Received 17 June 2002/Accepted 17 July 2003

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2003, p. 4583 4588 Vol. 41, No. 10 0095-1137/03/$08.00 0 DOI: 10.1128/JCM.41.10.4583 4588.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of Trichophyton mentagrophytes Takashi Mochizuki, 1 * Hiroshi Ishizaki, 1 Richard C. Barton, 2 Mary K. Moore, 3 Colin J. Jackson, 4 Steven L. Kelly, 4 and E. Glyn V. Evans 5 Department of Dermatology, Kanazawa Medical University, Uchinada, Japan, 1 and Division of Microbiology, University of Leeds, Leeds, 2 Department of Medical Mycology, St. Johns Institute of Dermatology, Kings College, London, 3 Institute of Biological Sciences, University of Wales, Aberystwyth, 4 and Welsh Mycology Reference Laboratory, Public Health Laboratory, Cardiff, 5 United Kingdom Received 17 June 2002/Accepted 17 July 2003 Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rdna). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rdna nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rdna NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates. Downloaded from http://jcm.asm.org/ * Corresponding author. Mailing address: Department of Dermatology, Kanazawa Medical University, Uchinada, Japan. Phone: 81 76 286 2211. Fax: 81 76 286 6369. E-mail: mocizuki@kanazawa-med.ac.jp. Dermatophytes are a specialized group of fungi which infect keratinized tissues of humans and animals. Among the dermatophytes, Trichophyton mentagrophytes is a cosmopolitan species and is one of the most common agents of dermatophytoses, together with Trichophyton rubrum (14). Identification of T. mentagrophytes is usually performed by examination of morphological properties, such as the macroscopic appearance of the colony and microscopic findings, and sometimes by physiological characters, such as the hair perforation and urease tests. T. mentagrophytes is known to be a species complex composed of three teleomorphic species, i.e., Arthroderma benhamiae, Arthroderma vanbreuseghemii, and Arthroderma simii, based on mating studies and the existence of several anamorphic varieties (14, 25). Many human isolates show substantial variety in morphological and physiological tests. Recent studies that have used molecular analysis have indicated that several markers are effective for discriminating members of the T. mentagrophytes complex from other dermatophyte species and between individual members of the complex. At present, restriction analysis of mitochondrial DNA (3, 13, 18, 19, 23), sequence analysis (5, 16, 17, 21), restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) regions of ribosomal DNA (rdna) (9), sequence analysis of chitin synthase 1 (12), restriction analysis of PCR amplicons of other regions (8), and random amplified polymorphic DNA (RAPD) analysis (11, 15, 20, 27) have all been proposed as useful markers for the identification of T. mentagrophytes and in some cases the differentiation of varieties of this species. The identification of species or varieties of dermatophytes is an important part of laboratory investigations of dermatophyte infections and is sufficient for the routine clinical diagnostic laboratory. However, there are occasions when subspecific or subvarietal differentiation of phenotypically identical isolates is required. Members of the T. mentagrophytes complex, particularly T. mentagrophytes var. interdigitale, are a common cause of tinea pedis, or athlete s foot. While these infections are easily treated, relapse of infection is common. This may arise from the original strain that survived treatment or from a new strain acquired, for example, from a communal bathing facility. Furthermore, a detailed understanding of the transmission of T. mentagrophytes strains between people and insights into the pathogenic mechanisms of this species will require a means of distinguishing between strains. The molecular methods developed so far do not distinguish between phenotypically identical isolates of a species or variety. Thus, more sensitive techniques are needed for strain identification or typing for each of the members of the T. mentagrophytes complex. Recently, RFLP analysis of the nontranscribed spacer (NTS) region of the rdna has been reported as a robust technique for the typing on August 31, 2018 by guest 4583

