Molecular Phylogeny of Sporothrix schenckii
http://www.100md.com
《微生物临床杂志》
Unitat de Microbiologia, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i Virgili, Reus, Tarragona, Spain
Servio de Micologia Medica, Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brazil
ABSTRACT
The pathogenic dimorphic fungus Sporothrix schenckii is the agent responsible for sporotrichosis, an important fungal infection with a worldwide distribution. Little is known about the population structure of S. schenckii, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to determine, by sequence analysis of three protein coding loci (chitin synthase, -tubulin, and calmodulin), whether this variability is due to species divergence or intraspecific diversity in S. schenckii. We included in the analysis 60 isolates (59 of clinical and 1 of environmental origin) of this species from a wide geographical range. DNA sequence data from the three nuclear regions were used in a phylogenetic analysis. The combined analysis of the three loci revealed the presence of three major clades, one grouping all of the European isolates, another with only Brazilian isolates, and the third with isolates from other South American countries and Africa. A total of 14 100% bootstrap-supported nodes were shown, 6 of them representing putative phylogenetic species. Our data also demonstrated that most of these species prevail in different geographical regions.
INTRODUCTION
Sporothrix schenckii is a thermally dimorphic fungus responsible for sporotrichosis, a chronic granulomatous infection of the skin and subcutaneous tissues, although it can disseminate, affecting any organ of the human body (3). The infection is distributed worldwide, although it is more common in tropical and subtropical areas. Despite the clinical importance of S. schenckii, little is known about its basic biology and population structure. S. schenckii has its natural habitat in soil and plants, although it has been isolated from a variety of other sources (5, 14, 26). Recent molecular studies have demonstrated the existence of a high level of intraspecific variability and that isolates are mainly grouped according to their geographical origin (10, 11, 14, 16). More recently, on the basis of internal transcribed spacer (ITS) region sequence analysis, it has been suggested that more than one species could exist within S. schenckii (27). Travassos and Lloyd (26) and Ghosh et al. (6) had also found morphological and physiological differences between isolates of clinical origin and those from other sources. Differences in virulence between clinical and environmental strains were reported, but no correlation was found with the different clinical forms of sporotrichosis (5, 16, 25). In this study, we have used DNA sequence data from multiple loci to assess the extent of clonality within a group of clinical isolates of S. schenckii from different geographic regions. Several molecular studies based on multiple gene sequences have demonstrated the existence of numerous phylogenetic species within well-established morphological species (4, 7, 17). However, the definition of phylogenetic species is controversial (13). Taylor et al. (24) argued that in the recognition of phylogenetic species, one-gene genealogy is not enough and the concordance of multiple-gene genealogies is required. The Rio de Janeiro region has been affected by a long-lasting outbreak of cat-transmitted sporotrichosis (22). The aim of the present study was to determine if these infections are caused by a single, highly virulent strain or by strains with multiple origins.
MATERIALS AND METHODS
Fungal isolates. Sixty isolates morphologically identified as S. schenckii (59 from clinical sources and 1 from an environmental source) were included in this study (Table 1). Clinical isolates were provided by different reference culture collections (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; Facultat de Medicina i Ciencies de la Salut, Reus, Spain; BCCM/IHEM Biomedical Fungi and Yeasts Collection, Brussels, Belgium; Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Brazil; Escola Paulista de Medicina, Universidade Federal de So Paulo, So Paulo, Brazil; and Biological Resource Center, Chiba, Japan). Most of the clinical isolates from Brazil (18 isolates) and the environmental isolate were related to a long-lasting outbreak of cat-transmitted sporotrichosis (22). Clinical isolates were from 11 patients, and the environmental isolate was from the house dust of one of these patients. Isolates were stored on potato dextrose agar (Difco Laboratories, Detroit, Mich.) at 4 to 7°C and in slant cultures submerged in mineral oil at room temperature.
DNA extraction, amplification, and sequencing. DNA was extracted and purified directly from fungal colonies by following the Fast DNA kit protocol (Bio 101, Inc., Vista, Calif.) with the homogenization step repeated three times with a FastPrep FP120 instrument (Thermo Savant, Holbrook, N.Y.). After each homogenization, the sample was kept in ice for 10 min. DNA was quantified with GeneQuant pro (Amersham Pharmacia Biotech, Cambridge, England). Regions of the following nuclear genes were amplified by PCR: the chitin synthase (CHS) gene with primers CHS-79F and CHS-354R (2), the calmodulin (CAL) gene with degenerated primers CL1 and CL2A (17), and the -tubulin (Bt2) gene with degenerated primers designed by us, i.e., Bt2-F [5'GG[CT]AACCA(AG)AT(ATC)GGTGC(CT)GC(CT)3'] and Bt2-R [5'ACCCTC(AG)GTGT AGTGACCCTTGGC3'] from primers Bt2a and Bt2b described by Glass and Donaldson (8). Amplifications were done by following the Ready-To-Go PCR bead protocol (Amersham Bioscience, Freiburg, Germany). For each reaction, we added 20 to 60 ng of DNA template and a 0.5 to 1 mM concentration of each primer in a total volume of 25 μl. The amplification program included an initial denaturation at 94°C for 5 min, followed by 35 cycles consisting of denaturation at 95°C for 30 s, annealing for 1 min at 55°C (CHS) or 60°C (CAL and Bt2), and extension for 1 min at 72°C. A final extension step of 72°C for 7 min was included. The PCR products were purified with a GFXTM PCR DNA purification kit (Pharmacia Biotech, Cerdanyola, Spain) and stored at –20°C until sequencing. PCR products were sequenced by using the above-mentioned primers and following the Taq DyeDeoxy Terminator cycle sequencing kit protocol (Applied Biosystems, Gouda, The Netherlands). DNA sequencing reaction mixtures were analyzed on a 310 DNA sequencer (Applied Biosystems).
