Trichophyton eboreum sp. nov. Isolated from Human Skin
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微生物临床杂志 2005年第10期
Department of Dermatology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany
Institute of Microbiology and Hygiene, Charite, Berlin, Germany
ABSTRACT
An unusual dermatophyte was isolated from the plantar scales of a human immunodeficiency virus-positive man with tinea pedis. Morphology, physiology, and molecular data provided evidence to support the new species Trichophyton eboreum. This dermatophyte is characterized by rapid growth on common mycological media, a flat powdery off-white colony, formation of clavate microconidia, smooth- and thin-walled cylindrical or club-shaped macroconidia with two to nine cells, the presence of hook-shaped hyphae, the production of cleistothecium-like structures and spiral hyphae in older cultures, positive hair perforation, the absence of pigmentation on potato glucose agar, the absence of a requirement for vitamins, a weak positive urease reaction, no growth at 37°C, resistance to 5% NaCl, resistance to fluconazole, good growth on human epidermal keratin, and the production of various enzymes on different media by the API-ZYM test. More than 5% divergence from any known species of dermatophyte was revealed by sequence analysis of the internal transcribed spacer of the rRNA gene.
INTRODUCTION
Dermatophytoses are among the most common communicable diseases in the world. They are caused by dermatophytes, which are highly specialized keratinophilic fungi capable of inducing typical skin infections in humans. The currently known species of dermatophytes are described in comprehensive textbooks and reviews (10, 12, 23, 32, 40). The identification of dermatophytes is based on methods that focus on morphological, physiologic, ecologic, and genetic features (20, 22, 23, 26-28, 31). A number of anthropophilic and zoophilic dermatophytes which had mostly been recognized in the past as separate species are now considered conspecific or varieties when molecular techniques, such as internal transcribed spacer (ITS) sequencing of the rRNA gene, are applied (3, 5, 7, 14, 16-19, 21, 25, 26, 29); nevertheless, separate species names may be justified, despite highly similar ITS sequences (15, 35, 41). These divergent assessments are mainly due to the asexual reproductive mode and to the recent evolution of anthropophilic and zoophilic dermatophytes. In contrast, geophilic dermatophytes can better be assigned to distinct species by the biological concept in correspondence with molecular data (16). Species in this group are more remote from each other. For example, the ITS sequences of the neighboring species Trichophyton vanbreuseghemii and Trichophyton gloriae show only 97% similarity, and those of Trichophyton terrestre (the anamorph of Arthroderma lenticularum) and Trichophyton ajelloi show only 95% similarity (16). Based on a polyphasic approach, we describe here a presumably geophilic dermatophyte that shows distinct morphological, physiologic, and also genetic characteristics that justify the proposal of a new species of Trichophyton.
CASE REPORT
A 42-year-old black man was admitted to the Department of Dermatology at the Clinic of the University of Schleswig-Holstein, Campus Kiel, Kiel, Germany, because of dry and scaling plantar lesions and slightly itching and scaling nummular lesions on the dorsal sides of his feet and on the extensor sides of the lower legs. The patient thought that these lesions had persisted for more than 10 years. The patient was born in Abidjan, Ivory Coast; had lived in Germany for the last 14 years; and had visited his home country occasionally. He had worked as a swimming pool attendant for many years, but his current occupation was as an electrician. He was human immunodeficiency virus positive and had renal dysfunction that required continuous medical treatment and dialysis.
A diagnosis of moccasin-type tinea pedis was made because of the typical clinical morphology of his plantar lesions. To verify this diagnosis mycologically, scales were scraped from his right planta pedis and used as clinical specimens for microscopic inspection and cultures following our routine procedure for the detection of fungi in lesional skin. Direct microscopic examination of the scales macerated with 15% KOH revealed colorless short hyphal elements. The scales were planted on Sabouraud glucose agar (bioMerieux, Marcy l'Etoile, France) with 400 mg liter–1 cycloheximide (AppliChem, Darmstadt, Germany) plus antibiotics and on a Sabouraud glucose agar slant with antibiotics only. Identical colonies developed at two of three points of inoculation only on the plate containing cycloheximide within 14 days at 26°C; the colonies were similar to those of T. terrestre or an unpigmented Trichophyton mentagrophytes strain. For an exact identification, our isolate was then submitted to extensive further mycological investigations (see below).
Treatment of our patient was initiated at his first visit with cyclopiroxolamine topically plus terbinafine 250 mg daily as an oral therapy for 10 weeks. The following visits showed a gradual clinical healing of the skin lesions, and repeated control specimens thereafter were negative for fungi.
