
DNA sequences
were assembled, edited, and subjected to phylogenetic analyses as
described previously (2). Sequence similarities were
expressed as the percentage of nucleotide differences determined by
pairwise sequence comparisons. Saccharomyces cerevisiae was used as the outgroup instead of Pneumocystis carinii
(2): the ITS1 sequence of S. cerevisiae is distant from all the other sequences (except
Candida glabrata) and thus forms a natural outgroup.
The branching order of the neighbor-joining (23)
dendrograms (Fig. 1) was
evaluated with 1,000 bootstrap analyses by using the SEQBOOT program in the PHYLIP software package (version 3.573) (J. Felsenstein, Department of Genetics, University of Washington, Seattle;
http://evolution.genetics.washington.edu/phylip.html).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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To determine if DNA polymorphisms in the ITS1 region contained diagnostically useful information, we analyzed single PCR products that had been amplified from 141 strains of yeasts, including 30 type and 5 reference strains. Each product contained the 3' end of the 18S rDNA gene, the entire ITS1 region, and the 5' end of the 5.8S rDNA gene. The PCR product length was analyzed for each strain by capillary electrophoresis under denaturing conditions (Table 1), a method that rapidly provides single-base-pair precision, low run-to-run variation, and an excellent correlation (R2 = 0.9992) with the actual number of nucleotides enumerated by direct sequencing (2). To confirm the specificity of length polymorphisms and identify species-specific ITS1 alleles, the forward and reverse strands of ITS1 region PCR products from 79 strains were directly sequenced (Table 2).
ITS1 sequence polymorphisms. ITS1 PCR product lengths determined by capillary electrophoresis ranged from 134.43 bp (Yarrowia lipolytica) to 475.24 bp (C. glabrata) with intraspecies strain variation generally less than 0.5 bp, except for Candida krusei (SD = 0.64 bp), Endomyces fibuliger (SD = 1.14 bp), and S. cerevisiae (SD = 2.09 bp) (Table 1). Similar intraspecies strain variation was observed among ITS2 alleles for Candida dubliniensis, Candida tropicalis, and S. cerevisiae (2), but the distribution of ITS2 region PCR product lengths among all tested species was more limited and ranged from 236.66 to 428.73 bp (Table 1). Of 40 species examined, 19 could be identified based solely on the apparent length of their ITS1 PCR products (species listed individually in Table 1).
Direct DNA sequence analysis of the ITS1 region PCR products (Table 2) confirmed a close correlation of the apparent ITS1 PCR product lengths measured by capillary electrophoresis with the actual product length (2) and revealed species-specific ITS1 DNA sequences for all 40 pathogenic species examined. Intraspecies sequence variability was detected among clinical strains and between clinical and type strains (Table 2). Nucleotide insertions or deletions in the ITS1 region were observed between type and clinical strains of C. albicans and E. fibuliger, as well as among clinical strains of E. fibuliger and S. cerevisiae. For these species, this variation accounts for the SD of
0.5 bp observed
for the PCR product lengths measured by capillary electrophoresis
(Table 1). Similar nucleotide insertions and deletions were found
previously in the ITS2 regions of type and clinical strains of C. glabrata and C. tropicalis, as well as among clinical
strains of C. tropicalis and S. cerevisiae
(2). Conspecific strains examined among a total of 79 isolates in this study, including 30 type strains and 5 reference
strains, generally displayed 99% sequence similarity (Table 2).
Together with the 53 new species-specific diagnostic alleles identified
(Table 2), these data provide two important measures of the diagnostic
utility of ITS1 sequences for identifying medically important yeasts.
Complementary ITS1 and ITS2 polymorphisms. Twenty-one species in Table 1 comprise five groups with nearly identical ITS1 PCR product lengths, yet 11 of these species can be identified if their ITS2 PCR product lengths are also determined: Candida rugosa, Candida lusitaniae; Pichia ohmeri, Candida lambica, Cryptococcus laurentii, Cryptococcus neoformans, Candida utilis, C. dubliniensis, C. albicans, C. tropicalis, and Rhodotorula glutinis. Notably, C. rugosa, P. ohmeri, C. neoformans, C. laurentii, and C. utilis could only be identified by using a combination of ITS1 and -2 length polymorphisms (Table 1) (2). That is, neither ITS PCR product length alone permitted unambiguous identification. ITS2 length polymorphisms alone identified 16 species of yeasts (2) compared with 19 identified by using ITS1 alone (Table 1). However, the combination of ITS1 and ITS2 length polymorphisms accurately identified 30 species of yeasts (Tables 1 and 2). The isolation rate of these 30 species in our clinical laboratory, which serves a spectrum of patients ranging from primary to tertiary care and provides reference laboratory services for five Western states, indicates that >98% of clinical isolates from our laboratory could be identified by ITS length polymorphisms.