Phylogenetic analysis. The sequences were aligned with the ClustalX (version 1.81) computer program (25), followed by manual adjustments with a text editor. The number of nonsynonymous and synonymous mutations was calculated by the method of Nei and Gojobori (15) implemented in the DnaSP program (19). The most parsimonious trees were produced in PAUP (phylogenetic analysis using parsimony and other methods), version 4.0b10 (23). One hundred heuristic searches were performed by random sequence addition and tree bisection-reconnection branch-swapping algorithms, collapsing zero-length branches, and saving all minimal-length trees (MulTrees) on different sets of data. Gaps were treated as missing data. Support for internal branches was assessed by a heuristic parsimony search of 500 bootstrapped sets of data. The combined data set was tested for incongruence with the partition homogeneity test as implemented in PAUP. To test alternative phylogenetic relationships, the Kishino-Hasegawa maximum-likelihood ratio test (12) was performed as implemented in PAUP.
Nucleotide sequence accession numbers. All of the sequences determined in this study were deposited in the EMBL database and assigned the accession numbers listed in Table 1.
RESULTS
With the primers used, we were able to amplify and sequence 280 bp, 410 bp, and 776 bp of the CHS, Bt2, and CAL loci, respectively, in 60 isolates of S. schenckii (Table 1). Of the 1,466 nucleotides sequenced, 1,311 were constant, 132 (9%) were parsimony informative, and 23 were variably parsimony noninformative. The lowest number was 11 in the CHS fragment, and the highest was 87 in the CAL fragment. The total numbers of nonsynonymous and synonymous changes were 0 and 23 (13 in CHS, 5 in Bt2, and 5 in CAL), respectively (Table 2). Sequences of the three genes were analyzed phylogenetically as separate and combined data sets.
In all of the trees obtained from the phylogenetic analysis of each locus, three main clades were shown. One comprised all of the isolates from Brazil (clade I), including also the only environmental isolate tested. Another clade (clade II) grouped the rest of the South American isolates and three more isolates, including the type strain. The third clade (clade III) grouped the European isolates, all from Spain, with the exception of two isolates of unknown origin.
The result of the partition homogeneity test showed that sequence data sets of the three loci were congruent (P = 0.11) and could therefore be combined. A total of 5,000 most parsimonious trees were produced by a heuristic search using the combined data set of 1,466 characters from the three loci. The trees had a consistency index of 0.925, a retention index of 0.991, and a homoplasy index of 0.074. A total of 29 different genotypes could be distinguished (Fig. 1). A total of 14 100% bootstrap-supported nodes were shown, representing six putative phylogenetic species. The clustering was similar to that observed in the particular trees of the different genes analyzed, especially with the CAL locus. The tree showed the same three main clades observed with the other loci, each of them receiving 100% bootstrap support.
The 18 isolates related to the long-lasting Brazilian outbreak of cat-transmitted sporotrichosis were located with the rest of the isolates from this country in clade I. They were isolated from 11 patients (Table 3). Four patients had more than one isolate. In two cases (P3 and P8), these belonged to a single genotype. Patients P4 and P9 were infected by two different genotypes each. The 18 isolates belonged to a total of six genotypes. Genotype G1 was the most common, infecting seven patients. Genotypes G1 and G2 were also present in other patients from Brazil not related to the outbreak.
Clade II was divided into two highly (100%) supported subclades (IIa and IIb). The Peruvian isolates, which were the most numerous after the Brazilian ones, were distributed into two different branches (IIa-1 and IIb-1), each with 100% bootstrap support. Subclade IIa was further divided into IIa-1, which included isolates from other countries (Bolivia, Colombia, and Argentina), and IIa-2, which had one isolate of unknown origin and the type strain. A similar distribution was observed in subclade IIb, in which the Peruvian branch (IIb-1) formed a phylogenetic group clearly separated from the other branch (IIb-2) comprising two isolates, one from South Africa and one from Argentina. This analysis showed the existence of a European clade (clade III). The latter clade was the most phylogenetically distant.
DISCUSSION
This report contains the results of a molecular phylogenetic analysis of the S. schenckii species complex inferred from DNA sequence data from three different loci. One of the most interesting results is the discovery that the isolates of S. schenckii, practically all of clinical origin, were grouped into six putative phylogenetic species. These cryptic species were further subdivided into a number of smaller groups that appear to be reproductively isolated in nature. This suggests not only that the existing S. schenckii populations are in the process of divergence but also that all of the resulting lineages are undergoing separation into distinct taxa. Our results were validated by the recent study of Neyra et al. (16), who analyzed the genetic diversity of the same Peruvian isolates that we tested, by using amplified fragment length polymorphism analysis and ITS2 sequencing. In that study, the isolates were grouped into two clades that were similar to those obtained in our phylogenetic trees.
