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Journal of Clinical Microbiology, October 2005, p. 5278-5280, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5278-5280.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of the DiversiLab System for Species and Strain Differentiation of Fusarium Species Isolates

M. Healy,1* K. Reece,1 D. Walton,1 J. Huong,1 S. Frye,1 I. I. Raad,2 and D. P. Kontoyiannis2*

Bacterial Barcodes, Inc.,1 Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas M. D. Anderson Cancer Center, Houston, Texas2

Received 13 April 2005/ Returned for modification 31 May 2005/ Accepted 12 July 2005


   ABSTRACT
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Advances in molecular typing of fusariosis would facilitate the study of its epidemiology. We tested 26 such isolates by the commercially available DiversiLab System. The system utilizes automated repetitive sequence-based PCR (rep-PCR) and web-based data analyses. rep-PCR dendrogram cluster analysis showed agreement with species sequence identification (elongation factor 1 alpha gene). Additionally, subtype differences within the same species were noted.


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Fusariosis, an emerging and severe opportunistic mold infection, is typically a community-acquired mycosis (15). However, the potential for nosocomial transmission has recently been raised (13). A reliable identification at the species and strain levels is important for epidemiological purposes and may be clinically useful, as there is differential susceptibility of different Fusarium species (18). Traditional determination of Fusarium species that is based on morphological methods is laborious and does not differentiate between strains, and only well-trained mycologists are able to ensure the diagnosis at the genus/species level (3, 18). Not surprisingly, the results are frequently inconclusive, with one-third to one-half of the Fusarium isolates not being identified to the species level (13, 15). Furthermore, the existing molecular methods for the typing of Fusarium either are laborious (e.g., sequencing), have low resolution, or are prone to artifacts (e.g., random amplified polymorphic DNA analysis).

Godoy et al. recently showed the clinical utility of manual repetitive sequence-based PCR (rep-PCR) for genotyping Fusarium strains (7). DiversiLab, an automated rep-PCR method (5), has standardized bacterial strain typing and has been used to characterize the genotypic relatedness among Candida, Aspergillus, Dermatophytes, Zygomycetes, and Mycobacterium isolates (4, 8, 11, 14, 16). For these reasons, we used automated rep-PCR to demonstrate discrimination of Fusarium species and strains.

(This work was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy [Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. D-463, 2004].)

We tested 21 clinical Fusarium isolates recovered from cancer patients (M. D. Anderson mycology laboratory strain collection) and five ATCC Fusarium isolates. Genomic DNA of each sample was extracted using the UltraClean microbial DNA isolation kit (Mo Bio Laboratories, CA). Since the common clinical laboratory identification method does not routinely discriminate between Fusarium species, each specimen was subjected to sequencing of both a 480-bp fragment of the 28S RNA gene region (9) and a 656-bp fragment of the elongation factor 1 alpha (EF-1a) gene region (12). Both sequencing methods were used because it has been reported that the 28S RNA gene region sequencing may be inadequate for identification of Fusarium species (6). Sequences were identified using BLAST on the NCBI website (www.ncbi.nlm.nih.gov/BLAST/) and on the Fusarium database (http://fusarium.cbio.psu.edu/). NCBI and the Index Fungorum of the CABI BioScience databases (www.indexfungorum.org) were used to resolve any discrepancies in the Fusarium nomenclature. The extracted DNA was also amplified using the DiversiLab Mold DNA fingerprinting kit (Bacterial Barcodes, Inc., Houston, TX) according to the manufacturer's instructions. Briefly, 50 ng of genomic DNA, the rep-PCR primer mix provided, 2.5 units AmpliTaq, and 1.5 µl 10x PCR buffer (Applied Biosystems, Foster City, CA) were added for a total of 25 µl per reaction. The thermal cycling parameters were as follows: initial denaturation of 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 70°C for 90 s and a final extension at 70°C for 3 min. The DNA amplicons were detected, and analyses were performed with the DiversiLab System and accompanying software (v. 2.1.66).

