MycologyGenotyping species of the Sporothrix schenckii complex by PCR-RFLP of calmodulin
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).
References (28)
- et al.
Rapid and specific identification of Sporothrix schenckii by PCR targeting the DNA topoisomerase II gene
J Dermatol Sci
(2005) - et al.
Detection of Sporothrix schenckii chitin synthase 1 (CHS1) gene in biopsy specimens from human patients with sporotrichosis
J Dermatol Sci
(2003) - et al.
A multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species
Mycoscience
(2000) - et al.
Cat-transmitted sporotrichosis epidemic in Rio de Janeiro, Brazil: description of a series of cases
Clin Infect Dis
(2004) - et al.
An epidemic of sporotrichosis in Rio de Janeiro, Brazil: epidemiological aspects of a series of cases
Epidemiol Infect
(2008) - et al.
Evaluation of internal transcribed spacer 2-RFLP analysis for the identification of dermatophytes
J Med Microbiol
(2010) - et al.
Phylogeny of the Ophiostoma stenoceras–Sporothrix schenckii complex
Mycologia
(2003) - et al.
Characterization of virulence profile, protein secretion and immunogenicity of different Sporothrix schenckii sensu stricto isolates compared with S. globosa and S. brasiliensis species
Virulence
(2013) - et al.
Detection of Sporothrix schenckii in clinical samples by a nested PCR assay
J Clin Microbiol
(2003) - et al.
Identification of Sporothrix schenckii based on sequences of the chitin synthase 1 gene
Mycoses
(2001)
Polymerase chain reaction–based assays for the diagnosis of invasive fungal infections
Clin Infect Dis
SATé-II: very fast and accurate simultaneous estimation of multiple sequence alignments and phylogenetic trees
Syst Biol
Epidemiological findings and laboratory evaluation of sporotrichosis: a description of 103 cases in cats and dogs in Southern Brazil
Mycopathologia
Molecular phylogeny of Sporothrix schenckii
J Clin Microbiol
Cited by (67)
Sporotrichosis
2022, Greene's Infectious Diseases of the Dog and Cat, Fifth EditionSporothrix and Sporotrichosis
2022, Encyclopedia of Infection and ImmunityCan the essential oil of rosemary (Rosmarinus officinalis Linn.) protect rats infected with itraconazole-resistant Sporothrix brasiliensis from fungal spread?
2021, Journal of Medical MycologyCitation Excerpt :Besides, the isolate obtained from a disseminated sporotrichosis case has shown melanogenic features. It was stored in the mycological collection of Centro de Diagnóstico e Pesquisa em Micologia Veterinária (UFPEL, Brazil; ID: “MV1710/S120”) and molecularly identified through PCR-restriction fragment length polymorphism-based analysis, according to Rodrigues et al. [22]. The inoculum was prepared based on young S. brasiliensis culture in Potato Dextrose Agar at 27° C, for seven days.
A new duplex PCR assay for the rapid screening of mating-type idiomorphs of pathogenic Sporothrix species
2021, Fungal BiologyCitation Excerpt :As a proof of concept, we demonstrated the importance of incorporating mating-type data in epidemiological studies in Sporothrix. As of the beginning of the epidemic, we have shown, by using molecular tools, that there are two populations of S. brasiliensis occurring in Brazil (Rodrigues et al., 2013a, 2013b, 2014a, 2014b, 2014c, 2016b). The major population is related to the cluster of genotypes circulating in Rio de Janeiro (Rodrigues et al., 2013b, 2014c).
New molecular marker for phylogenetic reconstruction of black yeast-like fungi (Chaetothyriales) with hypothetical EIF2AK2 kinase gene
2020, Fungal BiologyCitation Excerpt :Despite insufficient resolution in some fungal groups, the rDNA internal transcribed spacer is still in frequent use as a primary barcoding marker (de Hoog et al., 1998; Caligiorne et al., 2005; Rezaei-Matehkolaei et al., 2014). Introns in e.g. translation elongation factor 1-alpha subunit (TEF1), beta-tubulin (BT2), beta-actin (ACT1) or calmodulin (CAL) are available as more variable secondary barcoding genes in fungi with a relatively short evolutionary history (O’Donnell et al., 2015; Rodrigues et al., 2014). The search for novel markers has continued, resulting in genes that rather are useable for reconstructing phylogenies than for diagnostics of agents of disease and other species of medical interest, such as Fusarium (Stielow et al., 2015).