4584 MOCHIZUKI ET AL. J. CLIN. MICROBIOL. of T. rubrum strains (9, 10) and A. benhamiae (22). In the present study, we have attempted to evaluate the efficacy of this method for the differentiation of T. mentagrophytes strains, especially T. mentagrophytes var. interdigitale isolates. MATERIALS AND METHODS Dermatophyte isolates. Fifty-seven clinical isolates of T. mentagrophytes cultured from patients with dermatophytoses or animals in the United Kingdom at either the Mycology Reference Centre, Leeds (21 isolates), or St. Thomas s Hospital, London (36 isolates), and 10 isolates from either Japan or India maintained in the Department of Dermatology, Kanazawa Medical University, were used in the study (Table 1). In addition, DNA from the following isolates from strain collections were analyzed: Epidermophyton floccosum CBS 358.93; T. rubrum SM 8818; A. vanbreuseghemii RV 27960, A. benhamiae, Americano- European race, RV 26680; A. benhamiae, African race, RV 30001; T. mentagrophytes var. erinacei CBS 344.79; Trichophyton concentricum CBS 448.61; A. simii CBS 448.65; A. simii CBS 520.75; and T. mentagrophytes var. quinckeanum NCPF 309. All clinical isolates were identified on the basis of microscopy and colony characteristics and were classified as T. mentagrophytes var. mentagrophytes or T. mentagrophytes var. interdigitale. By working with primary isolates (since cultures maintained and subcultured over time often show atypical morphologies), T. mentagrophytes var. mentagrophytes colonies were characterized by the presence of a granular texture and a colony edge that was often stellate. T. mentagrophytes var. interdigitale colonies had a softer powdery or downy texture and a smoother colony margin. The microscopic features of both varieties were similar, with spherical conidia in clusters, occasionally spiral hyphae, and macroconidia, although the last two features were more often present in T. mentagrophytes var. mentagrophytes isolates. Downy T. mentagrophytes var. interdigitale isolates produced fewer conidia, which were often more elongate than those from powdery and granular isolates. In almost all cases T. mentagrophytes var. mentagrophytes isolates were from body sites other than the feet, often from patients with animal contacts, and in some cases from animals. In almost all cases T. mentagrophytes var. interdigitale isolates were from the feet. Preliminary screening by RFLP analysis of ITS regions. Preliminary screening by RFLP analysis of ITSs 1 and 2 was performed with the 67 T. mentagrophytes isolates. DNA was extracted from a small amount of mycelium torn from Sabouraud dextrose agar slants by a rapid preparation method (16). In brief, the mycelium was put into Eppendorf tubes containing 450 l of lysis buffer (200 mm Tris-HCl [ph 8], 0.5% sodium dodecyl sulfate, 250 mm NaCl, 25 mm EDTA) and ground with a conical grinder for 30 to 60 s. The homogenate was then incubated at 100 C for 15 min, 150 l of 3.0 M sodium acetate was added, and the mixture was centrifuged at 10,000 g for 5 min. The supernatant was extracted with phenol-chloroform-isoamyl alcohol (25:24:1), followed by extraction with chloroform, and the DNA was precipitated by adding the same volume of isopropanol. The precipitate was washed with 70% ethanol and then dissolved in 50 l of distilled water, and 2 l of the solution was used as a template in the following PCR. The PCR was performed with primers ITS-1 (5 -TCCGTAGG TGAACCTGCGG) and ITS-4 (5 -TCCTCCGCTTATTGATATGC) (26) under the PCR conditions described previously (9). The amplicon was digested with restriction enzyme MvaI (MBI Fermentas, IGI Ltd., Sunderland, United Kingdom) and then electrophoresed in a 5% polyacrylamide gel, stained with ethidium bromide, and observed under UV light. Isolates showing the same RFLP pattern as that of a typical T. mentagrophytes var. interdigitale isolate (SM 8796), which had been recovered from a