MATERIALS AND METHODS
Phenotypic features. In accordance with generally accepted guidelines for the identification and description of dermatophytes (4, 9, 12, 17, 23, 27, 30, 40), our strain was grown at 26°C on Sabouraud glucose agar, bromocresol purple-milk solids-glucose agar (36), Wort agar (Becton Dickinson Difco, Sparks, MD), potato dextrose agar (Difco), oatmeal-salts-agar (24), and phytone yeast-extract agar (Becton Dickinson BBL, Sparks, MD). The strain was tested for resistance to cycloheximide (400 mg liter–1) in Sabouraud agar, temperature resistance, osmotic resistance (Sabouraud agar supplemented with 3% and 5% NaCl), urease activity (urease agar; BBL), vitamin dependency (Trichophyton agars 1 to 7; Difco), and hair perforation (1). The strain was tested for growth on human hair and on human stratum corneum at 26°C. Natural (not dyed or otherwise treated) human scalp hair was cut into short pieces, washed, rinsed, defatted, and autoclaved. Stratum corneum was scraped from healthy and thoroughly cleaned human plantar skin and autoclaved. This material was placed in sterilized water without supplements in petri dishes and inoculated with a suspension of conidia obtained from Sabouraud cultures. Release of enzymes was tested as described previously (6, 8) by use of the API-ZYM test (bioMerieux), which is a standard system designed to semiquantitatively measure 20 different cellular enzymes by use of chromogenic substrates. The isolate was grown at room temperature in Sabouraud broth (bioMerieux), in a 2% neopeptone broth (neopeptone; Merck, Darmstadt, Germany), and on human hair and human stratum corneum as described above. Samples of cell-free culture broth were obtained, checked for bacterial contamination, and used for the enzyme assays following the manufacturer's instructions. The Etest (VIVA-Diagnostika, Cologne, Germany) was applied according to the manufacturer's instructions to determine susceptibility to fluconazole and itraconazole (13). All tests described above were done in duplicate or triplicate.
Molecular biology. (i) Strains. In total 45, anthropophilic, zoophilic, and geophilic dermatophyte species, including the new isolate, were analyzed (Table 1).
(ii) DNA isolation. DNA extractions were done with a GenomicPrep Cells and Tissue DNA isolation kit from Amersham Biosciences (Piscataway, NJ). The protocol of the manufacturer was slightly modified, as described elsewhere (33).
(iii) DNA amplification and sequencing. PCR of the ITS region was performed with the primer pair LSU266 and V9D (11). Sequence analysis was done with the internal primers ITS4 and ITS5 and an automated sequencing system (Beckman-Coulter, Fullerton, CA).
The sequences of the ITS1 and ITS2 regions of the new isolate were submitted to a BLASTn search (compares nucleotide similarities) by using the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/BLAST) to find the next most closely related fungal species. On the basis of this result and the phenotypic features, phylogenetically related species were chosen for the construction of the phylogenetic tree, inclusive of 27 Arthroderma spp.; 1 Ctenomyces sp.; and 16 species belonging to the anamorphic genera Trichophyton, Microsporum, Epidermophyton, and Chrysosporium.
(iv) Alignment and tree construction. Alignment was performed by using CLUSTAL V (DKFZ, Heidelberg, Germany), and tree construction was performed by using PAUP 4.0b10 (38). Parsimony and neighbor-joining analyses were conducted with unambiguously aligned sequences by using the heuristic search option and the Kimura two-parameter model, respectively. The robustness of the branches was assessed by bootstrap analysis with 1,000 replicates.
RESULTS
Culture morphology. No major morphological differences were seen between the colonies grown on the different agars. The main characteristics of our strain on Sabouraud glucose agar at 26°C were as follows: a flat spreading colony and a diameter of 50 to 60 mm within 14 days, with a granular or a slightly powdery surface, a radiating feathery margin, and off-white obverse and reverse sides (Fig. 1). Microconidia were abundant and were borne sessile alongside undifferentiated hyphae, which were one celled, clavate, ca. 2.5 μm wide, and 4 to 5 μm long (Fig. 2). Macroconidia were also abundant and were borne on short hyphae or sterigmata of various lengths with two to nine cells and were cylindrical or club shaped, ca. 4 μm wide, and 6 to 50 μm long, with thin and smooth walls and thin septa (Fig. 3 and 4). Hook-shaped short hyphae with blunt and thickened ends were borne on the sides of undifferentiated hyphae (Fig. 5). Cleistothecium-like structures composed of tangled and rather thick short mycelial elements developed on the surface of older cultures (Fig. 6). They showed protruding dumbbell-shaped, smooth-walled, short hook-shaped ascomatal hyphae but were devoid of ascospores. Many coiled and spiral hyphae (Fig. 7) and some chlamydospores occurred in older cultures.
Physiology. Rapid growth was seen on Sabouraud glucose agar with cycloheximide. No growth occurred on Sabouraud glucose agar at 37°C, but growth was not inhibited on Sabouraud glucose agar with 3% or 5% NaCl. Within 10 days colonies on bromocresol purple-milk solids-glucose agar showed no restriction of growth, slight alkalinity, no clearing beyond the colony margin, and no pigmentation. On potato dextrose agar, rapid growth was not accompanied by pigmentation. A weak positive urease reaction developed on Christensen urea agar within 7 days. Good growth on Trichophyton agars 1 to 7 indicated no requirement for vitamins. The hair perforation test was positive (Fig. 8). Cultures on human hair showed dense mycelia within 2 weeks, as did cultures on human stratum corneum.
