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Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding β-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati.
Aspergillus fumigatus continues to be the most common etiological agent of invasive aspergillosis. Identification of A. fumigatus has historically been based on morphological features; the organism is typified by green conidia produced in chains basipetally from uniseriate phialides (15). However, recent studies have shown that species identification on the basis of morphology alone is problematic; some isolates are pigmentless or sporulate poorly, and growth conditions can influence the morphology, making identification difficult (9). Moreover, some A. fumigatus-related species have similar phenotypic characteristics, making misidentification common when morphological examination alone is used (4, 5, 7).
Molecular phylogenetic typing methods based on the DNA sequences of gene sets have begun to complement and sometimes supersede traditional phenotypic species identification methods (for a review, see reference 6). For example, the conserved ribosomal internal transcribed spacer and the D1 and D2 (domains of the large ribosomal subunit) regions have been used to identify molds to the genus and species levels. At times, these regions are inadequate for the intrasection identification of species, and comparative sequence analyses of other loci are required to make an accurate identification. Recently, sequence data have been used to uncover new and previously described species among clinical isolates morphologically identified as “A. fumigatus” in clinical microbiology laboratories (3-5, 7, 10, 11). These studies frequently used partial sequence data from two or three genes to generate phylogenies that infer genetic relatedness and species boundaries. We previously identified A. lentulus as a unique species on the basis of its phylogenetic relationship to A. fumigatus and other Aspergillus spp. using multilocus sequence typing (MLST) (4). As MLST is not readily performed in clinical microbiology laboratories, we sought to develop a rapid molecular typing method based on PCR of evolutionary informative DNA regions. In the present study, we devised a species identification scheme based on the benA sequence to rapidly identify similar morphotypes in the section Fumigati (A. fumigatus, A. lentulus, and Neosartorya udagawae) using PCR-restriction length fragment polymorphisms (RFLPs). We validated this method by screening a collection of clinical and environmental isolates and by using DNA analysis computer software (i.e., in silico analysis) to examine additional A. lentulus and N. udagawae benA sequences deposited in the GenBank database.
Three hundred eighteen total Aspergillus isolates were used in this study. They included clinical and environmental isolates in our culture collection originating from the University of Washington and the Fred Hutchinson Cancer Research Center. We tested A. fumigatus, A. lentulus, Neosartorya udagawae, A. fumigatus var. ellipticus, N. pseudofischeri, and N. fischeri. Sixty-four strains were obtained from the Centers for Disease Control and Prevention (Atlanta, GA), and 12 were obtained from the University of Texas in San Antonio (Fungus Testing Laboratory, San Antonio, TX). N. fischeri (NRRL 4075) and A. fumigatus var. ellipticus (NRRL 5109) were obtained from S. W. Peterson (U.S. Department of Agriculture, Peoria, IL). Reference A. fumigatus strains Af293 and B-5233 were obtained from David Denning (University of Manchester, Manchester, United Kingdom) and June Kwon-Chung (National Institutes of Health, Bethesda, MD), respectively. The collection included three poorly sporulating strains that were previously identified as A. fumigatus (strains FH1, FH6, and FH219 (5) and that upon retesting with an expanded MLST scheme were found to be A. lentulus (data not shown) (7). The isolates were routinely maintained on potato dextrose agar (Difco, Becton Dickinson and Co.) at 37°C and frozen at −80°C in 8% skim milk for longer-term storage. All aspergilli were identified by morphology and/or previous MLST (3, 5) studies.
Chromosomal DNA was prepared from mycelia grown in Sabouraud dextrose broth (Sigma, St. Louis, MO) at 37°C for 48 h with shaking. Mycelia were harvested between sheets of Miracloth (Calbiochem, La Jolla, CA), and approximately 200 mg (wet weight) was used for DNA extraction by use of an Epicentre kit (MasterPure yeast DNA purification kit; Epicentre Biotech), as modified by Jin et al. (13). DNA quality and yield were determined by agarose gel electrophoresis by standard methods (17).