Another interesting aspect of our analysis was the finding that each of the main groups exhibited a degree of geographical specificity, which agrees with previous reports (14, 16). The possible existence of different species within S. schenckii was already suggested by Wilhelm de Beer et al. (27) on the basis of the analysis of the sequences of the ITS regions of 11 clinical and environmental isolates from South Africa. Our results disagree with those of Ishizaki (10), who found, by using mitochondrial DNA analysis, that all of the clinical isolates studied (more than 500) belonged to the same species.
In our study, the combined analysis of the three loci revealed the presence of two major groups, one including the European isolates and the other the South American and South African isolates (Fig. 1). In the latter group, two clades were observed, one formed by the 29 Brazilian isolates and the second by the rest. These results agree with those of Neyra et al. (16), in whose amplified fragment length polymorphism study the European isolates, two from Belgium and one from Italy, were also the most distantly placed and clearly separated from the American isolates. In our study, one South African isolate was close to one South American isolate. This result is in agreement with that of Ishizaki et al. (11), who found, by using restriction fragment length polymorphism analysis of mitochondrial DNA, that the South American isolates studied were related to the South African isolates studied. The 24 mitochondrial DNA restriction fragment length polymorphism types described were classified into two large phylogenetic groups, one predominant in South America and Africa and the other predominant in Australia and Asia. The biogeographical pattern shown in all of these studies seems to correlate with the events associated with the formation of natural barriers created by the fragmentation of the ancient supercontinent Gondwana in the upper Cretaceous period through the Paleocene period over the last 100 million years. O'Donnell et al. proposed this to account for the radiating speciation of the Gibberella fujikuroi species complex (18).
Although the genetic separation is considerable among the three major monophyletic clades, i.e., the Spanish clade, the Brazilian clade, and the clade made up of the rest of the South American isolates, each of them shows a high level of clonality. Primitive populations were probably isolated by the separation of the continents, and the formation of natural barriers facilitated their speciation as they became adapted to hosts endemic to the different regions. However, although geographical separation of the main clades is clearly evident, the different genotypes present within them are not related to geography (16), which seems to indicate that there has been interbreeding within these isolated populations.
Our analysis revealed that six different genotypes were present in a sample of 18 isolates from the Brazilian outbreak of cat-transmitted sporotrichosis, which demonstrates that the infections are not caused by a single, highly virulent genotype, as could be thought. In the city of Rio de Janeiro and the surrounding areas, from 1987 to 1997, only 13 cases of human sporotrichosis were recorded, but from 1998 to 2004, 759 human cases were reported (21). It is difficult to explain this dramatic increase in Sporothrix infections in this region. The researchers who have reported all of these cases are unable to provide an explanation for this epidemic (21). However, it should be noted that the highest number of cases occurred in an area characterized by underprivileged socioeconomic conditions and precarious health services. The typical human patients were female, mainly housewives, which is normal if we consider that members of this group are those most frequently exposed to the fungus because they care for cats (22). Barros et al. (1) explained the wide dissemination of the disease with factors related to the behavior of cats which, although cohabiting with human beings, do not always stay in the house but also circulate in the neighborhood, often getting involved in fights with other animals and coming into contact with soil and plants.
In conclusion, S. schenckii appears to be a complex of species, some prevailing in certain geographical regions. An accurate knowledge of species limits could be of high medical interest, as they may show different clinical patterns and respond differently to therapy. For instance, the Brazilian isolates present a distinctive clinical picture with immune manifestations (erythema multiforme) (9), disseminated cutaneous lesions, and atypical forms (20).
ACKNOWLEDGMENTS
We are indebted to the curators of the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands), the BCCM/IHEM Biomedical Fungi and Yeasts Collection (Brussels, Belgium), J. M. Torres (IMIM, Hospital del Mar, Barcelona, Spain), C. Rubio (Hospital Universitario Lozano Blesa, Zaragoza, Spain), R. Negroni (Hospital de Infecciosas Francisco Javier Muiz, Buenos Aires, Argentina), and P. Godoy (Escola Paulista de Medicina, Universidade Federal de So Paulo, So Paulo, Brazil) for supplying many of the strains used in this study.
This study was supported by Spanish Ministerio de Ciencia y Tecnología grant CGL 2004-00425/BOS.
FOOTNOTES
Corresponding author. Mailing address: Unitat de Microbiologia, Departament de Ciencies Mediques Basiques, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i Virgili, Carrer Sant Lloren 21, 43201 Reus, Tarragona, Spain. Phone: 34 977759359. Fax: 34 977759322. E-mail: josepa.gene@urv.cat.
