Species Identification of Clinical Zygomycetes Isolates by Automated Rep-PCR and DNA Sequencing Diversilab 44Th General Meeting, Washington DC, USA M

Poster # D-466 Interscience Conference on Antimicrobial Agents and Chemotherapy Species Identification of Clinical Zygomycetes Isolates by Automated rep-PCR and DNA Sequencing DiversiLab 44th General Meeting, Washington DC, USA M. HEALY 1, D. WALTON 1, K. REECE 1, M. LISING 1, T. BITTNER 1, S. FRYE1, D. P. KONTOYIANNIS 2 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

Figure 1. Automated rep-PCR process Figure 2. ITS Sequence BLAST results 94ºC for 2 min, 35 cycles of denaturation at 92ºC for 30 sec, annealing at 50ºC for 30 sec, extension at 70ºC for 90 sec, and a final ABSTRACT ITS Sequence NCBI BLAST Results extension at 70ºC for 3 min. Detection and analysis of rep-PCR products were implemented using the DiversiLab System in which rep-PCR primers rep-PCR primers bind to many key Genus Organism Accession# SS the amplified fragments were separated and detected using microfluidics chips with the Agilent 2100 Bioanalyzer (Agilent Background: Human zygomycosis, an emerging and severe invasive mold infection, is caused by members of the class Zygomycetes, specific repetitive sequences interspersed throughout the Figure 3. Consensus dendrogram,1 whichMucor includes a Mucortotal circinelloides of 67 isolates, comparinggb|AY213658.1| clinical 93.75 genome Technologies, Palo Alto, CA), and analysis was performed with the DiversiLab software version 2.1.66. Reports included the order Mucorales; among these, isolates belonging to the genera Mucor, Rhizopus, Rhizomucor and Cunninghamella have been most genome 2 Mucor Mucor circinelloides gb|AY213658.1| 96.64 and environmental isolates to the Burkholderia library. (#) illustrates number of isolates dendrogram, isolate information, and a virtual gel image of each sample. (Figure 1). frequently implicated in causing infections. Morphologic identification at the genus level is frequently imprecise. Although represented in each cluster. 3 Rhizopus Mucor circinelloides gb|AY213658.1| 96.94 zygomycosis is typically acquired in the community, nosocomial zygomycosis including outbreaks have been described. 4 Rhizopus Rhizopus microsporus var. oligosporus dbj|AB097395.1| 99.67 Amplicon separation and detection 4 Rhizopus oryzae dbj|AB097383.1| 99.67 RESULTS using lab-on-a-chip microfluidics Differentiating pseudo-outbreaks from zygomycosis is particularly challenging, since no molecular tools exist for epidemiologic 4 Rhizopus microsporus gb|AY243957.1| 99.67 technology investigation. Materials and Methods: We tested 16 Zygomycetes isolates recovered from cancer patients with definite or probable 5 Rhizopus Rhizopus homothallicus gb|AF115728.1|AF115728 99.81 DNA sequencing and molecular typing of Zygomycetes isolates. The initial identification of the 20 zygomycetes isolates by zygomycosis (EORTC/MSG criteria) including several different genera as determined by morphological analysis; Rhizopus (n=9), 5 Rhizopus microsporus var. oligosporus dbj|AB097395.1| 99.81 Electropherogram morphologic characteristics was as follows: 11 Rhizopus, 4 Rhizomucor, 1 Cunnighamella, 2 Mucor, 2 Zygomycetes of unknown genera. By 5 Rhizopus oryzae dbj|AB097383.1| 99.81 Mucor (n= 2), Rhizomucor (n=3), and Cunninghamella (n=1) and one unspecified zygomycetes. Isolates were cultured on yeast malt agar sequencing of the ITS1 and ITS2 regions, we determined the genus identification of 19 of 20 Zygomycetes isolates and we compared and DNA was extracted for amplification by rep-PCR using the DiversiLab DNA Fingerprinting Kit (Spectral Genomics, Inc). 