Previously reported data (4) together with the results of an in-house MLST screen (S. A. Balajee and K. A. Marr, unpublished results) were used to develop a PCR-RFLP based on a 492-bp region of A. fumigatus benA (GenBank accession number AFUA_2G14990) that spans three introns near the 5′ region of the orthologous genes in Emericella nidulans (anamorph, A. nidulans) and A. flavus (8). Amplification of benA of Aspergillus spp. was performed with approximately 75 ng of genomic DNA, forward primer βtub1 (5′-AATTGGTGCCGCTTTCTGG-3′), and reverse primer βtub2 (5′-AGTTGTCGGGACGGAATAG-3′) (4) as follows: the reactions were performed in a volume of 50 μl consisting of PCR buffer (25 mM TAPS [N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; pH 9.3], 50 mM KCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol); 0.2 mM each of dATP, dGTP, dCTP, and dTTP; 0.2 pmol (each) primer; and 2.5 of TaKaRa Ex Taq polymerase (Takara Bio Inc., Japan). After an initial denaturation step of 94°C for 2 min, the reactions were cycled 30 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s in a Techne TC-512 thermal cycler (Techne Inc.). The reactions were terminated with a final incubation at 72°C for 5 min following the last cycle. The amplification of benA was verified by agarose (1.5%) gel electrophoresis of a portion (5 μl) of each reaction mixture. Digestion of the amplicons was performed with 10 μl of the reaction mixtures in a 50-μl reaction volume containing 1× NE Buffer 1 (10 mM Bis-Tris-propane-HCl, 10 mM MgCl2, 1 mM dithiothreitol [pH 7.0]), 100 μg/ml bovine serum albumin, and 1.0 U of BccI enzyme (New England Biolabs) in a water bath at 37°C for 1 h. The resulting DNA fragments (25 μl) were separated in a 3.5% agarose gel and visualized by staining with ethidium bromide, according to standard methods (17). The gel electrophoresis images were captured with a GelDoc-ItTS imaging system (UVP, LLC, Upland, CA), and manipulated with ImageJ processing and analysis software (http://rsb.info.nih.gov/ij/).
Separate experiments investigated the star activity (nonspecific digestion outside a restriction enzyme's recognition site) of BccI that became apparent during the large-scale screening of Aspergillus isolates. Digestion of the benA amplicons with >5 U of BccI in a 50-μl volume resulted in restriction fragments whose sizes did not correlate with the predicted sizes (data not shown). Incubation of the A. fumigatus benA amplicon with XhoI (New England Biolabs) served as control for reactions with excess units of restriction enzyme. The latter digestions were performed with 10 μl of the strain Af293 benA amplicon in a 50-μl reaction mixture containing 1× NE Buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 MgCl2 1 mM dithiothreitol [pH 7.9]) and 10 U of XhoI at 37°C for 1 h. Digestion of the Af293 benA amplicon with XhoI cleaved the DNA once to produce two fragments of 426 bp and 66 bp, respectively.
The benA sequences from A. fumigatus Af293 (GenBank accession number AFUA_2G14990), A. lentulus FH5 (GenBank accession number AY738513 ), and N. udagawae CDC58/FH83 (GenBank accession number DQ058391 ) were aligned by using the ClustalW algorithm within MacVector (version 8.0) software (MacVector, Inc., Cary, NC). Restriction maps of the benA sequences were also generated with MacVector software. Other benA sequences deposited in the GenBank database were used for the in silico analysis of BccI sites (MacVector software) within the amplified region (GenBank accession numbers are given in parentheses): the benA sequences of A. lentulus isolates (total of 21) NRRL 35553 (EF669826), NRRL 35552 (EF669825), NRRL 35551 (EF669824), CBS 612.97 (DQ534081), IFM 41090 (AB248073), IFM 47063 (AB248074), CM-4428 (EU310870), CM-4426 (EU310869), CM-4420 (EU310868), CM-4387 (EU310868), CM-4387 (EU310866), CM-4370 (EU310865), CM-3583 (EU310853), CM-3537 (EU310851), CM-3364 (EU310850), CM-1290 (EU310839), CM-3134 (EU310842), CM-4330 (EU310864), CM-3599 (EU310854), CM-3538 (EU310852), and CM-4415 (EU310867) and N. udagawae isolates (total of 6) IBT 23363 (DQ534161), KACC 41683 (DQ534102), KACC F3759 (DQ534102), CBS 154.89 (DQ534080), CBM FA-0703 (AB248303), and FA-0702 (AB248302).