Japanese tinea pedis patient (20), were selected and used further. Larger-scale isolation of genomic DNA. The protocol used for the larger-scale isolation of fungal DNA for subtyping of T. mentagrophytes is basically the same as that reported previously (9). Fungal mycelium cultured in Sabouraud broth was harvested by filtration, and 200 to 300 mg of mycelium frozen in liquid nitrogen was ground and suspended in 600 l of lysis buffer (400 mm Tris-HCl [ph 8], 60 mm EDTA, 150 mm NaCl, 1% sodium dodecyl sulfate, 40 mg of proteinase K per ml). The homogenate was incubated at 60 C for 1 h, and then 100 l of 5 M sodium perchlorate was added and the mixture was incubated at 60 C for 15 min. The homogenate was then extracted with chloroform at 4 C, followed by extraction with phenol-chloroform-isoamyl alcohol (25:24:1) and then extraction with chloroform. DNA was precipitated with isopropanol, washed with 70% ethanol, and dissolved in 150 l of sterile water. The DNA concentration of the solution was approximately 0.5 g/ l. Detection of rdna polymorphisms in T. mentagrophytes isolates. Five to 10 g of each DNA sample was digested with restriction enzyme EcoRI (MBI Fermentas) and electrophoresed in 0.8% agarose gels, stained with ethidium bromide, and then observed under a UV lamp. The gels were denatured, and the DNAs were transferred onto nylon membranes by standard protocols for Southern transfer. The rdna probe used in the hybridization in the study was produced by a method similar to that used in a previous study of T. rubrum (9). Figure 1 shows a revised provisional restriction map of the rdna repeat proposed in the previous study of T. rubrum (9). The target region of the probe was the 3 end of 18S rdna, the adjacent ITS 1, 5.8S rdna, ITS 2, and the 5 end of 25S rdna in the genomic rdna repeat (9). The details of the probe system and synthesis have been described previously (8). In brief, DNA fragments were amplified with primers NS 5 (5 -AACTTAAAGGAATTGACGGAAG-3 ) (26) and ITS-4 with cesium chloride-purified chromosomal DNA of A. vanbreuseghemii (RV 27960 [ATCC 28145]) as the template. The DNA fragments were denatured by boiling; random hexanucleotide primers, deoxynucleoside triphosphates, Klenow DNA polymerase, and digoxigenin (DIG)-labeled dutp were added; and the fragments were labeled according to the instructions of the manufacturer of the DIG DNA Labeling kit (Boehringer Mannheim UK Ltd., Lewes, United Kingdom). The blotted membranes were hybridized with the probe for 18 h at 65 C, followed by two careful washes, and then the signals were detected with anti-dig-alkaline phosphatase conjugate and by an enzyme-catalyzed color reaction according to instructions of the manufacturer of the DIG Nucleic Acid Detection kit (Boehringer Mannheim UK Ltd.). RESULTS Preliminary screening of ITS regions by RFLP analysis. Sixty-seven isolates composed of 57 isolates from the United Kingdom and 10 isolates from Asia were screened by RFLP analysis of the ITS 1 and 2 regions with the restriction enzyme Mva I (Table 1; Fig. 2). Most species showed different patterns, as described by Jackson et al. (9), although T. concentricum, Trichophyton erinacei, and both strains of A. benhamiae appeared to be identical. The close relationship between these species has previously been shown by other investigators (6). Sixty isolates including a typical clinical isolate of T. mentagrophytes var. interdigitale, strain SM 8796, showed identical patterns composed of five bands; and these 60 isolates were used for the strain typing study. Geographically, 10 of these 60 isolates were from Asia and 50 isolates were from the United Kingdom. Seven isolates were excluded from further study because the patterns for five of seven isolates were the same as that for A. vanbreuseghemii and the pattern for one isolate (isolate EN 6349) was the same as that for A. benhamiae. One isolate (isolate EK 4260) showed a peculiar pattern not identical to those for any species in the T. mentagrophytes complex. DNA typing based on polymorphisms of the NTS regions. From the results of the probe hybridizations, the 