The remaining 10 species (Table 1) included four groups that could not be distinguished by ITS region PCR product lengths (Table 3). These isolates could be readily and accurately identified by DNA sequence analysis of either ITS allele (Table 3) or restriction endonuclease analysis of the PCR product containing both ITS1 and ITS2 (Table 3). Two exceptions were Cryptococcus liquefaciens and Cryptococcus diffluens. These closely related species (7) contained identical ITS2 alleles (Table 3) (2), provided indistinguishable BsaHI and HincII restriction analyses from their ITS1 and -2 PCR products, and displayed phenotypic characteristics of Cryptococcus albidus by routine automated biochemical methods and therefore were only distinguishable by the DNA sequences of their ITS1 alleles (Table 3).Identification of unusual isolates. In a previous study (2), we observed >98% concordance between biochemical and ITS2 genotypic identifications validated with over 400 clinical isolates. However, four clinical isolates could not be readily identified by automated biochemical systems (UWFP-345, -363, -366, and -367). Six additional clinical isolates (UWFP-348, -357, -359, -370, -373, and -380) had questionable designations: their identity predicted by biochemical characteristics was clearly not concordant with their ITS2 genotype. The identity of these 10 isolates was established in Table 4, which includes analysis of the hypervariable D1/D2 region of the 26S rRNA gene (5, 13). These data confirm the utility of ITS sequences for correctly identifying C. rugosa and C. pararugosa (Table 4, group 3), C. albidus, C. diffluens, and C. liquefaciens (group 4), R. glutinis and R. rubra (group 8), P. fabianii and P. veronae (group 10), and T. mucoides (group 11). Notably, P. veronae and P. fabianii have identical D1/D2 hypervariable sequences and could only be identified by using ITS alleles (Table 4, group 10).
Table 4 also contains analyses of isolates with ITS1 (Table 2) or ITS2 (2) alleles that showed99% sequence similarity between
type and clinical strains or among clinical strains. Interestingly, the
newly designated C. diffluens and C. liquefaciens
(7) strains share 99.6, 100, and 99.5% sequence
similarity at ITS1, ITS2, and the 26S rRNA hypervariable loci,
respectively (Table 4, group 4). Although >99% similarity is
generally typical of conspecific strains (2, 5, 13, 27),
Table 4 provides several examples where this may not be the case:
Candida kefyr (row 1), C. krusei (row 2),
P. farinosa (group 6), P. ohmeri (group 7), and
Y. lipolytica (group 9). Because species delineation now
encompasses the integration of both physiological and genotypic
characteristics, we use the term "sequevar" to distinguish clinical
isolates with the following characteristics: one diagnostic allele
<99% similar to the type strain while biochemical and two (or more)
other diagnostic loci (defined as 99% similar) concur for the
designation of a particular species
e.g., P. farinosa ITS1
sequevar 1, P. ohmeri ITS1 sequevar 1, and Y. lipolytica ITS1 sequevar 1 (Table 4, groups 6, 7, and 9). It is
possible that further investigation(s) will confirm these as new
species, considering the similarity of the three diagnostic loci
examined for C. diffluens and C. liquefaciens (Table 4). Furthermore, ITS2 sequevars, such as for C. keyfr (Table 4, row 1), may also exist. Together, these data indicate that
ITS analyses are useful for distinguishing closely related clinical isolates.
Phylogenetic analysis. In addition to discriminating among closely related species, we sought to determine if ITS1 alleles could accurately indicate the relationships among distantly related taxa. Phylogenetic trees were constructed with ITS1, ITS2, and 26S sequences (Fig. 1A, B, and C, respectively). Dendrogram topologies derived from the three sets of sequences were highly similar. Similar to the 26S tree, high bootstrap values were observed at the deep branches separating the heterogeneous ascomycetous yeasts and the different clades of the basidiomycetous yeasts. Within each clade, the relationships among species of monophyletic taxa with statistically well-supported peripheral branches were generally concordant between all three markers. Moreover, closely related species with identical 26S sequences could be separated by ITS sequences with strong bootstrap support (e.g., P. veronae and P. fabiani) and species with identical ITS2 sequences could be separated by examining their ITS1 alleles (e.g., C. diffluens and C. liquefaciens). We therefore conclude that ITS loci could be used for taxonomic placement and to infer phylogenetic relationships of previously undescribed species and novel pathogens isolated in the clinical laboratory.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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DNA-based methods have been used successfully to characterize pathogenic yeasts and provide evidence of novel species (1, 2, 5, 7, 10, 12, 14, 25, 27-29, 31). We have established an ITS database that provides accurate identification of at least 40 species of medically important yeasts. Nineteen of these can be rapidly identified by capillary electrophoresis to determine ITS1 PCR product lengths alone, compared with 16 identified by using ITS2 alone (Table 1). The use of both alleles identified 30 species (Table 1), including 4 species that could only be unambiguously designated by using both ITS alleles (see Results). Our diagnostic library of ITS length polymorphisms is validated with over 400 clinical strains (Tables 1 and 2) (2), and together, the ITS1 and -2 length polymorphisms identify >98% of the isolates received by our clinical laboratory.