6 Rhizopus Rhizopus homothallicus gb|AF115728.1|AF115728 99.12 6 Rhizopus microsporus var. oligosporus dbj|AB097395.1| 99.12 Virtual gel image to the morphology-based identification. We were unable to ascribe genus identification in one of the two Zygomycetes isolates that had Amplicon detection and analysis was completed using the DiversiLab System, which includes amplicon separation using lab-on-a- 6 Rhizopus oryzae dbj|AB097383.1| 99.12 unknown genus by morphological classification, the other was identified as a Rhizopus spp by sequence results. There was a 74% chip technology with the Agilent 2100 bioanalyzer and analysis and report generation using the web-based system software. The 7 Rhizopus Rhizopus homothallicus gb|AF115728.1|AF115728 99.81 concordance between morphologic and sequence-based identification (14/19) for the determination of the genus. Three of five of 7 Rhizopus microsporus var. oligosporus dbj|AB097395.1| 99.81 extracted DNA was additionally subjected to ITS gene sequencing (White et al, 1990, Chakrabarti et al, 2003). Results: The ITS the morphological misidentifications were actually Rhizopus spp. Based on the sequence analysis, 74% (14/19) were Rhizopus species. sequencing and the rep-PCR based-genus identification showed 100% concordance to each other but only a 60% concordance with Figure 3. Laboratory, Sequencing, and rep-PCR identification of Zygomycetes isolates 7 Rhizopus oryzae dbj|AB097383.1| 99.81 8 Rhizomucor Rhizopus homothallicus gb|AF115728.1|AF115728 99.82 The BLAST analysis did not allow for convincing species level identification within the Rhizopus isolates. The remaining were 3 morphological identification. Most of the morphological misidentifications were actually Rhizopus spp. Different molecular 8 Rhizopus microsporus gb|AY243957.1| 99.82 Mucor circinelloides, and Mucor racemosus, Cunninghamella bertolettiae, 1 each (Fig 2). Molecular typing analysis. Using automated rep- fingerprinting profiles were demonstrated. Conclusions: The DiversiLab System shows promise as a tool for identification at the 8 Rhizopus microsporus var. oligosporus dbj|AB097395.1| 99.82 PCR, the 20 Zygomycetes isolates showed several different fingerprint patterns (Fig 3). The dendrogram showed three clusters within 9 Rhizopus Rhizopus oryzae gb|AY211273.1| 99.63 genus level and for molecular typing of Zygomycetes. ITS Sequence Clinical Laboratory Virtual Gel Images the Rhizopus group, possibly indicating species or strain discrimination. The were two clusters within the Mucor group, possibly 9 Rhizopus microsporus var. chinensis dbj|AB097367.1| 99.63 Mucor 10 Rhizomucor Mucor racemosus emb|AJ271061.1|MRA271061 97.75 indicating species discrimination as one group contained all 3 M. circinelloides isolates and the other the single isolate of M. racemosus BACKGROUND 11 Zygomycetes Rhizopus oryzae dbj|AB181309.1| 94.44 (isolate 10, Fig 3). In addition, clear differences in all four-fingerprint patterns can be seen. Finally, the isolate identified only as Mucor 11 Amylomyces rouxii gb|AY228094.1| 94.44 Zygomycetes, both by morphological characterization and sequence analysis, grouped with a Rhizopus isolate, suggesting it may belong Zygomycetes are prototypes of refractory fungal pathogens; rhino-cerebral disease, sino-pulmonary infection, or disseminated 12 Rhizopus Rhizopus microsporus dbj|AB097348.1| 95.61 to this genus (isolates 11 and 12, Fig 3). The Cunninghamella isolate (#16) clustered closely with the Rhizopus isolate (#17 Fig 3), yet disease, and cutaneous disease are the most common.1 Human zygomycosis, an emerging and severe invasive mold infection, is *Rhizopus 12 Rhizopus oryzae dbj|AB097349.1| 95.61 13 Zygomycetes Rhizopus oryzae dbj|AB097345.1| 94.35 there are a number of band differences between the isolates as seen in the electropherogram overlay in Figure 4. caused by members of the class Zygomycetes, order Mucorales; among these, isolates belonging to the genera Mucor, Rhizopus, Rhizopus 13 Rhizopus microsporus dbj|AB097348.1| 94.35 Rhizomucor and Cunninghamella have been most frequently implicated in causing infections. This is a rather heterogeneous infection in 14 Rhizopus Rhizopus oryzae dbj|AB097324.1| 97.59 SUMMARY terms of presentation, patient population at risk, and outcome. 