60 isolates were first separated into two major groups on the basis of the presence (P) or absence (A) of a band at 2.5 kb. The P group comprised 49 isolates in which the 2.5 kb band was present, and the A group was composed of 11 isolates from which the 2.5-kb band was absent. The P and A groups were then divided into subgroups on the basis of other polymorphic bands observed on the blotted membrane. The P group was separated into 12 subgroups (DNA types) (Fig. 3). The largest subgroup, P-1, comprised 23 of 49 of isolates in the P group, including 9 of 10 isolates from Asia and 14 of 39 isolates from the United Kingdom. The second largest subgroup, P-2, comprised 5 of 49 of isolates in the P group. Two of 12 subgroups (subgroups P-7 and P-8) had two isolates, and 4 of 12 subgroups (subgroups P-9, P-10, P-11, and P-12) were represented only by single isolates. Each of the 11 isolates in the A group showed unique profiles, and the isolates were separated into 11 subgroups (Fig. 4). Subgroup A-2 showed a faint band about 2.5 kb in length. However, the banding pattern between 5 and 10 kb is charac-

VOL. 41, 2003 DIFFERENTIATION OF TRICHOPHYTON MENTAGROPHYTES STRAINS 4585 TABLE 1. Details and DNA types for fungal isolates used in the present study a Isolate Origin Site Variety b ITS profile c NTS subgroup SM 8796 Japan Foot T. mentagrophytes var. interdigitale T i P-1 Si 13 Japan Foot T. mentagrophytes var. interdigitale T i P-1 SM 8774 d India NK T. mentagrophytes var. interdigitale T i P-1 SM 8779 d India NK T. mentagrophytes var. interdigitale T i P-1 KMU Kdy Japan NK T. mentagrophytes var. interdigitale T i P-1 KMU Tkd Japan NK T. mentagrophytes var. interdigitale T i P-1 KMU Tnk Japan NK T. mentagrophytes var. interdigitale T i P-1 KMU Ymz Japan Foot T. mentagrophytes var. interdigitale T i P-1 KMU Ysh Japan Foot T. mentagrophytes var. interdigitale T i P-1 EP 0585 UK Toenail T. mentagrophytes var. interdigitale T i P-1 EP 0910 UK NK T. mentagrophytes var. interdigitale T i P-1 EP 0939 UK Foot T. mentagrophytes var. interdigitale T i P-1 EP 0940 UK Foot T. mentagrophytes var. interdigitale T i P-1 EP 1039 UK Toe T. mentagrophytes var. interdigitale T i P-1 EP 1119 UK Toe web T. mentagrophytes var. interdigitale T i P-1 EP 1120 UK Foot T. mentagrophytes var. interdigitale T i P-1 2111 UK NK T. mentagrophytes var. interdigitale T i P-1 2710 UK NK T. mentagrophytes var. interdigitale T i P-1 2882 UK NK T. mentagrophytes var. interdigitale T i P-1 3080 UK NK T. mentagrophytes var. interdigitale T i P-1 6679 UK NK T. mentagrophytes var. interdigitale T i P-1 8937 UK NK T. mentagrophytes var. interdigitale T i P-1 9294 UK NK T. mentagrophytes var. interdigitale T i P-1 EP 1174 UK Toenail T. mentagrophytes var. interdigitale T i P-2 0393 UK NK T. mentagrophytes var. interdigitale T i P-2 2675 UK NK T. mentagrophytes var. interdigitale T i P-2 4605 UK NK T. mentagrophytes var. interdigitale T i P-2 LM 49 UK NK T. mentagrophytes var. interdigitale T i P-2 EP 0914 UK Toenail T. mentagrophytes var. interdigitale T i P-3 EP 09421 UK Toenail T. mentagrophytes var. interdigitale T i P-3 EP 1127 UK NK T. mentagrophytes var. interdigitale T i P-3 5748-1 UK NK T. mentagrophytes var. interdigitale T i P-3 EP1159 UK Nail T. mentagrophytes var. interdigitale T i P-4 3083 UK NK T. mentagrophytes var. interdigitale T i P-4 3850 UK NK T. mentagrophytes var. interdigitale T i P-4 KMU Akw Japan Palm T. mentagrophytes var. interdigitale T i P-5 EP 0973 UK Foot T. mentagrophytes var. interdigitale T i P-5 4153 UK NK T. mentagrophytes var. interdigitale T i P-5 EP 0843 UK Foot T. mentagrophytes var. interdigitale T i P-6 EP 0852 UK Toe web T. mentagrophytes var. interdigitale T i P-6 T2.261 UK NK T. mentagrophytes var. interdigitale T i P-6 4217 UK NK T. mentagrophytes var. interdigitale T i P-7 8070 UK NK T. mentagrophytes var. interdigitale T i P-7 EP 1099 UK Toenail T. mentagrophytes var. interdigitale T i P-8 Tm atypical UK NK T. mentagrophytes var. interdigitale T i P-8 2035 UK NK T. mentagrophytes var. interdigitale T i P-9 EF 2608 UK Palm T. mentagrophytes