Poster # D-465 Interscience Conference on Antimicrobial Identification and Molecular Typing of Clinical Isolates by Two DNA Sequencing Methods and Automated rep-PCR Agents and Chemotherapy 1 1 1 1 1 2 2 th M. HEALY , D. WALTON , K. REECE , T. BITTNER , J. MANRY , I. RAAD , D. KONTOYIANNIS ; DiversiLab 44 General Meeting, Washington DC, USA www.bacbarcodes.com October 30 – November 2, 2004 Bacterial Barcodes – Spectral Genomics, Inc (1) and The University of Texas MD Anderson Cancer Center, Houston, TX (2). www.mdanderson.org

ABSTRACT Figure 2. Rep-PCR dendrogram. Figure 4. BLAST Results of 28S RNA and EF1 Alpha using NCBI and RESULTS Penn State database

Background: Infections caused by Fusarium species are increasing in frequency among immunocompromised hosts. Confirmation of EF1 alpha EF1 alpha 28s SEQ The DiversiLab System was used to generate rep-PCR DNA fingerprints for each isolate and the similarity comparison is shown as a dendrogram ID by EF1-a Virtual gel images Penn State Blast NCBI BLAST NCBI BLAST Similarity Similarity Similarity suspected fusariosis currently presents a challenge to the clinician, with difficulty in making diagnosis and frequently delaying treatment. The Key # ID Organism score Organism score Organism score for the 21 Fusarium samples. The rep-PCR dendrogram demonstrates species or strain discrimination within the three prominent Fusarium 1 spp. F. solani 93% Fusarium sp. 92.21 Nectria haematococca 99.56 10 F. proliferatum timely identification of these infections is poor and death occurs in up to 80% of the cases despite antifungal therapy. Furthermore the F. dimerum var. complexes(F. solani, F. oxysporum and Giberella fujikoroi), as shown in Figure 2. For example, five distinct rep-PCR fingerprints are seen within F. 2 spp. NM NM nectrioides 97.39 epidemiology of fusariosis (community acquired versus nosocomial) is controversial. The increase of infections and the severity of the 19 F. proliferatum 3 spp. F. solani 95% Fusarium sp. 91.94 Nectria haematococca 99.33 solani (Fig 2). Clustering of the Fusarium species by the DiversiLab System showed high similarity with manually generated EF1a and 28s rRNA Nectria haematococca 7 F. proliferatum mpVII 91.39 outcome now requires an accurate method of species discrimination. Traditional testing is based on colony morphology and molecular F. oxysporum f. sp. F. oxysporum f. sp. F. oxysporum f. sp. sequence dendrograms (Fig 2 and Fig 3). Sequencing data for each of 21 clinical Fusarium samples revealed five species: F. solani, F. oxysporum, F. 16 F. proliferatum 4 spp. cyclaminis 98% opuntiarum 98.52 batatas 96.91 methods, such as sequencing, are laborious. These methods of identifying Fusarium species do not differentiate between strains. This study F. inflexum 96.91 demirum, F. proliferatum and F. globosum (Fig 4). The species or strain discrimination observed in the rep-PCR patterns was expected due to the F. oxysporum f. sp. F. oxysporum f. sp. F. oxysporum f. sp. 6 F. proliferatum uses automated repetitive sequence-based PCR (rep-PCR) to demonstrate discrimination of Fusarium species and strains. Materials and 5 spp. cyclaminis 98% opuntiarum 94.06 batatas 96.92 dynamic complexity of the Fusarium genus. Fusarium is a large genus of hyphomycetes whose classification has been controversial and often F. inflexum 96.92 21 F. proliferatum 6 spp. F. proliferatum 96% F. proliferatum 97.93 Fusarium globosum 96.71 13 Methods: DNA was extracted from 15 Fusarium isolates including several different species, such as F. solani, F. oxysporum, F. proliferatum, and Fusarium confusing . Fusarium solani, Fusarium oxysporum and Giberella fujikoroi are three major complexes that encompasses numerous species and strains. F. pallidorum, previously characterized by morphology, 28S RNA gene sequencing, EF-1A sequencing and rep-PCR. Fifty nanograms of DNA 18 