+ Models MYCMED-592; No. of Pages 8 Journal de Mycologie Médicale (2016) xxx, xxx—xxx Available online at ScienceDirect www.sciencedirect.com ORIGINAL ARTICLE/ARTICLE ORIGINAL Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation Aspergillus tubingensis et Aspergillus niger comme Aspergillus noirs dominants, l’utilisation d’une simple PCR-RFLP pour la diffe´renciation pre´liminaire a, b b c H. Mirhendi *, F. Zarei , M. Motamedi , S. Nouripour-Sisakht a Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran b Department of Medical Parasitology and Mycology, School of Public Health, National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran c Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran Received 3 July 2015; received in revised form 1st December 2015; accepted 7 December 2015 KEYWORDS Summary This work aimed to identify the species distribution of common clinical and Black Aspergillus; environmental isolates of black Aspergilli based on simple restriction fragment length polymor- Aspergillus niger; phism (RFLP) analysis of the b-tubulin gene. A total of 149 clinical and environmental strains of Aspergillus tubingensis; black Aspergilli were collected and subjected to preliminary morphological examination. Total b-tubulin; genomic DNAs were extracted, and PCR was performed to amplify part of the b-tubulin gene. At PCR-RFLP; first, 52 randomly selected samples were species-delineated by sequence analysis. In order to Iran distinguish the most common species, PCR amplicons of 117 black Aspergillus strains were identified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28 were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates included Aspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and 17 BAL samples subjected to TasI-RFLP analysis of the b-tubulin gene, fell into two groups, consisting of about 59% (n = 69) A. tubingensis and 41% (n = 48) A. niger. Therefore, the method successfully and rapidly distinguished A. tubingensis and A. niger as the most common species * Corresponding author. E-mail address: [email protected] (H. Mirhendi). http://dx.doi.org/10.1016/j.mycmed.2015.12.004 1156-5233/# 2016 Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004 + Models MYCMED-592; No. of Pages 8 2 H. Mirhendi et al. among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able to differentiate A. tubingensis and A. niger according to the yellow color reaction specific to A. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical and environmental isolates in Iran. PCR-RFLP using TasI digestion of b-tubulin DNA enables rapid screening for these common species. # 2016 Elsevier Masson SAS. All rights reserved. Re´sume´ Ce travail avait pour but d’identifier la répartition des espèces des isolats cliniques et MOTS CLÉS environnementaux communs d’Aspergillus noirs en se basant sur le simple polymorphisme de longueur des fragments de restriction (RFLP) du gène de la -tubuline. Un total de 149 souches Aspergillus noir ; b cliniques et environnementales d’Aspergillus noirs ont été prélevées et soumises à l’examen Aspergillus niger ; morphologique préliminaire. L’ADN génomique total a été extrait et une PCR a été réalisée pour Aspergillus tubingensis ; amplifier une partie du gène de la -tubuline. Dans un premier temps, 52 échantillons choisis au b-tubuline ; b hasard ont été identifiés en espèces par analyse de séquence. Afin de distinguer les espèces les PCR-RFLP ; Iran plus communes, des amplicons de 117 souches d’Aspergillus noirs ont été identifiés par simple analyse PCR-RFLP en utilisant l’enzyme TasI. Parmi 52 isolats séquencés, 28 étaient Aspergillus tubingensis, 21 Aspergillus niger, et les trois isolats restants étaient : Aspergillus uvarum, Aspergillus awamori, et Aspergillus acidus. Tous les 100 échantillons de l’environnement et les 17 LBA soumis à l’analyse TasI-RFLP du gène de la b-tubuline se sont retrouvés en deux groupes, composés pour 59 % (n = 69) de A. tubingensis et 41 % (n = 48) de A. niger. Ainsi la méthode rapide distingue avec succès A. tubingensis et A. niger, espèces les plus communes chez les isolats cliniques et environnementaux. Bien que plus ancien, le test d’Ehrlich a également été en mesure de différencier A. tubingensis et A. niger selon la réaction de couleur jaune spécifique à A. niger. A. tubingensis et A. niger sont les Aspergillus noirs les plus