REFERENCES
Barros, M. B., A. O. Schubach, A. C. do Valle, M. C. Gutierrez Galhardo, F. Conceicao-Silva, T. M. Schubach, R. S. Reis, B. Wanke, K. B. Marzochi, and M. J. Conceicao. 2004. Cat-transmitted sporotrichosis epidemic in Rio de Janeiro, Brazil: description of a series of cases. Clin. Infect. Dis. 38:529-535.
Carbone, I., and L. M. Kohn. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553-556.
de Hoog, G. S., J. Guarro, J. Gene, and M. J. Figueras. 2000. Atlas of clinical fungi, 2nd ed. Centralbureau voor Schimmelcultures, Utrecht, The Netherlands.
Dettman, J. R., D. J. Jacobson, and J. W. Taylor. 2003. A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora. Evol. Int. J. Org. Evol. 57:2703-2720.
Dixon, D. M., I. F. Salkin, R. A. Duncan, N. J. Hurd, J. H. Haines, M. E. Kemna, and F. B. Coles. 1991. Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis. J. Clin. Microbiol. 29:1106-1113.
Ghosh, A., P. K. Maity, B. M. Hemashettar, V. K. Sharma, and A. Chakrabarti. 2002. Physiological characters of Sporothrix schenckii isolates. Mycoses 45:449-454.
Gilgado, F., J. Cano, J. Gene, and J. Guarro. 2005. Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species. J. Clin. Microbiol. 43:4930-4942.
Glass, N., and G. Donaldson. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61:1323-1330.
Gutierrez-Galhardo, M. C., M. B. Barros, A. O. Schubach, T. Cuzzi, T. M. Schubach, M. S. Lazera, and A. C. Valle. 2005. Erythema multiforme associated with sporotrichosis. J. Eur. Acad. Dermatol. Venereol. 19:507-509.
Ishizaki, H. 2003. Mitochondrial DNA analysis of Sporothrix schenckii. Jpn. J. Med. Mycol. 44:155-157.
Ishizaki, H., M. Kawasaki, M. Aoki, T. Matsumoto, A. A. Padhye, M. Mendoza, and R. Negroni. 1998. Mitochondrial DNA analysis of Sporothrix schenckii in North and South America. Mycopathologia 142:115-118.
Kishino, H., and M. Hasegawa. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29:170-179.
Mayden, R. L. 1997. A hierarchy of species concepts: the denouement in the saga of the species problem, p. 381-424. In M. F. Claridge, H. A. Dawah, and M. R. Wilson (ed.), Species: the units of biodiversity. Chapman & Hall, London, United Kingdom.
Mesa-Arango, A. C., M. R. Reyes-Montes, A. Perez-Mejía, H. Navarro-Barranco, V. Souza, G. Zúiga, and C. Toriello. 2002. Phenotyping and genotyping of Sporothrix schenckii isolates according to geographic origin and clinical form of sporotrichosis. J. Clin. Microbiol. 40:3004-3011.
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.
Neyra, E., P. Fonteyne, D. Swinne, F. Fauche, B. Bustamante, and N. Nolard. 2005. Epidemiology of human sporotrichosis investigated by amplified fragment length polymorphism. J. Clin. Microbiol. 43:1348-1352.
O'Donnell, K. 2000. Molecular phylogeny of the Nectria haematococca-Fusarium solani species complex. Mycologia 92:919-938.
O'Donnell, K., E. Cigelnik, and H. I. Nirenberg. 1998. Molecular systematics and phytogeography of the Gibberella fujikuroi species complex. Mycologia 90:465-493.
Rozas, J., J. C. Sanchez del Barrio, X. Messeguer, and R. Rozas. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496-2497.
Schubach, A., M. B. de Lima Barros, T. M. Schubach, A. C. Francesconi-do-Valle, M. C. Gutierrez-Galhardo, M. Sued, M. de Matos Salgueiro, P. C. Fialho-Monteiro, R. S. Reis, K. B. Marzochi, B. Wanke, and F. Conceicao-Silva. 2005. Primary conjunctival sporotrichosis: two cases from a zoonotic epidemic in Rio de Janeiro, Brazil. Cornea 24:491-493.
Schubach, A., T. M. Schubach, and M. B. L. Barros. 2005. Epidemic cat-transmitted sporotrichosis. N. Engl. J. Med. 353:1185-1186.
Schubach, A., T. M. Schubach, M. B. L. Barros, and B. Wanke. 2005. Cat-transmitted sporotrichosis, Rio de Janeiro, Brazil. Emerg. Infect. Dis. 11:1952-1954.
Swofford, D. L. 2001. PAUP. Phylogenetic analysis using parsimony (and other methods). (version 4.0). Sinauer Associates, Sunderland, Mass.
Taylor, J. W., D. J. Jacobson, S. Kroken, T. Kasuga, D. M. Geiser, D. S. Hibbett, and M. C. Fisher. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genet. Biol. 31:21-32.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.
Travassos, L. R., and K. O. Lloyd. 1980. Sporothrix schenckii and related species of Ceratocystis. Microbiol. Rev. 44:683-721.