var. mentagrophytes T i P-10 EK 4354 UK Chin T. mentagrophytes var. mentagrophytes T i P-11 EG 5704 UK Shin T. mentagrophytes var. mentagrophytes T i P-12 EE 412 UK Chin T. mentagrophytes var. mentagrophytes T i A-1 EG 5936 UK Animal T. mentagrophytes var. mentagrophytes T i A-2 EH 2078 UK Groin T. mentagrophytes var. mentagrophytes T i A-3 EH 4754 UK Buttock T. mentagrophytes var. mentagrophytes T i A-4 EH 5824 UK Animal T. mentagrophytes var. mentagrophytes T i A-5 EJ 6296 UK Neck T. mentagrophytes var. mentagrophytes T i A-6 EK 7724 UK Cat T. mentagrophytes var. mentagrophytes T i A-7 EL 3072 UK Face T. mentagrophytes var. mentagrophytes T i A-8 EZ 5146 UK Rabbit T. mentagrophytes var. mentagrophytes T i A-9 K 51 UK Neck T. mentagrophytes var. mentagrophytes T i A-10 P 31 UK Skin T. mentagrophytes var. mentagrophytes T i A-11 EN 6349 UK Wrist T. mentagrophytes var. mentagrophytes Ab ND EK 1471 UK Chest T. mentagrophytes var. mentagrophytes Av ND EK 4836 UK Face T. mentagrophytes var. mentagrophytes Av ND EK 827 UK Knee T. mentagrophytes var. mentagrophytes Av ND EZ 2640 UK Wrist T. mentagrophytes var. mentagrophytes Av ND 9218 UK NK T. mentagrophytes var. interdigitale Av ND EK 4260 UK Arm T. mentagrophytes var. mentagrophytes T? ND a Abbreviations: KMU, Kanazawa Medical University; UK, United Kingdom; NK, not known; ND, not determined. b Identifications were based on the morphology of primary cultures. c RFLP profiles of PCR-amplified ITS regions of rdna; T i, same profile as that of typical T. mentagrophytes var. interdigitale strain SM 8796; Av, same profile as that of A. vanbreuseghemii; Ab, same profile as that of A. benhamiae; T?, profile different from that any of the members in the T. mentagrophytes complex. d The original designations of these Indian isolates were Menon 187 for SM 8774 and Menon 302 for SM 8779.

4586 MOCHIZUKI ET AL. J. CLIN. MICROBIOL. FIG. 1. EcoRI restriction map of the rdnas of dermatophytes. The fragment between restriction sites Eco(n ) and Eco(n 1) represents the spacers that include the NTS region. The most likely explanation for the detectable length polymorphisms is the presence of repeating elements (?repeat) in the NTS region. The structures of ETSs in dermatophytes have not yet been determined. teristic for this subgroup; i.e., the banding pattern was not the same as that for any of the other A subgroups. Generally, the banding profiles for the A subgroups were more complex than those for the P subgroups. Geographically, all the Asian isolates were assigned to the P group and all the A group isolates were from the United Kingdom. All except one of the T. mentagrophytes var. mentagrophytes isolates were in the A group, and all of the T. mentagrophytes var. interdigitale isolates were in the P group. The most prevalent DNA type, type P-1, was found to be cosmopolitan. Reproducibility. Two separate DNA extractions were performed with eight isolates (isolates EH 2078, EH 4757, EK 4354, EK 7724, EL 3072, EZ 5146, K 51, P 31), and Southern blotting was performed for comparative purposes. The banding patterns of the NTS regions from the repeat extractions with these isolates were unchanged (data not shown). Since the banding profiles of subgroups P-3 and P-6 and subgroups P-6 and P-8 looked very similar, DNAs from representative isolates of subgroups P-3 (EP 0914), P-9 (2035), P-6 (T2.261), and P-8 (EP 1099) were loaded side by side on the same gel; and then the banding profiles were precisely compared (data not shown). The independent nature of subgroups P-3, P-6, P-8, and P-9 was confirmed. DISCUSSION Typing of dermatophyte strains has a number of potential epidemiological and clinical applications. The source of infection can be confirmed when the origins of the fungi are presumed to be other family members or public facilities, such as schools, swimming pools, hot spas, dormitories, or military establishments. At the clinical level, the cause of relapse after antimycotic therapy, that is, whether lesions are due to a recurrence of infection with the original strain or reinfection with a new strain, could be determined