Medical Mycology Advance Access published February 11, 2016 Medical Mycology, 2016, 0, 1–15 doi: 10.1093/mmy/myw004 Advance Access Publication Date: 0 2016 Original Article Original Article Phylogenetic analysis of dermatophyte species using DNA sequence polymorphism in calmodulin gene Bahram Ahmadi1,2, Hossein Mirhendi3,∗, Koichi Makimura4, G. Sybren de Hoog5, Mohammad Reza Shidfar2, 6 2 Sadegh Nouripour-Sisakht and Niloofar Jalalizand Downloaded from 1Department of Microbiology and Parasitology, School of Para-Medicine, Bushehr University of Medical Sciences, Bushehr, Iran, 2Departments of Medical Parasitology & Mycology, School of Public Health; National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran, 3Departments of Medical Parasitology & Mycology, School of Medicine, Isfahan University of Medical Sciences, http://mmy.oxfordjournals.org/ Isfahan, Iran, 4Teikyo University Institute of Medical Mycology and Genome Research Center, Tokyo, Japan, 5Fungal Biodiversity Center, Institute of the Royal Netherlands, Academy of Arts and Sciences, Centraalbureau voor Schimmelcultures-KNAW, Utrecht, The Netherlands and 6Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran ∗To whom correspondence should be addressed. Hossein Mirhendi, Professor, Department of Medical Parasitology and Mycology, School of Medicine; Isfahan University of Medical Sciences, Isfahan, Iran. Tel/Fax: +00982188951392; E-mail: [email protected]. by guest on February 12, 2016 Received 23 October 2015; Revised 23 December 2015; Accepted 5 January 2016 Abstract Use of phylogenetic species concepts based on rDNA internal transcribe spacer (ITS) regions have improved the taxonomy of dermatophyte species; however, confirmation and refinement using other genes are needed. Since the calmodulin gene has not been systematically used in dermatophyte taxonomy, we evaluated its intra- and interspecies sequence variation as well as its application in identification, phylogenetic analysis, and taxonomy of 202 strains of 29 dermatophyte species. A set of primers was designed and optimized to amplify the target followed by bilateral sequencing. Using pairwise nu- cleotide comparisons, a mean similarity of 81% was observed among 29 dermatophyte species, with inter-species diversity ranging from 0 to 200 nucleotides (nt). Intraspecies nt differences were found within strains of Trichophyton interdigitale, Arthroderma simii, T. rubrum and A. vanbreuseghemii, while T. tonsurans, T. violaceum, Epidermophyton floccosum, Microsporum canis, M. audouinii, M. cookei, M. racemosum, M. gypseum, T. mentagrophytes, T schoenleinii, and A. benhamiae were conserved. Strains of E. floc- cosum/M. racemosum/M. cookei, A. obtosum/A. gertleri, T. tonsurans/T. equinum and a genotype of T. interdigitale had identical calmodulin sequences. For the majority of the species, tree topology obtained for calmodulin gene showed a congruence with coding and non-coding regions including ITS, BT2,andTef-1α. Compared with the phylogenetic C The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. 1 All rights reserved. For permissions, please e-mail: [email protected] 2 Medical Mycology, 2016, Vol. 00, No. 00 tree derived from ITS, BT2,andTef-1α genes, some species such as E. floccosum and A. gertleri took relatively remote positions. Here, characterization and obtained dendro- gram of calmodulin gene on a broad range of dermatophyte species provide a basis for further discovery of relationships between species. Studies of other loci are necessary to confirm the results. Key words: Dermatophytes, Calmodulin gene, Phylogenetics. Introduction and phenotypically separated Trichophyton species.20,32,33 Annually, millions of humans and animals are infected Moreover, current advances in molecular taxonomy and in- by superficial fungal infections. As specialized filamentous sights into mating discovered that Trichophyton mentagro- fungi, dermatophytes have a specific ability to digest and phytes is a complex of anthropophilic and zoophilic species grow on keratinized host structures such as skin, nails, that produce different teleomorphs, leading to a confusion 34,35 and hair, causing the vast majority of mycotic infections.1,2 with regard to species denomination. Based on their major natural predilection sites, dermato- Regardless of the various advantages of ITS as the pri- phyte species are classified into anthropophilic, geophilic, mary genetic marker of dermatophyte identification, ad- Downloaded from and zoophilic groups. The asexual forms (anamorphs) of ditional verification and refinement using other genes is dermatophytes are classified into Trichophyton, Epidermo- critical to organizing sequence-based and classical species