communs dans les isolats cliniques et environnementaux en Iran. Une PCR-RFLP utilisant TasI avec digestion de l’ADN de la b-tubuline permet un dépistage rapide pour ces espèces communes. # 2016 Elsevier Masson SAS. Tous droits réservés. Introduction piperis, Aspergillus sclerotiicarbonarius, Aspergillus sclero- tioniger, Aspergillus tubingensis and Aspergillus vadensis) being only recently described [26]. Aspergillus species are main members of environmental Members of Aspergillus section Nigri are reported to be saprophytes and are typically included in fungal communities the third most common Aspergillus species associated with of both indoor and outdoor environments. They are normal invasive disease and aspergilloma [4,8,22]. Aspergillus niger components of organic debris, but can be life-threatening has also been reported as the most frequent etiological agent opportunistic agents in debilitated or immunocompromised of otomycosis [15]; other species are rarely reported and patients [3]. The genus Aspergillus includes several groups, may be miss-identified as A. niger [34]. Since different including Aspergillus section Nigri with several species [16], species may have dissimilar susceptibilities to antifungal some of which have been implicated in human disease [1]. drugs, species identification informs the choice of antifungal The taxonomy of Aspergillus section Nigri (known as black therapy [17,2,13]. In addition to their clinical significance, Aspergilli) remains somewhat ill-defined. It comprises a several black Aspergilli have agricultural importance, being closely related group of organisms that are difficult to dis- food spoilage organisms [21]. Ochratoxin A, produced by tinguish based on morphological characteristics such as some Aspergillus species in the section Nigri, is a potent colony morphology, conidial size, and ornamentation [29]. nephrotoxin and potential carcinogen, and concern has been Several approaches including morphological and physiologi- raised regarding the incorporation of this compound into the cal methods have been employed for studding this section human and animal food chain [24]. [29]. Development of molecular DNA-based techniques such We have already used the sequence analysis of b-tubulin as PCR-RFLP [7], RAPD-PCR [27], and nucleotide sequencing genes for species delineation of black Aspergilli isolates [37]. [23] for the identification of fungal strains has resulted in In the present study, the most common species of black reclassification of black Aspergilli, and so these tools are Aspergilli, i.e. A. tubingensis and A. niger, isolated from now being acknowledged as the gold standard [28]. About clinical and environmental samples, are differentiated by 26 species have been recognized within this section, with the use of simple PCR-RFLP analysis. some of them (Aspergillus aculeatinus, Aspergillus aculea- tus, Aspergillus japonicus, Aspergillus uvarum, Aspergillus brasiliensis, Aspergillus carbonarius, Aspergillus costari- Materials and methods caensis, Aspergillus ellipticus, Aspergillus foetidus, Asper- gillus heteromorphus, Aspergillus homomorphus, Strains. A total of 149 clinical and environmental isolates of Aspergillus ibericus, Aspergillus lacticoffeatus, Aspergillus black Aspergillus were used in this study. Forty-nine strains Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004 + Models MYCMED-592; No. of Pages 8 PCR-RFLP to differentiate A. tubingensis and A. niger 3 were isolated from patients with suspected fungal infections the amplicons was electrophoresed using a 1.5% agarose gel referred to diagnostic laboratories in Tehran, Iran, and in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, additional 100 strains were recovered from soil or air in pH 8.3) and visualized under UV irradiation after ethidium different climatic areas of the country or from some food bromide staining. Subsequently, PCR products from 52 sam- products. Air sampling was performed in hospitals and public ples comprising 32 clinical and 20 environmental Aspergillus places using the settled plate method on Dichloran Glycerol strains were purified and sequenced followed by species (DG-18) Agar. Strains were preliminarily identified as A. niger identifications by BLAST analysis (http://blast.ncbi.nlm. based on their macro-/microscopic colony appearance. nih.gov/Blast.cgi). Total genomic