Wilhelm de Beer, Z., T. C. Harrington, H. F. Vismer, B. D. Wingfield, and M. J. Wingfield. 2003. Phylogeny of the Ophiostoma stenoceras-Sporothrix schenckii complex. Mycologia 95:434-441.(Rita Marimon, Josepa Gene, Josep Cano, L)
Servio de Micologia Medica, Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brazil
ABSTRACT
The pathogenic dimorphic fungus Sporothrix schenckii is the agent responsible for sporotrichosis, an important fungal infection with a worldwide distribution. Little is known about the population structure of S. schenckii, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to determine, by sequence analysis of three protein coding loci (chitin synthase, -tubulin, and calmodulin), whether this variability is due to species divergence or intraspecific diversity in S. schenckii. We included in the analysis 60 isolates (59 of clinical and 1 of environmental origin) of this species from a wide geographical range. DNA sequence data from the three nuclear regions were used in a phylogenetic analysis. The combined analysis of the three loci revealed the presence of three major clades, one grouping all of the European isolates, another with only Brazilian isolates, and the third with isolates from other South American countries and Africa. A total of 14 100% bootstrap-supported nodes were shown, 6 of them representing putative phylogenetic species. Our data also demonstrated that most of these species prevail in different geographical regions.
INTRODUCTION
Sporothrix schenckii is a thermally dimorphic fungus responsible for sporotrichosis, a chronic granulomatous infection of the skin and subcutaneous tissues, although it can disseminate, affecting any organ of the human body (3). The infection is distributed worldwide, although it is more common in tropical and subtropical areas. Despite the clinical importance of S. schenckii, little is known about its basic biology and population structure. S. schenckii has its natural habitat in soil and plants, although it has been isolated from a variety of other sources (5, 14, 26). Recent molecular studies have demonstrated the existence of a high level of intraspecific variability and that isolates are mainly grouped according to their geographical origin (10, 11, 14, 16). More recently, on the basis of internal transcribed spacer (ITS) region sequence analysis, it has been suggested that more than one species could exist within S. schenckii (27). Travassos and Lloyd (26) and Ghosh et al. (6) had also found morphological and physiological differences between isolates of clinical origin and those from other sources. Differences in virulence between clinical and environmental strains were reported, but no correlation was found with the different clinical forms of sporotrichosis (5, 16, 25). In this study, we have used DNA sequence data from multiple loci to assess the extent of clonality within a group of clinical isolates of S. schenckii from different geographic regions. Several molecular studies based on multiple gene sequences have demonstrated the existence of numerous phylogenetic species within well-established morphological species (4, 7, 17). However, the definition of phylogenetic species is controversial (13). Taylor et al. (24) argued that in the recognition of phylogenetic species, one-gene genealogy is not enough and the concordance of multiple-gene genealogies is required. The Rio de Janeiro region has been affected by a long-lasting outbreak of cat-transmitted sporotrichosis (22). The aim of the present study was to determine if these infections are caused by a single, highly virulent strain or by strains with multiple origins.
MATERIALS AND METHODS
Fungal isolates. Sixty isolates morphologically identified as S. schenckii (59 from clinical sources and 1 from an environmental source) were included in this study (Table 1). Clinical isolates were provided by different reference culture collections (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; Facultat de Medicina i Ciencies de la Salut, Reus, Spain; BCCM/IHEM Biomedical Fungi and Yeasts Collection, Brussels, Belgium; Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Brazil; Escola Paulista de Medicina, Universidade Federal de So Paulo, So Paulo, Brazil; and Biological Resource Center, Chiba, Japan). Most of the clinical isolates from Brazil (18 isolates) and the environmental isolate were related to a long-lasting outbreak of cat-transmitted sporotrichosis (22). Clinical isolates were from 11 patients, and the environmental isolate was from the house dust of one of these patients. Isolates were stored on potato dextrose agar (Difco Laboratories, Detroit, Mich.) at 4 to 7°C and in slant cultures submerged in mineral oil at room temperature.
DNA extraction, amplification, and sequencing. DNA was extracted and purified directly from fungal colonies by following the Fast DNA kit protocol (Bio 101, Inc., Vista, Calif.) with the homogenization step repeated three times with a FastPrep FP120 instrument (Thermo Savant, Holbrook, N.Y.). After each homogenization, the sample was kept in ice for 10 min. DNA was quantified with GeneQuant pro (Amersham Pharmacia Biotech, Cambridge, England). Regions of the following nuclear genes were amplified by PCR: the chitin synthase (CHS) gene with primers CHS-79F and CHS-354R (2), the calmodulin (CAL) gene with degenerated primers CL1 and CL2A (17), and the -tubulin (Bt2) gene with degenerated primers designed by us, i.e., Bt2-F [5'GG[CT]AACCA(AG)AT(ATC)GGTGC(CT)GC(CT)3'] and Bt2-R [5'ACCCTC(AG)GTGT AGTGACCCTTGGC3'] from primers Bt2a and Bt2b described by Glass and Donaldson (8). Amplifications were done by following the Ready-To-Go PCR bead protocol (Amersham Bioscience, Freiburg, Germany). For each reaction, we added 20 to 60 ng of DNA template and a 0.5 to 1 mM concentration of each primer in a total volume of 25 μl. The amplification program included an initial denaturation at 94°C for 5 min, followed by 35 cycles consisting of denaturation at 95°C for 30 s, annealing for 1 min at 55°C (CHS) or 60°C (CAL and Bt2), and extension for 1 min at 72°C. A final extension step of 72°C for 7 min was included. The PCR products were purified with a GFXTM PCR DNA purification kit (Pharmacia Biotech, Cerdanyola, Spain) and stored at –20°C until sequencing. PCR products were sequenced by using the above-mentioned primers and following the Taq DyeDeoxy Terminator cycle sequencing kit protocol (Applied Biosystems, Gouda, The Netherlands). DNA sequencing reaction mixtures were analyzed on a 310 DNA sequencer (Applied Biosystems).