The direct sequence analysis of 79 isolates confirmed the accuracy of estimating PCR product lengths by capillary electrophoresis (2) and provided 53 new diagnostic ITS1 alleles from a well-characterized group of clinical isolates with concordant biochemical analysis-, ITS1-, and ITS2-based identifications (Table 2). ITS1 alleles showed species specificity and permitted 40 species of pathogenic yeasts to be unambiguously identified by restriction endonuclease or DNA sequence analysis of ITS1 region PCR products (Tables 2 and 3). In combination with sequences from ITS2 (55 alleles total from reference 2 and Table 2) and the D1/D2 hypervariable region of the 26S rRNA gene, unusual clinical isolates were also readily identified (Table 4). Together with the ITS1 alleles, the additional ITS2 and D1/D2 region sequences from this study provide a total of 67 new diagnostic alleles (Table 2, footnote d, and Table 4).
Data from three diagnostic loci, the D1/D2 hypervariable region of the
26S rRNA gene, ITS1, and ITS2, allowed ambiguous biochemical and
genotypic designations to be clarified (Table 4). In this respect, we
found ITS1 to be most useful, because it distinguished among closely
related isolates with identical sequences at either the 26S rRNA gene
or ITS2 loci. Similarly, others have found ITS sequences particularly
useful for distinguishing among closely related yeasts (5, 12,
14, 27, 28, 31). Furthermore, our data indicating that ATCC
11066 (originally designated Candida stellatoidea) is
C. albicans (Table 2) agrees with other molecular data
(20) that C. stellatoidea does not merit
species status (15, 32). Although we observed intraspecies
ITS allelic variation, as has been reported previously (2,
27), conspecific strains generally demonstrated 99% sequence
similarity (Table 2). This result is in agreement with studies
including other loci and large numbers of diverse isolates (2, 5,
12, 14, 27, 28). However, the possibility exists for exceptions
to this benchmark (Table 4), because the converse argument implies that
99% sequence similarity connotes different species: for example,
Cryptococcus diffluens and C. liquefaciens
demonstrate >99% similarity at three diagnostic loci (Table 4 [see
also Results and reference 7]). To facilitate accurate
reporting of genetic data and to maintain the integrity of public
databases for the purposes of identifying clinically significant
yeasts, we use the term "sequevar" to indicate isolates that differ
at one locus (99% sequence similarity) but share two (or more) loci
(99% similarity) concordantly with biochemical and morphological
indicators for a particular species designation. Clearly, additional
investigation will be necessary to accurately determine the species
relationships among taxa containing such isolates.
The data in Table 4 indicate the utility of ITS sequences for distinguishing among closely related yeasts, and we also determined the relevance of ITS sequences for establishing the relationships among distantly related taxa. The phylogenetic relationships observed in the ITS1 and ITS2 trees were highly concordant with those inferred from 26S rDNA sequences (Fig. 1). ITS2 and ITS1 were better than 26S at resolving the taxonomic position of Cryptococcus humicolus. In agreement with our data, this species has been previously shown to belong to the Trichosporon clade by analysis of 18S rDNA and ITS sequences (29), as well as 26S rDNA (10). In the ascomycetous group, Pichia species were polyphyletic in all three dendrograms. These results are concordant with the data of Kurtzman et al., who also showed that Pichia species are polyphyletic and widely distributed among the ascomycetous yeasts based on 26S rDNA phylogenies (13). Thus, our data confirm that ITS1 and ITS2 regions are not only phylogenetically informative, as shown by other investigators (5, 28, 29, 31), but are also useful in distinguishing closely as well as distantly related taxa (Fig. 1). By extrapolation, an expanded ITS sequence database could be used for taxonomic placement and to infer phylogenetic relationships of previously undescribed species and novel pathogens. Ready access to these species-specific DNA sequences, amplified with universal primers, predicts that array-based hybridization schemes will become useful diagnostic tools in the clinical microbiology laboratory.
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FOOTNOTES |
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* Corresponding author. Mailing address: Departments of Laboratory Medicine and Microbiology, University of Washington, 1959 NE Pacific St., NW 120, Box 357110, Seattle, WA 98195. Phone: (206) 598-6131. Fax: (206) 598-6189. E-mail: cookson{at}u.washington.edu.
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Abstract
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Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, November 2001, p. 4042-4051, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4042-4051.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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