Previously (5), we developed a PCR-RFLP screen based on rodlet A to discriminate between A. fumigatus and potential A. lentulus isolates. However, that RFLP is based on a single polymorphism within the rodA amplicon, and it does not distinguish other Aspergillus spp. from A. fumigatus. MLST data evaluating seven loci (calmodulin, carboxypeptidase 5, glyceraldehyde 3-phosphate dehydrogenase, class III chitinase G, rodlet A, β-tubulin, and catalase) revealed that benA (the gene encoding β-tubulin) produced the most useful phylogenetic data (4, 5; unpublished data). We elected to utilize BccI to digest the benA amplicon in a PCR-RFLP scheme based on the availability of the enzyme, the sizes of the restriction fragments, and the number of polymorphic sites encoded by more than 1 nucleotide difference among species; the benA homology alignment between A. fumigatus, A. lentulus, and N. udagawae and the BccI restriction digest map are shown in Fig. 1A and B, respectively. This benA PCR-RFLP is predicted to be useful for distinguishing most known section Fumigati members, including A. fumigatus, A. lentulus, N. udagawae, and N. pseudofischeri (Table (Table1).1). However, the digestion of benA amplicons with BccI from N. fischeri or A. fumigatus var. ellipticus is not expected to differentiate these organisms from A. fumigatus (Table (Table11).
Digestion of the benA amplicons produced the expected DNA fragments for each Aspergillus species (Fig. (Fig.2A).2A). The benA digestion pattern for each organism was stable, as judged with independent DNA samples prepared from biological replicates (at least two independent cultures; data not shown). The benA PCR-RFLP was further tested with 284 A. fumigatus isolates, 15 A. lentulus isolates, and 10 N. udagawae/A. udagawae isolates (A. udagawae is the anamorph of N. udagawae; seven of the nine isolates were previously classified as A. udagawae ) and was found to generate results 100% concordant with the predicted DNA banding patterns for each species. We also tested three additional N. pseudofischeri isolates (isolates FH240, FH242, and FH274 ) and confirmed the digestion fragment pattern predicted by the benA sequence data.
During the large-scale screening of our isolates, we found the need to optimize the BccI digestions. The enzyme exhibits a high level of star activity and tends to lose activity over time (P. Zhang, production manager, New England Biolabs, personal communication). Fresh enzyme used at the indicated concentration (see Materials and Methods; 1 U/50 μl digestion) for 1 h at 37°C consistently produced complete digestion of the benA amplicons. The number of units per digest was increased to 5 U as the enzyme aged for several months during storage at −20°C, as determined by the detection of an intact amplicon, in addition to digestion products. The use of >5 U of BccI per digest increased the star activity and generated DNA fragments reminiscent of degradation (Fig. (Fig.2B2B).
All of our A. lentulus and N. udagawae clinical isolates originated from sites within the United States. To determine if the benA BccI polymorphic sites are conserved in other geographically distinct isolates and to increase the robustness of our analysis, we examined A. lentulus (21 entries) and N. udagawae (6 entries) benA sequences deposited in the GenBank database. This in silico analysis of A. lentulus and N. udagawae sequences revealed that the BccI polymorphisms were conserved within each species and that the predicted DNA fragment sizes were in 100% agreement with the experimental results generated with our isolates (data not shown). This suggests that the BccI polymorphisms are genetically stable and characteristic of each species and are thus a useful marker for species discrimination of A. fumigatus morphotypes.
In this study, we have developed a PCR-RFLP method that can be employed to differentiate Aspergillus species within the section Fumigati, with the BccI polymorphisms at a 5′ region of benA distinguishing between 285 A. fumigatus strains (clinical and environmental), 37 A. lentulus strains, and 17 N. udagawae strains and sequences examined. Although the majority of cases of invasive aspergillosis appear to be attributed to A. fumigatus, other related and often misidentified Aspergillus spp. have been uncovered as causes of invasive infections (4, 5, 12); the true burden of these infections remains to be fully elucidated. However, accurate identification may have clinical relevance, as different species are not equally susceptible to antifungal drugs in vitro. For example, A. lentulus exhibits low and variable in vitro susceptibilities to itraconazole, voriconazole, amphotericin B, and the echinocandins (4, 7, 18). In addition, others have reported decreased in vitro susceptibilities of A. lentulus isolates to ravuconazole, posaconazole, and terbinafine (1).