by typing. Some patients suffer from chronic infections which may be caused by certain strains, identifiable by the DNA type. For the purpose of evaluating intraspecies polymorphisms, conventional criteria such as colony morphology are limited because dermatophytes typically undergo changes in colony morphology during maintenance of the isolates. Very sensitive molecular markers are required for strain identification. RAPD analysis has recently been used to differentiate T. mentagrophytes strains and has demonstrated some degree of intraspecies variation among the isolates examined (11, 27). However, the method is known to FIG. 2. RFLP patterns of the PCR-amplified ITS 1 and 2 regions of rdna digested with the restriction enzyme MvaI. All products were electrophoresed in a 5% polyacrylamide gel and stained with ethidium bromide. Lanes: Ef, E. floccosum CBS 358.93; Tr, T. rubrum SM 8818; Av, A. vanbreuseghemii RV 27960, Ab (Am-Eu); A. benhamiae, Americano-European race, RV 26680; Ab (Af) A. benhamiae, African race, RV 30001; Te, T. mentagrophytes var. erinacei CBS 344.79; Tc, T. concentricum CBS 448.61; As (448.65), A. simii CBS 448.65; As (520.75), A. simii CBS 520.75; Tq, T. mentagrophytes var. quinckeanum NCPF 309; Ti, T. mentagrophytes var. interdigitale SM 8796; EK 4836, EN 6349, EP 0585, and P31, representative isolates of T. mentagrophytes from humans with dermatophytoses used in the study; M, molecular size marker. Isolates such as EK 4836 and EN 6349, which showed patterns different from those for the T. mentagrophytes var. interdigitale isolates, were eliminated from the subsequent DNA typing study based on NTS regions.

VOL. 41, 2003 DIFFERENTIATION OF TRICHOPHYTON MENTAGROPHYTES STRAINS 4587 FIG. 3. Southern blotting of EcoRI-digested genomic DNA from the 12 subgroups (DNA types P-1 to P-12) of the P group of human T. mentagrophytes isolates, in which a 2.5-kb band was present. Subgroups are defined by the banding patterns of bands between 5 and 10 kb. Lanes M, molecular size markers. have problems with reproducibility (20), and therefore, more robust methods are required. In the present study, we have applied a molecular method which was previously used with T. rubrum (9) to detect intraspecies polymorphisms in the NTS region of T. mentagrophytes rdna. In the previous study with T. rubrum, analysis of the NTS region showed 14 DNA types among 50 isolates (9), whereas no intraspecies polymorphisms were found in RAPD analysis studies (15, 20) or by sequence analysis of the rdna ITS 1 region (17), and only two subtypes were found in studies of mitochondrial DNA (3, 23). In a preliminary study of Japanese A. benhamiae isolates, five NTS types were found among eight isolates, whereas no intraspecies polymorphisms were found by RFLP analysis of ITS regions (22). Analysis of the NTS region is thus considered a sensitive method of typing. It could not, however, be assumed that T. mentagrophytes strains could be distinguished by NTS analysis. Candida krusei exhibits extensive variation in the NTS region (2), while Candida albicans does not (R. C. Barton, unpublished data). We therefore adopted a similar approach to search for NTS polymorphisms in T. mentagrophytes isolates showing T. mentagrophytes var. interdigitale-type DNA by RFLP analysis of ITS regions. PCR systems for the detection of polymorphisms in the NTS region have been already applied for identification T. rubrum (10), C. krusei (2), and Aspergillus fumigatus (24) strains. However, effective PCR primers for amplification of the NTS regions of T. mentagrophytes have yet to be developed, and Southern blotting was needed to detect the polymorphisms in this intergenic spacer. The isolates used for the present hybridization study were selected by RFLP analysis of PCR-amplified ITS regions. Morphologically identified T. mentagrophytes is composed of several biologically different taxa (14, 25); therefore, we wanted to use a biologically homogeneous group of isolates to apply this highly sensitive molecular method to study intraspecies polymorphisms. Since the complete nucleotide sequences