The Etest revealed resistance to fluconazole up to 256 μg ml–1 and inhibition by 0.032 μg ml–1 itraconazole.
The API-ZYM test detected the following enzymes: with cultures in Sabouraud broth, clearly positive proof of esterase (C4), esterase lipase (C8), leucine arylamidase, phosphatase acid, -glucosidase, N-acetyl--glucosaminidase, and -mannosidase; with cultures in neopeptone broth, clearly positive proof of phosphatase alkaline, leucine arylamidase, and -glucosidase; with cultures on hair, clearly positive proof of phosphatase alkaline, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, phosphatase acid, naphthol-AS-BI-phosphohydrolase, -glucosidase, N-acetyl--glucosaminidase, and -mannosidase; and with cultures on stratum corneum, clearly positive proof of phosphatase alkaline, phosphatase acid, naphtol-AS-BI-phosphohydrolase, and -glucosidase.
Distinctive moldy or mossy odors were perceptible even from closed but unsealed petri dishes after 2 weeks of growth.
Molecular data. The ITS1, ITS2, and 5.8S rRNA sequences of our isolate were compared with the sequences of up-to-date validly described dermatophyte species of the genus Arthroderma, of which the anamorphs belong to the genera Trichophyton, Epidermophyton, Chrysosporium, and Microsporum (Table 1, Fig. 9, and Fig. 10) (10). Before the tree was calculated, a BLASTn search determined that the nearest neighbor of the isolated strain was Chrysosporium vespertilium (94% similarity) (39). As shown in Fig. 9, clustering of the new isolate amid the geophilic group of Trichophyton and Chrysosporium species is supported by a high bootstrap value of 98%. The nearest teleomorph species were Arthroderma flavescens and Arthroderma multifidum.
DISCUSSION
The fungus that we describe here was the only microbial agent isolated from lesional skin in our patient. Clinical and mycological healing occurred with antimycotic therapy. However, because this strain was isolated only once (attempts to reisolate it again failed after the initiation of treatment), our data do not allow determination of whether it was just a contaminant that might have overgrown the true cause of infection or whether it was the pathogenic agent responsible for the case of tinea pedis described here.
On Sabouraud agar, the colony of our isolate was macroscopically typical for cultures of dermatophytes, and microscopically, many macroconidia that showed all the characteristics of Trichophyton macroconidia (smooth and thin walled, club shaped, multicellular) were seen. Therefore, our strain was identified as a dermatophyte of the genus Trichophyton (2, 12, 26, 32, 37, 40).
Further characterization of this strain revealed some similarities with T. mentagrophytes and T. terrestre. Similar to both of these species, it had a flat and granular colony with no distinct surface pigmentation. Like T. mentagrophytes, it produced many macroconidia and spiral hyphae and was positive for hair perforation and urease activity. Similar to T. terrestre, it developed conidia of various lengths with two or more cells and had no marked pigmentation of its reverse side. However, in contrast to T. mentagrophytes, our strain lacked pigmentation and the shape and arrangement of its conidia resembled those of Trichophyton rubrum conidia rather than those of T. mentagrophytes conidia. In addition, it developed peculiar hook-shaped hyphae and cleistothecium-like mycelial structures on its surface as distinguishing features. Similar characteristics are known as "pseudogymnothecia" and are found among some heterothallic members of the geophilic clade (10, 34). The physiological properties of our isolate are consistent with dermatophytes (6, 8, 13, 36), but we could not identify a singular feature as a hallmark of Trichophyton eboreum.
The molecular analysis of the ITS region clearly supports the grouping of the isolated strain amid the geophilic Arthroderma species (bootstrap support, 98%). By ITS sequence analysis, the distance to the next asexual species, C. vespertilium, is 6%; and the distance to the next sexual species, e.g., A. flavescens, is even larger. The low similarity (94%) clearly separates the new species from any Arthroderma species known to date. By comparison of ITS sequence similarities among dermatophytes, nearly all of the species in Arthroderma, which are biological species, are less than 98% related. This implies that the whole group of species stems from the same adaptive radiation event in the past. Exceptions are the gross of asexual species that are anthropophilic or zoophilic and two geophilic Microsporum species, Microsporum ripariae and Microsporum gallinae, which are 98% and 100% similar to their next relatives, Arthroderma fulvum and Arthroderma grubyi, respectively (16, 18, 28, 29). The relatedness in some of the morphological features between T. eboreum and T. mentagrophytes or T. rubrum is clearly not supported by the molecular analysis (Fig. 10). Taken together, the data presented unequivocally indicate that our isolate belongs to a hitherto unknown anamorphic species in the Arthroderma clade. There is reason to assume that it is a geophilic species, although its ecological niche remains unknown.