F. proliferatum acutatum 96.71 Fungi named F. solani are now known to belong to at least 26 separate phylogenetic species14 that share anamorphic and telemorphic relationships. ® 17 F. proliferatum Fusarium proliferatum 96.71 was amplified using the DiversiLab Mold Kit. The amplicons were separated using lab-on-a-chip technology and the Agilent 2100 7 spp. F. proliferatum 96% F. proliferatum 94.74 Fusarium globosum 96.49 This can be observed in samples 1,3,9,13 and 26 (Fig 4), where N. haematococca and N africana are anamorphs of F. solani. The Fusarium oxysporum 20 F. proliferatum Gibberella Fusarium Bioanalyzer. Results were analyzed using the system software. Results: The results showed multiple, visually distinct fingerprint patterns. acutatum 96.49 complex is composed of numerous strains classed into several formae speciales based on pathogenic criteria. Each forma specialis groups strains that 13 F. solani Fusarium proliferatum 96.49 15,16 The software classified the organisms into the appropriate Fusarium species groups, concordant with previous characterization. In addition, 8 spp. NM NM Fusarium sp. 96.49 are pathogenic to one particular species or group of plants. Therefore the forma specialis is more indicative of the host rather than phylogenetic 1 F. solani Fusarium buharicum 96.27 subtype differences within samples of the same species were also noted by variations in the patterns and as were displayed by the dendrogram Fusarium camptoceras 96.05 differences. This may explain BLAST results that can differ between two different databases as shown by sample 23 in Fig 4. EF1a BLAST 3 F. solani Neocosmospora 9 spp. africana 94% F. solani f. batatas 93.98 Nectria haematococca 100 indicating pattern similarities. Conclusions: The results indicate that the DiversiLab System can be a useful tool to differentiate Fusarium 9 F. solani Nectria ipomoeae 93.45 data from the PSU Fusarium database return a match for F. oxysporum f. sp. batatas, while EF1a data from NCBI return a F. oxysporum f. sp. Asparagi 10 spp. F. proliferatum 97% F. proliferatum 95.81 Fusarium globosum 96.49 isolates to the species level as well as differentiate samples within a subspecies. Because of the differences seen within a species, this approach 2 F. species Gibberella Fusarium (Fig 4). The 28s rRNA BLAST results returned from NCBI (Fig 4) show that, in some cases, 28s rRNA gene region could only identify Fusarium shows strong promise for studying the epidemiology of fusariosis. acutatum 96.49 complexes and not the individual species or strains that exist within those complexes. Each sample’s 28s rRNA sequence data return multiple 26 F. verticillioides Fusarium proliferatum 96.49 F. oxysporum f. sp. F. oxysporum f. sp. F. oxysporum f. sp. 23 F. oxysporum 11 spp. cyclaminis 98% opuntiarum 92.7 batatas 96.92 matches with identical similarity percentage scores. However, all multiple matches for each respective sample shared a common Fusarium species F. oxysporum f. sp. BACKGROUND 5 F. oxysporum asparagi 92.05 F. inflexum 96.92 complex (F. solani, F. oxysporum or Giberella fujikoroi). Also, both 28s rRNA and EF1a sequence data was needed to identify 20 of the 21 Fusarium F. oxysporum f. sp. F. oxysporum f. sp. F. oxysporum f. sp. 4 F. oxysporum 12 spp. cyclaminis 98% opuntiarum 92.7 batatas 96.92 samples. Samples 2 and 26 (Fig 4) did not return a EF1a BLAST match from either the PSU Fusarium or NCBI databases. However, 28s rRNA F. oxysporum f. sp. Fusariosis, an emerging and severe opportunistic mold infection, is typically a community-acquired mycosis. However, the potential for asparagi 92.03 F. inflexum 96.92 12 F. oxysporum F. solani f. sp. Nectria haematococca Nectria haematococca sequencing indicated that both samples are F. demirum, which for sample 26 is concordant with ATCC identification. nosocomial transmission has been raised, even