Phylogenetic analysis. The sequences were aligned with the ClustalX (version 1.81) computer program (25), followed by manual adjustments with a text editor. The number of nonsynonymous and synonymous mutations was calculated by the method of Nei and Gojobori (15) implemented in the DnaSP program (19). The most parsimonious trees were produced in PAUP (phylogenetic analysis using parsimony and other methods), version 4.0b10 (23). One hundred heuristic searches were performed by random sequence addition and tree bisection-reconnection branch-swapping algorithms, collapsing zero-length branches, and saving all minimal-length trees (MulTrees) on different sets of data. Gaps were treated as missing data. Support for internal branches was assessed by a heuristic parsimony search of 500 bootstrapped sets of data. The combined data set was tested for incongruence with the partition homogeneity test as implemented in PAUP. To test alternative phylogenetic relationships, the Kishino-Hasegawa maximum-likelihood ratio test (12) was performed as implemented in PAUP.
Nucleotide sequence accession numbers. All of the sequences determined in this study were deposited in the EMBL database and assigned the accession numbers listed in Table 1.
RESULTS
With the primers used, we were able to amplify and sequence 280 bp, 410 bp, and 776 bp of the CHS, Bt2, and CAL loci, respectively, in 60 isolates of S. schenckii (Table 1). Of the 1,466 nucleotides sequenced, 1,311 were constant, 132 (9%) were parsimony informative, and 23 were variably parsimony noninformative. The lowest number was 11 in the CHS fragment, and the highest was 87 in the CAL fragment. The total numbers of nonsynonymous and synonymous changes were 0 and 23 (13 in CHS, 5 in Bt2, and 5 in CAL), respectively (Table 2). Sequences of the three genes were analyzed phylogenetically as separate and combined data sets.
In all of the trees obtained from the phylogenetic analysis of each locus, three main clades were shown. One comprised all of the isolates from Brazil (clade I), including also the only environmental isolate tested. Another clade (clade II) grouped the rest of the South American isolates and three more isolates, including the type strain. The third clade (clade III) grouped the European isolates, all from Spain, with the exception of two isolates of unknown origin.
The result of the partition homogeneity test showed that sequence data sets of the three loci were congruent (P = 0.11) and could therefore be combined. A total of 5,000 most parsimonious trees were produced by a heuristic search using the combined data set of 1,466 characters from the three loci. The trees had a consistency index of 0.925, a retention index of 0.991, and a homoplasy index of 0.074. A total of 29 different genotypes could be distinguished (Fig. 1). A total of 14 100% bootstrap-supported nodes were shown, representing six putative phylogenetic species. The clustering was similar to that observed in the particular trees of the different genes analyzed, especially with the CAL locus. The tree showed the same three main clades observed with the other loci, each of them receiving 100% bootstrap support.
The 18 isolates related to the long-lasting Brazilian outbreak of cat-transmitted sporotrichosis were located with the rest of the isolates from this country in clade I. They were isolated from 11 patients (Table 3). Four patients had more than one isolate. In two cases (P3 and P8), these belonged to a single genotype. Patients P4 and P9 were infected by two different genotypes each. The 18 isolates belonged to a total of six genotypes. Genotype G1 was the most common, infecting seven patients. Genotypes G1 and G2 were also present in other patients from Brazil not related to the outbreak.
Clade II was divided into two highly (100%) supported subclades (IIa and IIb). The Peruvian isolates, which were the most numerous after the Brazilian ones, were distributed into two different branches (IIa-1 and IIb-1), each with 100% bootstrap support. Subclade IIa was further divided into IIa-1, which included isolates from other countries (Bolivia, Colombia, and Argentina), and IIa-2, which had one isolate of unknown origin and the type strain. A similar distribution was observed in subclade IIb, in which the Peruvian branch (IIb-1) formed a phylogenetic group clearly separated from the other branch (IIb-2) comprising two isolates, one from South Africa and one from Argentina. This analysis showed the existence of a European clade (clade III). The latter clade was the most phylogenetically distant.
DISCUSSION
This report contains the results of a molecular phylogenetic analysis of the S. schenckii species complex inferred from DNA sequence data from three different loci. One of the most interesting results is the discovery that the isolates of S. schenckii, practically all of clinical origin, were grouped into six putative phylogenetic species. These cryptic species were further subdivided into a number of smaller groups that appear to be reproductively isolated in nature. This suggests not only that the existing S. schenckii populations are in the process of divergence but also that all of the resulting lineages are undergoing separation into distinct taxa. Our results were validated by the recent study of Neyra et al. (16), who analyzed the genetic diversity of the same Peruvian isolates that we tested, by using amplified fragment length polymorphism analysis and ITS2 sequencing. In that study, the isolates were grouped into two clades that were similar to those obtained in our phylogenetic trees.