2-4 There are some subgroups of patients who are at very high risk of Rhizopus 14 Rhizopus japonicus dbj|AB097347.1| 97.39 rapidly progressive, fatal zygomycosis: pancytopenic patients, patients treated with high dose corticosteroids, patients with persistent 15 Rhizopus Rhizopus oryzae dbj|AB097358.1| 97.39 Rhizopus 15 Rhizopus japonicus dbj|AB097356.1| 97.39 • Characterization of the Zygomycetes by clinical laboratory morphology may be assisted by using sequence analysis for identification severe metabolic acidosis, patients with end-stage renal disease, acidosis and iron overload. Not uncommonly this infection has a 16 Cunninghamella Cunninghamella bertholletiae gb|AF254931.1|AF254931 90.64 of respective genera. Even more powerful is the combination of data obtained from sequencing and molecular typing. 5,6 rapidly fatal course in this patient population. Morphologic identification at the genus level is frequently imprecise. Although Rhizopus 17 Rhizopus Rhizopus oryzae gb|AY211273.1| 99.82 zygomycosis is typically acquired in the community, nosocomial zygomycosis including outbreaks have been described. 17 Rhizopus microsporus var. chinensis dbj|AB097367.1| 99.82 • As illustrated in Figure 2, automated rep-PCR was further able to distinguish within genera possibly to the species and/or strain *Rhizomucor Differentiating pseudo-outbreaks from zygomycosis is particularly challenging, since no molecular tools exist for epidemiologic 18 Rhizomucor Rhizopus sexualis var. americanus dbj|AB113012.1| 99.75 level. It may be possible to utilize a comprehensive rep-PCR fingerprint library of well characterized isolates for additional 7,8 18 Rhizopus oryzae gb|AY211273.1| 99.75 characterization of Zygomycetes in the clinical laboratory. investigation. This study presents the use of automated rep-PCR (Fig 1) and ITS sequencing as molecular tools for zygomycetes Rhizopus identification and strain discrimination. 19 Rhizomucor Rhizopus oryzae dbj|AB109758.1| 99.43 19 Rhizopus microsporus var. chinensis dbj|AB097367.1| 99.43 • Although zygomycosis is typically acquired in the community, nosocomial zygomycosis including outbreaks have been described. *Rhizomucor 20 Rhizopus Rhizopus oryzae dbj|AB109758.1| 99.83 Differentiating pseudo-outbreaks from zygomycosis is particularly challenging, since no molecular tools exist for epidemiologic 8,11-13 METHODS Zygomycetes investigation. This study and others show that the DiversiLab System has promise as a molecular typing tool for identification at the genus, species, sub species and strain level and for microorganisms, in particular, fungi. We tested 20 Zygomycetes isolates recovered from cancer patients with definite or probable zygomycosis (EORTC/MSG criteria) Rhizopus including several different genera as determined by morphological analysis; Rhizopus (n=11), Rhizomucor (n= 4), Mucor (n= 2), and *Zygomycetes REFERENCES Cunninghamella (n=1) and two unspecified zygomycetes. Isolates were cultured on yeast malt agar and DNA was extracted from approximately 10µL hyphae using the UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc., CA). DNA Rhizopus Figure 4. rep-PCR curve overlay of sample 16 and 17 1. Gonzalez C and walsh TJ: zygomycosis. In: yu, V, merigan T, barriere S (editors). Antimicrobial therapy. 2nd ed. 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