The clinical picture in our patient was a characteristic chronic moccasin-type tinea pedis. This type of aphlegmasic tinea is typically caused by anthropophilic but not geophilic dermatophytes. It is quite possible, therefore, that in our case T. eboreum was a contaminant with no causal relation to the skin lesions and that we missed the true causative agent. On the other hand, it cannot be excluded that due to the immunosuppression of the patient, a usually nonpathogenic species had caused a commensal superficial colonization. The good in vitro growth of T. eboreum on human stratum corneum and on human hair clearly demonstrates that this species has the capability to utilize human keratins for its nutrition.
Since T. eboreum had not been detected previously either as a contaminant or as a pathogen in medical microbiological laboratories, it must be a very rare species, at least in countries in which cultures of dermatophytes are done routinely. It is possibly a strictly geophilic species and/or a species with an extraordinarily limited geographic distribution. With our report we want to draw attention to this species. It is hoped that a raised awareness will lead to more observations in the future that will help to clarify the distribution and pathogenic potential of T. eboreum. Our strain is deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen and at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (strains DSMZ 16978 and CBS 117155, respectively). The Latin word "eboreum" in its name means "ivory" and is reminiscent of the origin of its carrier and the whitish color of its colony.
Descriptio Trichophytonis eborei Brasch et Grser sp. nov. In agaro Sabouraud (2% glucose) ad 26°C: colonia per 14 dies crescit ad diametrum 50 ac 60 mm; plane se extendentis thalli superficies granea vel leviter pulverulenta, margo radiatus et pennatus, obversa et aversa pars albida. Nullum pigmentum exsudatum vel se diffundens. Microconidia in speciem clavae formata, circa 2.5 μm lata et 4 ac 5 μm longa, lateraliter hyphis sublata. Copiosa macroconidia in speciem clavae formata ex 9 vel paucioribus cellulis composita, cylindrata vel in speciem clavae formata, cum rotundatis non adhaerentibus apicibus, tenuibus ac mollibus parietibus et tenuibus saeptis, circa 4 μm lata et usque ad 50 μm longa brevibus subtentis ramis sublata. Hyphae in speciem hami curvatae, cum terminis obtusis atque crassatis, lateribus hypharum indifferentium sublatae. Conspicui myceliales globi cleistotheciaformes dense internexi in superficie vetustiorum culturarum cum eminentibus brevibus hyphis manipuliformibus tenuitunicatis in speciem hami curvatis. Multae spirales hyphae et nonnullae chlamydospores in vetustioribus culturis.
In aliis usitatis mycologicis culturis similiter crescit ac in agaro Sabouraud. Non crescit ad 37°C, non inhibetur per 400 mg liter–1 cycloheximidem vel 5% NaCl. In agaro bromocresol lacte purpureo solidi-glucose levis alcalinitas per 10 dies; agarum non translucent ultra marginem coloniae, nullum pigmentum apparet, incrementum non inhibetur. Nullum pigmentum apparet in patata-dextrose agaro. Bene crescit in humano strato corneo et in humano capillo; experimentum perforationis capillaris positivum. Leviter positiva urease-reactio in Christensen uretico agaro per 7 dies. Vitaminis opus non est. Resistit contra fluconazole usque ad 256 μg ml–1 (Etest). Ampla varietas effusorum encymorum in cultura capillari.
Si cum notis dermatophytibus et cum specie Chrysosporium comparare velis, simillimum est 94% ac Chrysosporium verpertilium, ut apparet ex integra analysi sequentiarum spatiorum 1 ac 2 internorum transcriptorum et spatii intermedii 5.8S ribosomalis RNA. Proxima teleomorph species: A. flavescens et A. multifidum.
Teleomorph ignotum. Conservatur in Collectione Germana Microorganismorum et Culturarum Cellularum (DSMZ 16978) et in Administratione Centrali Culturarum Mucescentium in Hollandia (CBS 117155).
Holotypus. Holotypus segregata est in Departemento Dermatologiae Universitatis Slesvico-Holsaticae, in Campo Chiloniensi in urbe Chiloniensium (Germania) ex tinea pedis generis moccasinensis viri adulti ex urbe Abidjan (Litus Ebureum); identitas a J. Brasch et Y. Grser mense Decembri anni 2004 cognita est. In herbario Instituti Botanici Universitatis Slesvico-Holsaticae, in Campo Chiloniensi in urbe Chiloniensium (Germania) praeservatus (KIEL MuscArth200501).
ACKNOWLEDGMENTS
Our thanks go to J. O. Busch for his clinical cooperation; to V. Beck-Jendroschek, Janine Frhlich, and Annett Petrich for their very committed technical assistance; to R. Summerbell for helpful comments on the manuscript; and to B. Gauly for the Latin translation of the species description.