Another interesting aspect of our analysis was the finding that each of the main groups exhibited a degree of geographical specificity, which agrees with previous reports (14, 16). The possible existence of different species within S. schenckii was already suggested by Wilhelm de Beer et al. (27) on the basis of the analysis of the sequences of the ITS regions of 11 clinical and environmental isolates from South Africa. Our results disagree with those of Ishizaki (10), who found, by using mitochondrial DNA analysis, that all of the clinical isolates studied (more than 500) belonged to the same species.
In our study, the combined analysis of the three loci revealed the presence of two major groups, one including the European isolates and the other the South American and South African isolates (Fig. 1). In the latter group, two clades were observed, one formed by the 29 Brazilian isolates and the second by the rest. These results agree with those of Neyra et al. (16), in whose amplified fragment length polymorphism study the European isolates, two from Belgium and one from Italy, were also the most distantly placed and clearly separated from the American isolates. In our study, one South African isolate was close to one South American isolate. This result is in agreement with that of Ishizaki et al. (11), who found, by using restriction fragment length polymorphism analysis of mitochondrial DNA, that the South American isolates studied were related to the South African isolates studied. The 24 mitochondrial DNA restriction fragment length polymorphism types described were classified into two large phylogenetic groups, one predominant in South America and Africa and the other predominant in Australia and Asia. The biogeographical pattern shown in all of these studies seems to correlate with the events associated with the formation of natural barriers created by the fragmentation of the ancient supercontinent Gondwana in the upper Cretaceous period through the Paleocene period over the last 100 million years. O'Donnell et al. proposed this to account for the radiating speciation of the Gibberella fujikuroi species complex (18).
Although the genetic separation is considerable among the three major monophyletic clades, i.e., the Spanish clade, the Brazilian clade, and the clade made up of the rest of the South American isolates, each of them shows a high level of clonality. Primitive populations were probably isolated by the separation of the continents, and the formation of natural barriers facilitated their speciation as they became adapted to hosts endemic to the different regions. However, although geographical separation of the main clades is clearly evident, the different genotypes present within them are not related to geography (16), which seems to indicate that there has been interbreeding within these isolated populations.
Our analysis revealed that six different genotypes were present in a sample of 18 isolates from the Brazilian outbreak of cat-transmitted sporotrichosis, which demonstrates that the infections are not caused by a single, highly virulent genotype, as could be thought. In the city of Rio de Janeiro and the surrounding areas, from 1987 to 1997, only 13 cases of human sporotrichosis were recorded, but from 1998 to 2004, 759 human cases were reported (21). It is difficult to explain this dramatic increase in Sporothrix infections in this region. The researchers who have reported all of these cases are unable to provide an explanation for this epidemic (21). However, it should be noted that the highest number of cases occurred in an area characterized by underprivileged socioeconomic conditions and precarious health services. The typical human patients were female, mainly housewives, which is normal if we consider that members of this group are those most frequently exposed to the fungus because they care for cats (22). Barros et al. (1) explained the wide dissemination of the disease with factors related to the behavior of cats which, although cohabiting with human beings, do not always stay in the house but also circulate in the neighborhood, often getting involved in fights with other animals and coming into contact with soil and plants.
In conclusion, S. schenckii appears to be a complex of species, some prevailing in certain geographical regions. An accurate knowledge of species limits could be of high medical interest, as they may show different clinical patterns and respond differently to therapy. For instance, the Brazilian isolates present a distinctive clinical picture with immune manifestations (erythema multiforme) (9), disseminated cutaneous lesions, and atypical forms (20).
ACKNOWLEDGMENTS
We are indebted to the curators of the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands), the BCCM/IHEM Biomedical Fungi and Yeasts Collection (Brussels, Belgium), J. M. Torres (IMIM, Hospital del Mar, Barcelona, Spain), C. Rubio (Hospital Universitario Lozano Blesa, Zaragoza, Spain), R. Negroni (Hospital de Infecciosas Francisco Javier Muiz, Buenos Aires, Argentina), and P. Godoy (Escola Paulista de Medicina, Universidade Federal de So Paulo, So Paulo, Brazil) for supplying many of the strains used in this study.
This study was supported by Spanish Ministerio de Ciencia y Tecnología grant CGL 2004-00425/BOS.
FOOTNOTES
Corresponding author. Mailing address: Unitat de Microbiologia, Departament de Ciencies Mediques Basiques, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i Virgili, Carrer Sant Lloren 21, 43201 Reus, Tarragona, Spain. Phone: 34 977759359. Fax: 34 977759322. E-mail: josepa.gene@urv.cat.
REFERENCES
Barros, M. B., A. O. Schubach, A. C. do Valle, M. C. Gutierrez Galhardo, F. Conceicao-Silva, T. M. Schubach, R. S. Reis, B. Wanke, K. B. Marzochi, and M. J. Conceicao. 2004. Cat-transmitted sporotrichosis epidemic in Rio de Janeiro, Brazil: description of a series of cases. Clin. Infect. Dis. 38:529-535.