The DiversiLab System generated rep-PCR DNA fingerprints for each of the 26 Fusarium isolates, and the similarity comparisons are shown as a dendrogram (Fig. 1). The Fusarium samples generally clustered by species according to the EF-1a identification using a 90% similarity cutoff for the dendrogram, which has been described for the identification of other fungi (8, 14). Sixteen of the 24 samples clustered with same-species isolates. Seven of the 24 samples were outliers (did not cluster within the 90% cutoff), and one sample clustered within the 10% cutoff with a different species. The F. proliferatum group showed two clusters and two outliers, indicating several strain patterns identified for this species. Three of the five F. solani isolates cluster together, yet there are five patterns identified. Four of the five F. oxysporum isolates cluster together, while two patterns are seen. Samples 2 and 8 were identified as Fusarium species because the identification could not be determined by 28S or EF-1a sequencing methods. Sample 2 was not similar to any other sample, while sample 8 might be identified as F. oxysporum according to rep-PCR; however, further testing is needed. The ATCC F. dimerum isolate has a distinct fingerprint. Two of the clinical F. globosum isolates clustered together, and the ATCC F. fujikuroi appears similar to, but not indistinguishable from, these.



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  		FIG. 1. The dendrogram and virtual gel images indicate species and strain discrimination of the 26 Fusarium samples subjected to rep-PCR. Percent similarity was determined by Pearson correlation coefficient, and the dendrogram was constructed by the unweighted pair group method with arithmetic mean. Species identification (ID) is defined in the figure as determined by EF-1a sequencing. A 90% similarity cutoff (vertical line) was chosen for Fusarium species identification using rep-PCR, and strain differences can be appreciated at up to 95% similarity.

 
The subspecies and strain discrimination observed in the rep-PCR patterns was expected due to the dynamic complexity of the Fusarium genus. Fusarium is a large genus of hyalohyphomycetes whose classification has been controversial and often confusing (17). Fungi named F. solani are now known to belong to at least 26 separate phylogenetic species (1) that share anamorphic and telemorphic relationships. The Fusarium oxysporum complex is composed of numerous (over 150) strains classed into several formae speciales based on pathogenic criteria (2). Each forma specialis groups strains that are pathogenic to one particular species or group of plants (2, 10). Therefore, the forma specialis is more indicative of the host rather than phylogenetic differences. As a result of the conflicting (or confusing) nomenclature of formae speciales, EF-1a BLAST data from the PSU Fusarium database returned a match for F. oxysporum f. sp. batatas, while EF-1a data from NCBI returned F. oxysporum f. sp. asparagi (Fig. 1, sample 23).

The DiversiLab System shows promise as a tool for the identification of Fusarium isolates to the species level using sequencing identification as a "gold standard." Its automation permitted time-efficient, easy-to-use, novel genotyping for the identification and strain typing of these fungi. For example, all 26 Fusarium isolates were processed in 1 day by a single technician with rep-PCR, whereas sequencing of the 26 samples required 3 days.

Epidemiologic investigations could be facilitated by the use of the DiversiLab System to aid in resolving the source of outbreaks and the strains involved in those outbreaks, as well as in surveillance by determining the existence and frequency of pathogenic species/strains of Fusarium. Thus, building a large, robust rep-PCR library that includes Fusarium and other, phylogenetically close relatives such as nonpigmented isolates from the Ascomycetes would be crucial to providing identification of a large, diverse genus such as Fusarium. Finally, developing a standardized nomenclature should be a priority, as the complexes that exist in Fusarium along with the numerous anamorphic species provide a very dynamic and at times confusing nomenclature.


   FOOTNOTES
 
* Corresponding author. Mailing address for D. P. Kontoyiannis: Department of Infectious Diseases, Infection Control and Employee Health, Unit 402, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-6237. Fax: (713) 745-6839. E-mail: dkontoyi{at}mdanderson.org. Mailing address for M. Healy: Bacterial Barcodes, Inc., 8080 North Stadium Drive, Suite 1200, Houston, TX 77054. Phone: (713) 467-8500. Fax: (713) 467-7766. E-mail: mhealy{at}bacbarcodes.com. Back


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Journal of Clinical Microbiology, October 2005, p. 5278-5280, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5278-5280.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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