Cultures of strains were provided by the Centraalbureau voor Schimmelcultures.
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Institute of Microbiology and Hygiene, Charite, Berlin, Germany
ABSTRACT
An unusual dermatophyte was isolated from the plantar scales of a human immunodeficiency virus-positive man with tinea pedis. Morphology, physiology, and molecular data provided evidence to support the new species Trichophyton eboreum. This dermatophyte is characterized by rapid growth on common mycological media, a flat powdery off-white colony, formation of clavate microconidia, smooth- and thin-walled cylindrical or club-shaped macroconidia with two to nine cells, the presence of hook-shaped hyphae, the production of cleistothecium-like structures and spiral hyphae in older cultures, positive hair perforation, the absence of pigmentation on potato glucose agar, the absence of a requirement for vitamins, a weak positive urease reaction, no growth at 37°C, resistance to 5% NaCl, resistance to fluconazole, good growth on human epidermal keratin, and the production of various enzymes on different media by the API-ZYM test. More than 5% divergence from any known species of dermatophyte was revealed by sequence analysis of the internal transcribed spacer of the rRNA gene.
INTRODUCTION
Dermatophytoses are among the most common communicable diseases in the world. They are caused by dermatophytes, which are highly specialized keratinophilic fungi capable of inducing typical skin infections in humans. The currently known species of dermatophytes are described in comprehensive textbooks and reviews (10, 12, 23, 32, 40). The identification of dermatophytes is based on methods that focus on morphological, physiologic, ecologic, and genetic features (20, 22, 23, 26-28, 31). A number of anthropophilic and zoophilic dermatophytes which had mostly been recognized in the past as separate species are now considered conspecific or varieties when molecular techniques, such as internal transcribed spacer (ITS) sequencing of the rRNA gene, are applied (3, 5, 7, 14, 16-19, 21, 25, 26, 29); nevertheless, separate species names may be justified, despite highly similar ITS sequences (15, 35, 41). These divergent assessments are mainly due to the asexual reproductive mode and to the recent evolution of anthropophilic and zoophilic dermatophytes. In contrast, geophilic dermatophytes can better be assigned to distinct species by the biological concept in correspondence with molecular data (16). Species in this group are more remote from each other. For example, the ITS sequences of the neighboring species Trichophyton vanbreuseghemii and Trichophyton gloriae show only 97% similarity, and those of Trichophyton terrestre (the anamorph of Arthroderma lenticularum) and Trichophyton ajelloi show only 95% similarity (16). Based on a polyphasic approach, we describe here a presumably geophilic dermatophyte that shows distinct morphological, physiologic, and also genetic characteristics that justify the proposal of a new species of Trichophyton.
CASE REPORT
A 42-year-old black man was admitted to the Department of Dermatology at the Clinic of the University of Schleswig-Holstein, Campus Kiel, Kiel, Germany, because of dry and scaling plantar lesions and slightly itching and scaling nummular lesions on the dorsal sides of his feet and on the extensor sides of the lower legs. The patient thought that these lesions had persisted for more than 10 years. The patient was born in Abidjan, Ivory Coast; had lived in Germany for the last 14 years; and had visited his home country occasionally. He had worked as a swimming pool attendant for many years, but his current occupation was as an electrician. He was human immunodeficiency virus positive and had renal dysfunction that required continuous medical treatment and dialysis.
A diagnosis of moccasin-type tinea pedis was made because of the typical clinical morphology of his plantar lesions. To verify this diagnosis mycologically, scales were scraped from his right planta pedis and used as clinical specimens for microscopic inspection and cultures following our routine procedure for the detection of fungi in lesional skin. Direct microscopic examination of the scales macerated with 15% KOH revealed colorless short hyphal elements. The scales were planted on Sabouraud glucose agar (bioMerieux, Marcy l'Etoile, France) with 400 mg liter–1 cycloheximide (AppliChem, Darmstadt, Germany) plus antibiotics and on a Sabouraud glucose agar slant with antibiotics only. Identical colonies developed at two of three points of inoculation only on the plate containing cycloheximide within 14 days at 26°C; the colonies were similar to those of T. terrestre or an unpigmented Trichophyton mentagrophytes strain. For an exact identification, our isolate was then submitted to extensive further mycological investigations (see below).
Treatment of our patient was initiated at his first visit with cyclopiroxolamine topically plus terbinafine 250 mg daily as an oral therapy for 10 weeks. The following visits showed a gradual clinical healing of the skin lesions, and repeated control specimens thereafter were negative for fungi.
