Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes

Medical Mycology, 2014, 52, 674–688 doi: 10.1093/mmy/myu033 Advance Access Publication Date: 30 July 2014 Original Article Original Article Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes 1 Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, 2 Departments of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, 3 Teikyo University Institute of Medical Mycology, Tokyo, Japan, 4 Fungal Biodiversity Center, Institute of the Royal Netherlands, Academy of Arts and Sciences, Centraalbureau voor Schimmelcultures-KNAW, Utrecht, The Netherlands and 5 Department of Parasitology and Mycology, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran *To whom correspondence should be addressed. Hossein Mirhendi, Department of Medical Parasitology & Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. E-mail: Received 6 February 2014; Revised 29 March 2014; Accepted 15 April 2014 Abstract We investigated the resolving power of the beta tubulin protein-coding gene (BT2) for systematic study of dermatophyte fungi. Initially, 144 standard and clinical strains belonging to 26 species in the genera Trichophyton, Microsporum, and Epidermophyton were identified by internal transcribe spacer (ITS) sequencing. Subsequently, BT2 was partially amplified in all strains, and sequence analysis performed after construction of a BT2 database that showed length ranged from approximately 723 (T. ajelloi) to 808 nucleotides (M. persicolor) in different species. Intraspecific sequence variation was found in some species, but T. tonsurans, T. equinum, T. concentricum, T. verrucosum, T. rubrum, T. violaceum, T. eriotrephon, E. floccosum, M. canis, M. ferrugineum, and M. audouinii were invariant. The sequences were found to be relatively conserved among different strains of the same species. The species with the closest resemblance were Arthroderma benhamiae and T. concentricum and T. tonsurans and T. equinum with 100% and 99.8% identity, respectively; the most distant species were M. persicolor and M. amazonicum. The dendrogram obtained from BT2 topology was almost compatible with the species concept based on ITS sequencing, and similar clades and species were distinguished in the BT2 tree. Here, beta tubulin was characterized in a wide range of dermatophytes in order to assess intra- and interspecies variation and resolution and was found to be a taxonomically valuable gene. Key words: dermatophyte, beta tubulin, phylogeny, species identification. 674  C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: Ali Rezaei-Matehkolaei1 , Hossein Mirhendi2,∗ , Koichi Makimura3 , G. Sybren de Hoog4 , Kazuo Satoh3 , Mohammad Javad Najafzadeh5 and Mohammad Reza Shidfar1 Rezaei-Matehkolaei et al. Introduction refinement using other genes are overdue [7]. Therefore, characterization of new genetic markers for dermatophyte group is needed. Beta tubulin is a monomeric globular protein involved in the generation of microfilaments; it has been used successfully for species delineation in other groups of fungi such as Aspergillus, Penicillium, Scedosporium, and Phaeoacremonium [14–17]. The locus includes some introns, which are known to be good estimators for distinction of closely related species. Since the beta tubulin gene has not systematically been used in dermatophyte taxonomy, our aim was to investigate the beta tubulin protein-coding gene (BT2) as a new genetic marker in order to evaluate its intra- and interspecies variation in dermatophytes. Several reference strains and clinical isolates, including a wide range of common and rare pathogenic species, were used for this purpose. Materials and methods Strains and isolates A total of 144 strains consisting of 26 species of dermatophytes were used in sequence analysis of partial beta tubulin, comprising 109 reference and 35 clinical isolates (Table 1). The reference strains were obtained from the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands, and the Teikyo University Institute of Medical Mycology (TIMM), Tokyo, Japan. The clinical isolates were recovered from a variety of specimens, including skin, nail, and hair, with proven dermatophytosis submitted to two medical mycology laboratories in Tehran, Iran. Species names used in this study were in concordance with the molecular-based taxonomy introduced by Gräser et al. [7]. DNA extraction DNA was extracted and purified from fungal colonies using a previously described method [18]. Briefly, 10–20 mm3 of the fresh colonies grown on Sabouraud glucose agar (Difco, Detroit, MI, USA) were added to 1.5-ml tubes that contained 300 µl of lysis buffer (200 M Tris-HCl, pH 7.5; 25 mM ethylenediaminetetraacetic acid (EDTA); 0.5% w/v sodium dodecyl sulfate; and 250 mM NaCl) and crushed with a conical grinder (Micro Multi Mixer; IEDA Co. Ltd., Tokyo, Japan) for 1 min. The samples were incubated in a boiling water bath for 10 min, mixed with 150 µl of 3.0 M sodium acetate, kept at −20◦ C for 10 min, and centrifuged at 12 000 rpm for 10 min. The supernatant was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and once more with chloroform. The DNA in supernatant was precipitated with 250 µl isopropanol, washed with 300 ml of 70% ethanol, air dried, and Dermatophytes are currently classified in the genera Trichophyton, Microsporum, and Epidermophyton and are ecologically categorized as geophilic, zoophilic, or anthropophilic. About 30 of the known 40 species of dermatophytes can be listed as potential pathogens of humans and other mammals [1]. They have the ability to degrade keratin of hair, nails, and feathers; can produce superficial infections (dermatophytosis) of the skin of healthy human hosts; and can penetrate into deeper tissues in immunocompromised hosts [2]. Dermatophytosis is the most common fungal infection and one of the most common infectious diseases. Scales from the infected skin and nails transmit Anthropophilic species. In recent years, the incidence of nail dermatophytosis (onychomycosis) has increased and now accounts for up to 90% of toenail and 50% of fingernail disorders [3]. In industrialized nations, about 20% of the population aged >60 years and 50% of the population aged >70 years carry a fungal infection of the foot [4,5]. Treatment of onychomycosis imposes $43 million annually to the US healthcare system [6]. Identification of a dermatophyte to the species level is epidemiologically, ecologically, and therapeutically significant because of routes of infection (eg, M. canis infection originates from cats and dogs, while M. audouinii is transmitted between humans) and different treatment regimens (eg, (...truncated)