Carbone, I., and L. M. Kohn. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553-556.
de Hoog, G. S., J. Guarro, J. Gene, and M. J. Figueras. 2000. Atlas of clinical fungi, 2nd ed. Centralbureau voor Schimmelcultures, Utrecht, The Netherlands.
Dettman, J. R., D. J. Jacobson, and J. W. Taylor. 2003. A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora. Evol. Int. J. Org. Evol. 57:2703-2720.
Dixon, D. M., I. F. Salkin, R. A. Duncan, N. J. Hurd, J. H. Haines, M. E. Kemna, and F. B. Coles. 1991. Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis. J. Clin. Microbiol. 29:1106-1113.
Ghosh, A., P. K. Maity, B. M. Hemashettar, V. K. Sharma, and A. Chakrabarti. 2002. Physiological characters of Sporothrix schenckii isolates. Mycoses 45:449-454.
Gilgado, F., J. Cano, J. Gene, and J. Guarro. 2005. Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species. J. Clin. Microbiol. 43:4930-4942.
Glass, N., and G. Donaldson. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61:1323-1330.
Gutierrez-Galhardo, M. C., M. B. Barros, A. O. Schubach, T. Cuzzi, T. M. Schubach, M. S. Lazera, and A. C. Valle. 2005. Erythema multiforme associated with sporotrichosis. J. Eur. Acad. Dermatol. Venereol. 19:507-509.
Ishizaki, H. 2003. Mitochondrial DNA analysis of Sporothrix schenckii. Jpn. J. Med. Mycol. 44:155-157.
Ishizaki, H., M. Kawasaki, M. Aoki, T. Matsumoto, A. A. Padhye, M. Mendoza, and R. Negroni. 1998. Mitochondrial DNA analysis of Sporothrix schenckii in North and South America. Mycopathologia 142:115-118.
Kishino, H., and M. Hasegawa. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29:170-179.
Mayden, R. L. 1997. A hierarchy of species concepts: the denouement in the saga of the species problem, p. 381-424. In M. F. Claridge, H. A. Dawah, and M. R. Wilson (ed.), Species: the units of biodiversity. Chapman & Hall, London, United Kingdom.
Mesa-Arango, A. C., M. R. Reyes-Montes, A. Perez-Mejía, H. Navarro-Barranco, V. Souza, G. Zúiga, and C. Toriello. 2002. Phenotyping and genotyping of Sporothrix schenckii isolates according to geographic origin and clinical form of sporotrichosis. J. Clin. Microbiol. 40:3004-3011.
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.
Neyra, E., P. Fonteyne, D. Swinne, F. Fauche, B. Bustamante, and N. Nolard. 2005. Epidemiology of human sporotrichosis investigated by amplified fragment length polymorphism. J. Clin. Microbiol. 43:1348-1352.
O'Donnell, K. 2000. Molecular phylogeny of the Nectria haematococca-Fusarium solani species complex. Mycologia 92:919-938.
O'Donnell, K., E. Cigelnik, and H. I. Nirenberg. 1998. Molecular systematics and phytogeography of the Gibberella fujikuroi species complex. Mycologia 90:465-493.
Rozas, J., J. C. Sanchez del Barrio, X. Messeguer, and R. Rozas. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496-2497.
Schubach, A., M. B. de Lima Barros, T. M. Schubach, A. C. Francesconi-do-Valle, M. C. Gutierrez-Galhardo, M. Sued, M. de Matos Salgueiro, P. C. Fialho-Monteiro, R. S. Reis, K. B. Marzochi, B. Wanke, and F. Conceicao-Silva. 2005. Primary conjunctival sporotrichosis: two cases from a zoonotic epidemic in Rio de Janeiro, Brazil. Cornea 24:491-493.
Schubach, A., T. M. Schubach, and M. B. L. Barros. 2005. Epidemic cat-transmitted sporotrichosis. N. Engl. J. Med. 353:1185-1186.
Schubach, A., T. M. Schubach, M. B. L. Barros, and B. Wanke. 2005. Cat-transmitted sporotrichosis, Rio de Janeiro, Brazil. Emerg. Infect. Dis. 11:1952-1954.
Swofford, D. L. 2001. PAUP. Phylogenetic analysis using parsimony (and other methods). (version 4.0). Sinauer Associates, Sunderland, Mass.
Taylor, J. W., D. J. Jacobson, S. Kroken, T. Kasuga, D. M. Geiser, D. S. Hibbett, and M. C. Fisher. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genet. Biol. 31:21-32.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.
Travassos, L. R., and K. O. Lloyd. 1980. Sporothrix schenckii and related species of Ceratocystis. Microbiol. Rev. 44:683-721.
Wilhelm de Beer, Z., T. C. Harrington, H. F. Vismer, B. D. Wingfield, and M. J. Wingfield. 2003. Phylogeny of the Ophiostoma stenoceras-Sporothrix schenckii complex. Mycologia 95:434-441.(Rita Marimon, Josepa Gene, Josep Cano, L)