MATERIALS AND METHODS
Phenotypic features. In accordance with generally accepted guidelines for the identification and description of dermatophytes (4, 9, 12, 17, 23, 27, 30, 40), our strain was grown at 26°C on Sabouraud glucose agar, bromocresol purple-milk solids-glucose agar (36), Wort agar (Becton Dickinson Difco, Sparks, MD), potato dextrose agar (Difco), oatmeal-salts-agar (24), and phytone yeast-extract agar (Becton Dickinson BBL, Sparks, MD). The strain was tested for resistance to cycloheximide (400 mg liter–1) in Sabouraud agar, temperature resistance, osmotic resistance (Sabouraud agar supplemented with 3% and 5% NaCl), urease activity (urease agar; BBL), vitamin dependency (Trichophyton agars 1 to 7; Difco), and hair perforation (1). The strain was tested for growth on human hair and on human stratum corneum at 26°C. Natural (not dyed or otherwise treated) human scalp hair was cut into short pieces, washed, rinsed, defatted, and autoclaved. Stratum corneum was scraped from healthy and thoroughly cleaned human plantar skin and autoclaved. This material was placed in sterilized water without supplements in petri dishes and inoculated with a suspension of conidia obtained from Sabouraud cultures. Release of enzymes was tested as described previously (6, 8) by use of the API-ZYM test (bioMerieux), which is a standard system designed to semiquantitatively measure 20 different cellular enzymes by use of chromogenic substrates. The isolate was grown at room temperature in Sabouraud broth (bioMerieux), in a 2% neopeptone broth (neopeptone; Merck, Darmstadt, Germany), and on human hair and human stratum corneum as described above. Samples of cell-free culture broth were obtained, checked for bacterial contamination, and used for the enzyme assays following the manufacturer's instructions. The Etest (VIVA-Diagnostika, Cologne, Germany) was applied according to the manufacturer's instructions to determine susceptibility to fluconazole and itraconazole (13). All tests described above were done in duplicate or triplicate.
Molecular biology. (i) Strains. In total 45, anthropophilic, zoophilic, and geophilic dermatophyte species, including the new isolate, were analyzed (Table 1).
(ii) DNA isolation. DNA extractions were done with a GenomicPrep Cells and Tissue DNA isolation kit from Amersham Biosciences (Piscataway, NJ). The protocol of the manufacturer was slightly modified, as described elsewhere (33).
(iii) DNA amplification and sequencing. PCR of the ITS region was performed with the primer pair LSU266 and V9D (11). Sequence analysis was done with the internal primers ITS4 and ITS5 and an automated sequencing system (Beckman-Coulter, Fullerton, CA).
The sequences of the ITS1 and ITS2 regions of the new isolate were submitted to a BLASTn search (compares nucleotide similarities) by using the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/BLAST) to find the next most closely related fungal species. On the basis of this result and the phenotypic features, phylogenetically related species were chosen for the construction of the phylogenetic tree, inclusive of 27 Arthroderma spp.; 1 Ctenomyces sp.; and 16 species belonging to the anamorphic genera Trichophyton, Microsporum, Epidermophyton, and Chrysosporium.
(iv) Alignment and tree construction. Alignment was performed by using CLUSTAL V (DKFZ, Heidelberg, Germany), and tree construction was performed by using PAUP 4.0b10 (38). Parsimony and neighbor-joining analyses were conducted with unambiguously aligned sequences by using the heuristic search option and the Kimura two-parameter model, respectively. The robustness of the branches was assessed by bootstrap analysis with 1,000 replicates.
RESULTS
Culture morphology. No major morphological differences were seen between the colonies grown on the different agars. The main characteristics of our strain on Sabouraud glucose agar at 26°C were as follows: a flat spreading colony and a diameter of 50 to 60 mm within 14 days, with a granular or a slightly powdery surface, a radiating feathery margin, and off-white obverse and reverse sides (Fig. 1). Microconidia were abundant and were borne sessile alongside undifferentiated hyphae, which were one celled, clavate, ca. 2.5 μm wide, and 4 to 5 μm long (Fig. 2). Macroconidia were also abundant and were borne on short hyphae or sterigmata of various lengths with two to nine cells and were cylindrical or club shaped, ca. 4 μm wide, and 6 to 50 μm long, with thin and smooth walls and thin septa (Fig. 3 and 4). Hook-shaped short hyphae with blunt and thickened ends were borne on the sides of undifferentiated hyphae (Fig. 5). Cleistothecium-like structures composed of tangled and rather thick short mycelial elements developed on the surface of older cultures (Fig. 6). They showed protruding dumbbell-shaped, smooth-walled, short hook-shaped ascomatal hyphae but were devoid of ascospores. Many coiled and spiral hyphae (Fig. 7) and some chlamydospores occurred in older cultures.
Physiology. Rapid growth was seen on Sabouraud glucose agar with cycloheximide. No growth occurred on Sabouraud glucose agar at 37°C, but growth was not inhibited on Sabouraud glucose agar with 3% or 5% NaCl. Within 10 days colonies on bromocresol purple-milk solids-glucose agar showed no restriction of growth, slight alkalinity, no clearing beyond the colony margin, and no pigmentation. On potato dextrose agar, rapid growth was not accompanied by pigmentation. A weak positive urease reaction developed on Christensen urea agar within 7 days. Good growth on Trichophyton agars 1 to 7 indicated no requirement for vitamins. The hair perforation test was positive (Fig. 8). Cultures on human hair showed dense mycelia within 2 weeks, as did cultures on human stratum corneum.