phyton,andMicrosporum, and their sexual forms (teleo- concepts. Since the calmodulin gene has not been systemati- morphs) are members of the genus Arthroderma in the sub- cally used in dermatophyte taxonomy, in this study, our aim http://mmy.oxfordjournals.org/ phylum Ascomycotina.1,3 was to evaluate nucleotide sequence analysis of calmodulin Diagnosis of dermatophyte infections is essential for ap- gene as a new genetic marker for a subset of dermatophyte propriate antifungal therapy because of the length of treat- species, as well as to assess its application with regard to ment, potential side effects of the drugs, and their high identification, phylogenetic analysis, and taxonomy stud- cost. Moreover, having information on zoophilic or an- ies based on intra- and interspecies variation. Several ref- thropophilic sources of the causative dermatophyte agent erence strains and clinical isolates including a wide range may allow prophylactic measures such as treatment of both of common and rare pathogenic species were used for this 4 purpose. animal or human reservoirs. Precise definition and clas- by guest on February 12, 2016 sification of microorganisms including bacteria, parasites, viruses, and fungi have always been of great relevance to Materials and methods taxonomic identity, phylogenetic analysis, epidemiology, and clinical microbiology.5 Reference strains and clinical isolates To resolve the evolutionary relationships between A total of 202 strains of 29 dermatophyte species com- dermatophytes, nucleic acid-based methods have been prising 60 reference strains and 142 clinical isolates were used since early 1980 s. Consequently, several ge- used for sequence analysis of partial calmodulin gene nomic and molecular researches for characterization (Table 1). The reference strains were obtained from Cen- of dermatophytes has revealed a homogeneous group traalbureau voor Schimmelcultures (CBS), Utrecht, the of species with very low genetic diversity in com- Netherlands, and Teikyo University, Institute of Medical parison with overall high phenotypic heterogeneity in Mycology (TIMM), Tokyo, Japan. The clinical isolates dermatophytes.5–8 were collected from a variety of specimens, including skin, The following DNA fragments have been noticed as nail, and hair submitted to two medical mycology labo- the main dermatophyte genetic markers: ribosomal DNA ratories in Tehran, Iran. The clinical isolates were identi- (rDNA) regions,9–13 chitin synthase 1 (CHS1),14–17 DNA fied to species level by an already described PCR-restriction topoisomerase II (TOP-II),18,19 beta tubulin (BT2),20,21 fragment length polymorphism (PCR-RFLP) method9 and and translation elongation factor 1-α (Tef-1α) genes.20,22 in some cases based on ITS-sequencing. Species names Phylogenetic analysis and identification of dermatophytes were determined according to Graser et al.35 Hence, T. based on sequencing of the internal transcribed spacer (ITS) mentagrophytes strains CBS 435.73, NBRC 5809, CBS regions of rDNA has proved to be useful as a gold standard 119445, and NBRC5974, were all identified as T. interdig- method.23–31 Only a small number of nucleotide differences itale by ITS-PCR-RFLP analysis. Also, A. gypseum strain in the ITS regions have been observed in several ecologically CBS 161.69 were identified as A. incurvatum. Ahmadi et al. 3 Ta b l e 1 . Reference and clinical strains of dermatophytes used in this study for partial sequence analysis of the calmodulin gene. GenBank accession numbers, fragment size, and the range of intraspecies variations within the species, are shown. Species (total number Length of calmodulin Range of intra-species of tested strains) Strains (accession numbers) sequence (bp) variation A. benhamiae (4) Ci1947 (KM678214), Ji362 (KP781967), Ji363 (KP781968), 517 0 Ji364 (KP781969) T. concentricum (1) CBS 563.83 (KM387107) 517 – T. erinacei (1) CBS 344.79 (KM387155) 518 – T. eriotrephon (1) CBS 220.25 (KM387109) 518 – T. verrucosum (1) CBS 554.84 (KM387123) 518 – M. audouinii (2) Ji324 (KP781965), Ji325 (KP781966) 488 0 M. canis (9) CBS 130922 (KM387125), CBS 131110 (KM387126), CBS 488 0 130818 (KM387127) Ci 622 (KM387145), Ci 1335 (KM387146), Ci 675 (KM387147), Ci 9219 (KM387148), Ci987 (KM387149), Ci 9938 (KM387150) Downloaded from M. ferrugineum (1) TIMM 445.51 (KP781964) 488 – A. grubyi (1) CBS 243.66 (KM387098) 525 – A. simii (3) CBS 417.65 (KM387128), Ji 77009 (KP781974), Ji 77010 519 0–2 (KP781975) http://mmy.oxfordjournals.org/ T. mentagrophytes (2) CBS 318.56 (KM387110), CBS 101546 (KM387113) 518 0 T. schoenleinii (4) CBS 434.63 (KM387117), CBS 564.94 (KM387118), NBRC 518 0 8192 (KM387119), CBS 130812 (KM387262) A. vanbreuseghmii (3) Ji 405 (KP781970),