though this remains unclear. Fusarium species are considered the third most common fungal 13 spp. cucurbitae 98 mpV 93.22 mpV 99.56 11 F. oxysporum Fusarium solani 99.56 1 14 spp. F. globosum 94% F. proliferatum 95.46 Fusarium globosum 96.49 genus (after Candida and Aspergillus) isolated from systemic infections in bone marrow transplant patients . More than 90% of Fusarium 8 F. species Gibberella Fusarium acutatum 96.49 infections are caused by three species: F. solani, F. verticilloides (F. moliniforme), and F. oxysporum, each responsible for about 30% of clinical 15 F. globosum SUMMARY 2 3 Fusarium proliferatum 96.49 cases . The diagnosis of Fusarium infection is principally based on mycology and histopathology . These traditional methods of identification 14 F. globosum 15 spp. F. globosum 95% F. proliferatum 95.74 Fusarium globosum 96.92 Gibberella Fusarium 2 for Fusarium species are time-consuming, and only well-trained mycologists are able to ensure the diagnosis at the genus/species level . 25 F. fujikuroi acutatum 96.92 • The DiversiLab System shows promise as the tool for the identification of Fusarium isolates to the species level as confirmed by two sequencing Fusarium proliferatum 96.92 Furthermore, as noted in various reports, the results are frequently inconclusive with one-third to one-half of 22 F. solani 16 spp. F. proliferatum 97% F. proliferatum 97.64 Fusarium globosum 96.71 methods, in particular EF1a, which was able to provide a more definitive identification. 4,5 Gibberella Fusarium 7 the isolates not identified to the species level . Identification at the species level is important for Figure 1. DiversiLab System. 24 F. species acutatum 96.71 • We experienced that rep-PCR is easier and faster than 28S rDNA gene sequencing. All 21 Fusarium isolates were processed in one day by a Fusarium proliferatum 96.71 epidemiological purposes and may be absolutely necessary as some new antifungal agents show variable 65 70 75 80 85 90 95 100 17 spp. F. proliferatum 95% F. proliferatum 96.71 Fusarium globosum 96.49 single technician with rep-PCR, whereas sequencing the 26 samples required three days. 6 Gibberella Fusarium = ATTC Sample activity against Fusarium, depending on the species . Methods for the molecular typing for Fusarium are either % Similarity acutatum 96.49 • A large, robust rep-PCR library would be necessary to provide identification of a large diverse genus such as Fusarium, where a complex such as 16 laborious (e.g., sequencing), have low resolution or are prone to artifacts (e.g., RAPD analysis). A repetitive Fusarium proliferatum 96.49 Fusarium oxysporum contains more than 150 host-specific formae speciales strains. 7 18 spp. F. proliferatum 95% F. proliferatum 97.77 Fusarium globosum 96.71 sequence-based PCR (rep-PCR) method has been used for bacterial strain typing and for characterizing the Figure 3. Fusarium 28S rRNA gene and elongation factor one alpha phylogenetic tree. Gibberella Fusarium acutatum 96.71 • Epidemiologic investigations could be facilitated by the use of the Diversilab System to aid in resolving the source of outbreaks and the strains 8,9,10 genotypic relatedness among Candida, Aspergillus as well as Mycobacteria isolates . These results support EF1a Fusarium Database from PSU 28S RNA-NCBI Database Fusarium proliferatum 96.71 involved in those outbreaks. Additionally, other uses may include determining the existence and frequency of pathogenic species/strains of 19 spp. F. proliferatum 95% F. proliferatum 96.55 Fusarium globosum 96.92 rep-PCR as a possible method for identification of Fusarium species. We present the use of a commercial Gibberella Fusarium 24 F.species 24 F. dimerum acutatum 96.92 Fusarium. automated rep-PCR assay system (Fig. 1) for the rapid and accurate identification of Fusarium species and the 2 F.species 2 F. dimerum Fusarium proliferatum 