Mycology
Genotyping species of the Sporothrix schenckii complex by PCR-RFLP of calmodulin

https://doi.org/10.1016/j.diagmicrobio.2014.01.004Get rights and content

Abstract

Sporotrichosis is one of the most common subcutaneous mycosis in Latin America and is caused by 4 pathogenic thermodimorphic fungi in the genus Sporothrix. From both therapeutic and epidemiological perspectives, it is essential to identify the causative agents down to the species level. Traditional parameters may overlap among closely related species, and we propose polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) as an alternative approach. In the present study, the calmodulin gene was amplified and digested with HhaI to yield 5 different electrophoretic patterns representing all medically important Sporothrix species: Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, and Sporothrix luriei. The PCR-RFLP protocol described here is a simple and inexpensive method and is highly suitable for accurate routine genotyping of relevant Sporothrix species.

Introduction

Sporothrix schenckii sensu lato (s.l.) comprise a group of thermodimorphic pathogenic fungi that may infect subcutaneous tissues of humans and animals (Rodrigues et al., 2013a, Rodrigues et al., 2013b). It is commonly thought that fungal propagules are traumatically introduced from soil and plant debris. The resulting disease is known as sporotrichosis and is found worldwide but is most common in tropical and subtropical regions (Rodrigues et al., 2013a, Silva-Vergara et al., 2012, Verma et al., 2012). Prevalence may take epidemic proportions (Pappas et al., 2000, Rodrigues et al., 2013a, Rodrigues et al., 2013b, Song et al., 2011).

Several potentially pathogenic species are recognized (Marimon et al., 2006, Marimon et al., 2007, Marimon et al., 2008a) including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa, and S. luriei (Zhou et al., 2013). Recently, Sporothrix mexicana (Rodrigues et al., 2013a) and Sporothrix pallida (de Beer et al., 2003, Zhou et al., 2013) were described as rare agents of human sporotrichosis, but both species are placed at a relatively large distance from the clinical clade by phylogenetic analysis, and infections by these species are exceptional.

From clinical and epidemiological perspectives, it is important to have a reliable method for fast and accurate identification of Sporothrix species. Conventional identification based on morphology together with physiological and biochemical characteristics (Marimon et al., 2007, Marimon et al., 2008a) is tedious and unreliable because of the occurrence of deviating strains (Rodrigues et al., 2013a). A wide variety of PCR-based molecular techniques, such as random amplified polymorphic DNA-PCR, nested PCR, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), PCR-enzyme immunoassay, real-time PCR, and microarray technology have been investigated as possible alternatives for routine identification of fungi (De Baere et al., 2010, Kourkoumpetis et al., 2012, Tsui et al., 2011). PCR-RFLP-based screening assays are cheap, fast, easy to perform, simple to interpret, and can be highly discriminatory depending on the target gene.

The aim of the present study was to develop a simple molecular diagnostic technique for the differentiation of clinical Sporothrix isolates. Based on calmodulin (CAL) gene sequence information, an optimal restriction enzyme was identified to distinguish isolates in the S. schenckii complex. A PCR-RFLP assay was tested for specificity against DNA from Sporothrix isolates representing different species of medical interest.

Section snippets

Strains and DNA extraction

The Sporothrix isolates used in this study are shown in Table 1. Type strains were included in all experiments. DNA was extracted and purified directly from fungal colonies using the FastDNA kit protocol (MP Biomedicals, Vista, CA, USA) as described by Rodrigues et al. (2013a). The CAL locus was selected as a target gene due to the large number of sequences available in public databases. In doing so, we attempt to cover most of the genetic diversity described so far for clinical strains. The

Results

PCR amplification of the CAL gene using the CL1 and CL2A primers yielded a single amplicon of approximately 850–900 bp (Fig. 1). The HhaI restriction sites for the species S. brasiliensis are in intron 2 (single site unique to S. brasiliensis) and in exons 4 (single site) and 5 (3 sites). Remaining restriction sites in the exon regions are shared with the sister species S. schenckii s. str. The restriction map for S. schenckii s. str. was conserved intraspecifically, with the exception of 2

Discussion

In general, culture-based methods for identification of fungi are time consuming and often inconclusive due to fungal phenotypic variability. This holds true for identifying species belonging to the S. schenckii complex. In this study, PCR-RFLP analysis of CAL gene sequences was used to distinguish species in a clade of clinically relevant species, i.e., S. brasiliensis, S. schenckii s. str., S. globosa, and S. luriei. The technique was also used to distinguish members of this clade from 2

Acknowledgments

AMR acknowledges financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2011/07350-1). ZPC thanks FAPESP (Proc. 2009/54024-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. 472600/2011-7) for funding. This work was supported in part by grants from FAPESP, CNPq, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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