The Etest revealed resistance to fluconazole up to 256 μg ml–1 and inhibition by 0.032 μg ml–1 itraconazole.
The API-ZYM test detected the following enzymes: with cultures in Sabouraud broth, clearly positive proof of esterase (C4), esterase lipase (C8), leucine arylamidase, phosphatase acid, -glucosidase, N-acetyl--glucosaminidase, and -mannosidase; with cultures in neopeptone broth, clearly positive proof of phosphatase alkaline, leucine arylamidase, and -glucosidase; with cultures on hair, clearly positive proof of phosphatase alkaline, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, phosphatase acid, naphthol-AS-BI-phosphohydrolase, -glucosidase, N-acetyl--glucosaminidase, and -mannosidase; and with cultures on stratum corneum, clearly positive proof of phosphatase alkaline, phosphatase acid, naphtol-AS-BI-phosphohydrolase, and -glucosidase.
Distinctive moldy or mossy odors were perceptible even from closed but unsealed petri dishes after 2 weeks of growth.
Molecular data. The ITS1, ITS2, and 5.8S rRNA sequences of our isolate were compared with the sequences of up-to-date validly described dermatophyte species of the genus Arthroderma, of which the anamorphs belong to the genera Trichophyton, Epidermophyton, Chrysosporium, and Microsporum (Table 1, Fig. 9, and Fig. 10) (10). Before the tree was calculated, a BLASTn search determined that the nearest neighbor of the isolated strain was Chrysosporium vespertilium (94% similarity) (39). As shown in Fig. 9, clustering of the new isolate amid the geophilic group of Trichophyton and Chrysosporium species is supported by a high bootstrap value of 98%. The nearest teleomorph species were Arthroderma flavescens and Arthroderma multifidum.
DISCUSSION
The fungus that we describe here was the only microbial agent isolated from lesional skin in our patient. Clinical and mycological healing occurred with antimycotic therapy. However, because this strain was isolated only once (attempts to reisolate it again failed after the initiation of treatment), our data do not allow determination of whether it was just a contaminant that might have overgrown the true cause of infection or whether it was the pathogenic agent responsible for the case of tinea pedis described here.
On Sabouraud agar, the colony of our isolate was macroscopically typical for cultures of dermatophytes, and microscopically, many macroconidia that showed all the characteristics of Trichophyton macroconidia (smooth and thin walled, club shaped, multicellular) were seen. Therefore, our strain was identified as a dermatophyte of the genus Trichophyton (2, 12, 26, 32, 37, 40).
Further characterization of this strain revealed some similarities with T. mentagrophytes and T. terrestre. Similar to both of these species, it had a flat and granular colony with no distinct surface pigmentation. Like T. mentagrophytes, it produced many macroconidia and spiral hyphae and was positive for hair perforation and urease activity. Similar to T. terrestre, it developed conidia of various lengths with two or more cells and had no marked pigmentation of its reverse side. However, in contrast to T. mentagrophytes, our strain lacked pigmentation and the shape and arrangement of its conidia resembled those of Trichophyton rubrum conidia rather than those of T. mentagrophytes conidia. In addition, it developed peculiar hook-shaped hyphae and cleistothecium-like mycelial structures on its surface as distinguishing features. Similar characteristics are known as "pseudogymnothecia" and are found among some heterothallic members of the geophilic clade (10, 34). The physiological properties of our isolate are consistent with dermatophytes (6, 8, 13, 36), but we could not identify a singular feature as a hallmark of Trichophyton eboreum.
The molecular analysis of the ITS region clearly supports the grouping of the isolated strain amid the geophilic Arthroderma species (bootstrap support, 98%). By ITS sequence analysis, the distance to the next asexual species, C. vespertilium, is 6%; and the distance to the next sexual species, e.g., A. flavescens, is even larger. The low similarity (94%) clearly separates the new species from any Arthroderma species known to date. By comparison of ITS sequence similarities among dermatophytes, nearly all of the species in Arthroderma, which are biological species, are less than 98% related. This implies that the whole group of species stems from the same adaptive radiation event in the past. Exceptions are the gross of asexual species that are anthropophilic or zoophilic and two geophilic Microsporum species, Microsporum ripariae and Microsporum gallinae, which are 98% and 100% similar to their next relatives, Arthroderma fulvum and Arthroderma grubyi, respectively (16, 18, 28, 29). The relatedness in some of the morphological features between T. eboreum and T. mentagrophytes or T. rubrum is clearly not supported by the molecular analysis (Fig. 10). Taken together, the data presented unequivocally indicate that our isolate belongs to a hitherto unknown anamorphic species in the Arthroderma clade. There is reason to assume that it is a geophilic species, although its ecological niche remains unknown.