96.92 • The complexes that exist in Fusarium along with the numerous anamorphic species provide a very dynamic and at times confusing 10 F. solani 13 F. solani 20 spp. F. proliferatum 97% F. proliferatum 95.5 Fusarium globosum 96.92 potential to obtain strain-level differentiation. Gibberella Fusarium 1 F. solani 7 F. solani acutatum 96.92 nomenclature. A rep-PCR library with standardized nomenclature might allow easier epidemiological tracking of Fusarium infections. In this 20 F. proliferatum 9 F. solani Fusarium proliferatum 96.92 regard, the Diversilab System may be useful for epidemiological studies, surveillance, and differentiation of fungal strains. 6 F. proliferatum 3 F. solani 21 spp. F. proliferatum 97% F. proliferatum 98.27 Fusarium globosum 96.92 METHOD 21 G. fujikuroi Gibberella Fusarium 25 F. fujikuroi • The automation of rep-PCR permits time-efficient, easy-to-use, novel genotyping for the identification and strain typing of fungi. These and 15 G. fujikuroi acutatum 96.92 23 F. oxysporum 8,9,10 20 G. fujikuroi Fusarium proliferatum 96.92 other data suggest that rep-PCR methodology may be a useful tool for reliable identification of Fusarium. 11 F. oxysporum We tested 21 clinical Fusarium species isolates recovered from cancer patients as determined by morphological analysis along with 5 ATCC 19 G. fujikuroi F. solani f. sp. Nectria haematococca Neocosmospora 26 F.verticillioides 22 solani cucurbitae 98% mp I 98.16 vasinfecta 97.01 25 G. fujikuroi Fusarium solani f. sp. Fusarium isolates. Genomic DNA of each sample was extracted using the Mo Bio UltraClean™ Microbial DNA Isolation Kit (Mo Bio 22 F. solani piperis 97.01 16 G. fujikuroi 21 F. proliferatum Neocosmospora REFERENCES Laboratories, CA). Rep-PCR was performed using the Diversilab Mold Kit (Spectral Genomics, Inc) for DNA fingerprinting. The amplicons 10 G. fujikuroi africana 97.01 18 F. proliferatum 18 G. fujikuroi Neocosmospora 1. Guarro J and Gene L. (1992) Fusarium infections. Criteria for the identification of the responsible species. Mycoses 35:109-114. ornamentata 97.01 (1 µl) were separated using microfluidics technology on the Agilent® 2100 Bioanalyzer (Agilent Technologies, Inc.) and the results were 16 F. proliferatum 6 G. fujikuroi F. oxysporum f. sp. F. oxysporum f. sp. F. oxysporum f. sp. 2. Cocuroccia B, et al. (2003) Case Reports: Localized cutaneous hyalohyphomycosis caused by a Fusarium species infection in a renal transplant patient. J. Clin. Microbiol. 41:905-7. 23 oxysporum batatas 98% asparagi 98.44 batatas 96.68 3. Boutati EI and Anaissie EJ. (1997) Fusarium, a significant emerging pathogen in patients with hematologic malignancy: Ten years’ experience at a cancer center and implications for management. Blood. 90:999-1008. 17 F. proliferatum 7 G. fujikuroi analyzed by the Diversilab software v2.1 (Fig 2). Each specimen was additionally subjected to sequencing of both the 28S RNA gene region F. oxysporum f. sp. 19 F. proliferatum 4. Hennequin C, et al. (1997) Invasive Fusarium infections: a retrospective survey of 31 cases. The French Groupe d’Etudes des Mycoses Opportunities. J. Med. Vet. Mycol. 35:107-14. 11 17 G. fujikuroi radicis-lycopersici 98% F. inflexum 96.68 5. Arikan S, et al. (1998) Activity of amphotericin, itraconazole and voriconazole against Aspergillus and Fusarium, adstr. J-19. In Program and abstracts of the 38th ICAAC, ASM, Washington, D.C. and a portion of the elongation factor one alpha region. Two separate amplicons were generated using ITS1 and ITS4 primers and EF1 and 15 F. globosum 14 G. fujikuroi F. dimerum var. 24 dimerum NM NM nectrioides 100 6. Versalovic J, deBruijn FL, Lupski JR. 1998. Bacterial Genomes: Physical Structure and Analysis. Repetitive sequence-based PCR (rep-PCR) DNA Fingerprinting of Bacterial Genomes, Chapman and Hall, New York, NY. 34:437-53. 