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Genotyping and in vitro antifungal susceptibility of Neoscytalidium dimidiatum isolates from different origins

https://doi.org/10.1016/j.ijantimicag.2009.05.006Get rights and content

Abstract

This study evaluated the genetic variability and in vitro susceptibility patterns of isolates of Neoscytalidium dimidiatum and Scytalidium hyalinum from different geographical origins. Partial sequences of four loci (the ITS region and D1/D2 domains of the 28S rRNA gene and the tubulin and chitin synthase genes) were analysed. Among a total of 1970 bp sequenced in 24 isolates, 7 polymorphic positions (0.36%) were detected, representing five different sequence types (ST1–ST5), from which two (ST2 and ST3) were detected exclusively in isolates from plants, two (ST1 and ST5) were found only in clinical isolates and one (ST4) was observed in isolates from humans and from a mango tree. We propose subordinating S. hyalinum as a variety of N. dimidiatum. Amphotericin B was the most active drug, but low minimum inhibitory concentrations were also detected for voriconazole, terbinafine and anidulafungin.

Introduction

Neoscytalidium dimidiatum is a dematiaceous fungus causing diseases in numerous plant hosts [1]. In humans it produces mostly chronic superficial infections of the hands and feet, clinically resembling dermatophytosis [2]. Cases of mycetoma, subcutaneous lesions, cerebral phaeohyphomycosis and other deep-seated infections have been reported less frequently, mainly affecting immunocompromised patients [2], [3], [4], [5]. The fungus is endemic in tropical and subtropical areas of South America, the Caribbean, Asia and Africa but has been increasingly reported from other non-endemic regions owing to immigration and travel [3].

In culture, N. dimidiatum shows cottony, fast-growing colonies that may appear hyaline at first but soon become dark brown to blackish. Cylindrical to ellipsoidal both hyaline and darkly pigmented arthroconidia are invariably observed. In old cultures, a pycnidial synanamorph may also be produced [1], [2]. Owing to the presence of arthroconidia, earlier literature included this mould in the genus Scytalidium as Scytalidium dimidiatum [3], [4]. A very similar but non-pigmented fungus, Scytalidium hyalinum, has also been reported from human skin and nail infections [2]. Previous molecular studies have suggested that both taxa might be conspecific [6], [7]. Nevertheless, the synonymy has yet not been formally proposed. Recent phylogenetic analyses using sequences of rDNA demonstrated that the type species of the genus Scytalidium, Scytalidium lignicola, and S. dimidiatum grouped into different classes [1]. Therefore, the new genus Neoscytalidium was proposed to accommodate S. dimidiatum, under the name N. dimidiatum [1]. The list of synonyms also included Hendersonula toruloidea and Fusicoccum dimidiatum, but S. hyalinum was not mentioned. Nattrassia mangiferae, another binomial widely used in earlier literature to refer to N. dimidiatum isolates, was considered a different species, which differs from the latter taxon in lacking an arthroconidial anamorph.

Genotyping of pathogenic fungi can provide useful information regarding their epidemiology, routes and sources of infection, cryptic speciation, and the possible association between particular genotypes and medically relevant traits such as resistance to antifungal drugs and virulence. In recent years, several multilocus sequence approaches have been developed for pathogenic fungi [8], [9]. These methods overcome the drawbacks of low reproducibility of image-based methods such as restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP), and facilitate global sharing of genetic data via the Internet. Optimal therapy has not been developed for diseases caused by Neoscytalidium [2]. Newer antifungal agents and alternative therapeutic options need to be explored. This study aimed to (i) assess the genetic relationships among isolates of N. dimidiatum and its hyaline counterpart by analysing sequences of four nuclear loci and (ii) determine whether the sequence types (STs) identified showed different antifungal susceptibility patterns.

Section snippets

Fungal isolates

Twenty-four isolates morphologically identified as N. dimidiatum or S. hyalinum were studied (Table 1). The isolates were cultured on potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI) and were incubated at 25 °C for 5–6 days.

DNA extraction, amplification and sequencing

DNA extraction was performed directly from fungal colonies following the FastDNA® Kit protocol (BIO 101 Inc., Vista, CA), with the homogenisation step repeated five times. Portions of the following loci were chosen for genotyping: the internal transcribed spacer

Nucleotide sequence variability

With the primers used, it was possible to amplify and sequence 284, 444, 434 and 807–808 bp of the CHS, TUB, D1/D2 and ITS loci, respectively. Among a total of 1970 aligned characters of these loci, only 7 polymorphic positions (0.36%) were detected (Table 2), 4 of them belonging to the ITS locus, which exhibited a T  C transition, a G  A transition and one A  T transversion as well as a single-site indel. The CHS locus had two variable positions, consisting of a synonymous T  C transition in the

Discussion

With the aim of clarifying the taxonomy and genetic relationships between N. dimidiatum and S. hyalinum, Roeijmans et al. [6] studied their interspecific and intraspecific variability using rDNA RFLP. Although differences in amplicon lengths were detected in the 18S and 28S rRNA genes among isolates of N. dimidiatum, the RFLP profiles did not distinguish this taxon clearly from S. hyalinum. These results agree with the high nucleotide sequence conservation observed in this study in regions of

Acknowledgments

The authors are indebted to the curators of the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands) for supplying some of the isolates studied.

Funding: This work was supported by the Spanish Ministerio de Educación y Ciencia (grant CGL2007-65669/BOS).

Competing interests: None declared.

Ethical approval: Not required.

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