The clinical picture in our patient was a characteristic chronic moccasin-type tinea pedis. This type of aphlegmasic tinea is typically caused by anthropophilic but not geophilic dermatophytes. It is quite possible, therefore, that in our case T. eboreum was a contaminant with no causal relation to the skin lesions and that we missed the true causative agent. On the other hand, it cannot be excluded that due to the immunosuppression of the patient, a usually nonpathogenic species had caused a commensal superficial colonization. The good in vitro growth of T. eboreum on human stratum corneum and on human hair clearly demonstrates that this species has the capability to utilize human keratins for its nutrition.
Since T. eboreum had not been detected previously either as a contaminant or as a pathogen in medical microbiological laboratories, it must be a very rare species, at least in countries in which cultures of dermatophytes are done routinely. It is possibly a strictly geophilic species and/or a species with an extraordinarily limited geographic distribution. With our report we want to draw attention to this species. It is hoped that a raised awareness will lead to more observations in the future that will help to clarify the distribution and pathogenic potential of T. eboreum. Our strain is deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen and at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (strains DSMZ 16978 and CBS 117155, respectively). The Latin word "eboreum" in its name means "ivory" and is reminiscent of the origin of its carrier and the whitish color of its colony.
Descriptio Trichophytonis eborei Brasch et Grser sp. nov. In agaro Sabouraud (2% glucose) ad 26°C: colonia per 14 dies crescit ad diametrum 50 ac 60 mm; plane se extendentis thalli superficies granea vel leviter pulverulenta, margo radiatus et pennatus, obversa et aversa pars albida. Nullum pigmentum exsudatum vel se diffundens. Microconidia in speciem clavae formata, circa 2.5 μm lata et 4 ac 5 μm longa, lateraliter hyphis sublata. Copiosa macroconidia in speciem clavae formata ex 9 vel paucioribus cellulis composita, cylindrata vel in speciem clavae formata, cum rotundatis non adhaerentibus apicibus, tenuibus ac mollibus parietibus et tenuibus saeptis, circa 4 μm lata et usque ad 50 μm longa brevibus subtentis ramis sublata. Hyphae in speciem hami curvatae, cum terminis obtusis atque crassatis, lateribus hypharum indifferentium sublatae. Conspicui myceliales globi cleistotheciaformes dense internexi in superficie vetustiorum culturarum cum eminentibus brevibus hyphis manipuliformibus tenuitunicatis in speciem hami curvatis. Multae spirales hyphae et nonnullae chlamydospores in vetustioribus culturis.
In aliis usitatis mycologicis culturis similiter crescit ac in agaro Sabouraud. Non crescit ad 37°C, non inhibetur per 400 mg liter–1 cycloheximidem vel 5% NaCl. In agaro bromocresol lacte purpureo solidi-glucose levis alcalinitas per 10 dies; agarum non translucent ultra marginem coloniae, nullum pigmentum apparet, incrementum non inhibetur. Nullum pigmentum apparet in patata-dextrose agaro. Bene crescit in humano strato corneo et in humano capillo; experimentum perforationis capillaris positivum. Leviter positiva urease-reactio in Christensen uretico agaro per 7 dies. Vitaminis opus non est. Resistit contra fluconazole usque ad 256 μg ml–1 (Etest). Ampla varietas effusorum encymorum in cultura capillari.
Si cum notis dermatophytibus et cum specie Chrysosporium comparare velis, simillimum est 94% ac Chrysosporium verpertilium, ut apparet ex integra analysi sequentiarum spatiorum 1 ac 2 internorum transcriptorum et spatii intermedii 5.8S ribosomalis RNA. Proxima teleomorph species: A. flavescens et A. multifidum.
Teleomorph ignotum. Conservatur in Collectione Germana Microorganismorum et Culturarum Cellularum (DSMZ 16978) et in Administratione Centrali Culturarum Mucescentium in Hollandia (CBS 117155).
Holotypus. Holotypus segregata est in Departemento Dermatologiae Universitatis Slesvico-Holsaticae, in Campo Chiloniensi in urbe Chiloniensium (Germania) ex tinea pedis generis moccasinensis viri adulti ex urbe Abidjan (Litus Ebureum); identitas a J. Brasch et Y. Grser mense Decembri anni 2004 cognita est. In herbario Instituti Botanici Universitatis Slesvico-Holsaticae, in Campo Chiloniensi in urbe Chiloniensium (Germania) praeservatus (KIEL MuscArth200501).
ACKNOWLEDGMENTS
Our thanks go to J. O. Busch for his clinical cooperation; to V. Beck-Jendroschek, Janine Frhlich, and Annett Petrich for their very committed technical assistance; to R. Summerbell for helpful comments on the manuscript; and to B. Gauly for the Latin translation of the species description.
Cultures of strains were provided by the Centraalbureau voor Schimmelcultures.
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