The recognition that Aspergillus species identification solely on the basis of morphological features is insufficient has pushed the development of molecular methods for the purpose of species identification. The current strategy of using sequenced-based identification methods relies upon the accuracy of banked sequence data together with the quality of the data generated from the unknown isolate. Once the sequence data are aligned, a homology score is used to make the identification; and this score may be influenced by the quality of the sequences, the length of the sequence data, and the software used to make the alignment. However, the use of a restriction site polymorphism within a conserved gene for species identification does not depend upon sequence quality and, most importantly, does not rely upon arbitrary cutoff homology scores. Furthermore, same-species homology score cutoff values have not been standardized to date, and there is a lack of consensus on how best to use sequence homology data to identify a species (2). Thus, sequence homology-based methods for the identification of fungi may be approaching the “gold standard,” but a critical parameter for the determination of relatedness, the percent standard homology value, has not been standardized. Other molecular methods that do not rely on pure sequence data can complement or altogether circumvent the need for the sequencing of genetic loci.
We previously developed a molecular identification method that uncovered A. lentulus on the basis of sequence data for five genetic loci that included β-tubulin (benA) (4). β-Tubulin has proven to be useful for phylogenetic relatedness studies of Aspergillus and related species (3-5, 8, 11, 16), because it appears to be a slowly evolving, conserved gene with a high degree of interspecies variability. Recently, an editorial addressing the molecular identification of Aspergillus spp. recommended the use of comparative sequence analysis of β-tubulin for species identification, once isolates are assigned to a species complex or section (e.g., the section Fumigati) on the basis of the nuclear ribosomal internal transcribed region and/or traditional morphological identification methods (2). We have taken this identification method a step further and simplified it by avoiding sequence-based homology caveats. The PCR-RFLP described here takes advantage of BccI restriction site polymorphisms within benA that are unique to A. fumigatus, A. lentulus, and N. udagawae. This methodology can be used together with other identification methods to confirm the identities of A. fumigatus isolates and discriminate those isolates from A. lentulus and N. udagawae. However, one limitation that we encountered was the inability to distinguish A. fumigatus from N. fischeri and A. fumigatus var. ellipticus. This is not surprising, as N. fischeri and A. fumigatus var. ellipticus are closely related to A. fumigatus phylogenetically (8) and perhaps do not warrant separate species designations. N. fischeri has been reported to cause less than five human infections, and A. fumigatus var. ellipticus has not been associated with disease in humans; thus, the clinical importance of these isolates remains undefined. Another related but apparently unusual causative agent of aspergillosis, N. pseudofischeri, which displays decreased in vitro susceptibilities to voriconazole and amphotericin B (3), was readily identified by using BccI benA polymorphisms (Table (Table11 and Fig. Fig.1A1A).
Other previously unknown agents of aspergillosis have emerged from the analysis of atypical A. fumigatus clinical isolates that were reclassified by molecular identification methods (1, 14, 18). These A. fumigatus-related organisms include A. arvii, A. fumisynnematus, A. viridinutans, A. fumigatiaffinis, and N. hiratsukae, which may be of some clinical importance, as several of these species display reduced susceptibilities to multiple antifungal drugs (1, 18). Because these organisms appear to be rare etiologic agents of aspergillosis, the sequence data from these isolates are limited and not available in sufficient numbers for the robust evaluation of our benA PCR-RFLP method at this time.
The true prevalence of these closely related, non-A. fumigatus organisms as causes of human disease is unknown, and we do not yet understand the potential importance of their variable in vitro susceptibility profiles. However, an increased number of studies have reported these isolates, particularly A. lentulus, among clinical culture collections (1, 18). The method described herein will be useful both for microbiology laboratories and for investigations evaluating the prevalence and significance of these newly described opportunistic pathogens.
This study was supported by a National Institutes of Health grant (R21 AI067971) to K.A.M.
We thank Leon W. Razai for technical assistance.
Published ahead of print on 29 April 2009.