12 14 F. globosum EF2 primers . The amplicon was purified using High Pure PCR Product Purification Kit (Roche Diagnostics Co., IN) and the subsequent 8 F. species Trichothecium 7. Redkar, RJ, et al. (1996) DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR. J. Clin. Microbiol. 34:1677-81. 12 F. oxysporum 5 F.oxysporum domesticum 96.26 8. Healy, M., K. Reece, D. Walton, J. Huong, K. Shah, and D. P. Kontoyiannis. In Press Sept 2004. Species Identification and Strain Differentiation of Aspergillus Clinical Isolates Using Automated Rep-PCR. J Clin Microbiol 42 Fusarium falciforme 96.04 sequencing reaction was performed with the BigDye® Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, CA) along with ITS1 and 5 F. oxysporum 11 F.oxysporum 25 proliferatum F. fujikuroi 96% Gibberella fujikuroi 96.48 Fusarium globosum 96.44 9. Cangelosi, G. A., et al (2004). Evaluation of ahigh-throughput repetitive-sequence-based PCR system for DNA fingerprinting of Mycobacterium tuberculosis and Mycobacterium avium complex strains. J Clin Microbiol 42:2685-93. 10. Hennequin C, et al. (1999) Identification of Fusarium species involved in human infections by 28S rRNA gene sequencing. J. Clin. Microbiol. 37:3586-89 4 F. oxysporum 12 F.oxysporum ITS4 and EF1and EF2 primers . The products were purified using PERFORMA® DTR Gel Filtration Cartridges (EdgeBioSystems, MD) and Fusarium proliferatum 96.44 11. O'Donnell K, et al. (1998) Multiple evolutionary origins of the causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies. PNAS. 95: 2044 - 2049. 23 F.oxysporum 3 F. solani Gibberella fujikuroi 96.44 12. Daboussi MJ et al. (2002) Evolution of the Fot1 transposons in the genus Fusarium: discontinuous distribution and epigenetic inactivation. Mol Biol Evol. (4):510-20 Gibberella Gibberella sequenced on an ABI-377 (Applied Biosystems, CA). The results were analyzed using Sequencing Analysis v3.3. The contigs were 1 F. solani 4 F.oxysporum 26 fujikuroi F. verticillioides 99% moniliformis 95.67 moniliformis 98.47 13. Summerbell, RC. H.J. Schroers. (2002) Analysis of Phylogenetic Relationship of Cylindrocarpon lichenicola and Acremonium falciforme to the Fusarium solani Species Complex and a Review of Similarities in the Spectrum of Opportunistic Infections constructed using SeqMan (DNA Star, Inc., WI) and identified using BLAST on the NCBI website (www.ncbi.nlm.nih.gov/BLAST/). The 13 F. solani 26 F. species Fusarium Caused by These Fungi J Clin Microbiol. (8):286675. pseudonygamai 98.47 14. Armstrong G. M., J. K. Armstrong, (1981) Formae speciales and races in Fusarium oxysporum causing wilt diseases Pp. 391–399 in P. Nelson, T. A. Tousson, and R. J. Cook, eds. Fusarium: diseases, biology, and . Pennsylvania State 9 F. solani 22 F. solani University Park, University Park, Pa EF1a contigs were also identified using BLAST on the Fusarium Database (http://fusarium.cbio.psu.edu/). NCBI and the Index Fungorum *NM=No BLAST Match return that passed bit score and %similarity parameters 30 40 50 60 70 80 90 100 40 50 60 70 80 90 100 = ATTC Sample 15. Hua-Van A, Langin T, Daboussi MJ. (2001) Evolutionary history of the impala transposon in Fusarium oxysporum. Mol Biol Evol. (10):1959-69. % Similarity % Similarity of the CABI BioScience Databases (www.indexfungorum.org) were used to resolve any discrepancies in the Fusarium nomenclature. 16. Baayen R. P. , K. O’Donnell, P. J. M. Bonants, E. Cigelnik, L. P. N. M. Kroon, E. J. A. Roebroeck, and C. Waalwijk. (2000) Gene Genealogies and AFLP Analyses in the Fusarium oxysporum Complex Identify Monophyletic and Nonmonophyletic Formae Speciales Causing Wilt and Rot Disease. American Phytopathological Society. (90): 891-900. The rep-PCR technology and rep-PCR primers are covered by U.S. patents (5,691,136 and 5,523,217) and by international patents for Canada and Europe. *For Research Use Only. Not For Use In Diagnostic Procedures.