of the ITS 1 and 2 regions of almost all the members of the T. mentagrophytes complex have been reported (4, 5, 16, 17), the RFLP patterns of the regions for each of the members were easily anticipated. MvaI digestion was applied to discriminate T. mentagrophytes var. interdigitale from the other members in the T. mentagrophytes complex (9). By RFLP analysis of the ITS regions, the most closely related taxa, T. mentagrophytes var. interdigitale and A. vanbreuseghemii, which have 98.8% DNA homology in their ITS 1 sequences (251 of 255 bases are common) (16), are easily distinguishable. Therefore, the 60 isolates selected were highly homogeneous and were suitable for use in the evaluation of intraspecies polymorphisms. The present study placed 60 isolates of T. mentagrophytes into 23 DNA subgroups. There was a good correlation between the anamorph variety, based on colony morphology, source of infection, and clinical pattern, and the DNA subtype. All 14 isolates originally identified as T. mentagrophytes var. mentagrophytes showed characteristic and highly variable patterns. The profiles for the 11 isolates in the A groups were observed only among these T. mentagrophytes var. mentagrophytes isolates, while three strains (EF 2608, EG 5704, and EK 4354) showed unique P types. The genotypes of the last three isolates appear to be closely related to the T. mentagrophytes var. interdigitale genotypes, although they are phenotypically T. mentagrophytes var. mentagrophytes. On the other hand, T. mentagrophytes var. interdigitale isolates showed less variety. A total of 46 T. mentagrophytes var. interdigitale isolates were members of nine subgroups (subgroups P-1 to P-9), and P-1 isolates made up half of the P group. The evolutionary relationships among T. mentagrophytes varieties are still being worked out; but in our study many T. mentagrophytes var. interdigitale isolates showed similar P-type profiles, and this variety therefore FIG. 4. Southern blotting of EcoRI-digested genomic DNA from the 11 subgroups (DNA types A-1 to A-11) of the A group of human T. mentagrophytes isolates, in which the 2.5-kb band was absent. Lanes M, molecular size markers.

4588 MOCHIZUKI ET AL. J. CLIN. MICROBIOL. appears to be more clonal than T. mentagrophytes var. mentagrophytes. For strain typing purposes, RFLP analysis of the rdna, including the NTS region, is highly discriminatory for the detection of T. mentagrophytes var. mentagrophytes isolates. However, another more sensitive marker is needed to subtype the common P types of T. mentagrophytes var. interdigitale. Geographically, P-1 strains were isolated from both Asia (9 of 10 isolates) and the United Kingdom (14 of 50 isolates), but there were no T. mentagrophytes var. mentagrophytes isolates from Japan. The incidence of the subgroups appears to be very different, and additional population studies with isolates from Asia and other geographical regions are needed. Dermatophyte rdna, including the NTS region, also includes external transcribed spacers (ETSs) (Fig. 1). Although the structures of ETSs in dermatophytes have yet to be reported, ETSs are known to have many functions in the maturation of rrna in yeast (1, 7). The major source of the rdna length polymorphisms is unlikely to be the conserved ETS and probably results from a repetitive element(s) elsewhere in the NTS present in various copy numbers. Such elements are responsible for length variations of the NTSs in T. rubrum (9, 10), C. krusei (2), and A. fumigatus (25). The tandemly repetitive subelement 1 and 2 sequences in T. rubrum have recently been examined in detail, and genetically homologous elements do not appear to be present in T. mentagrophytes (10). Base substitution or methylation (even in some of the copies) at the EcoRI restriction enzyme recognition site is an alternative cause of the polymorphisms. A heterokaryotic state, in which cells contain multiple nuclei with different genotypes, may also be a source of variation. In addition, the intensities of the largest bands are generally very faint compared with those of the major bands, which may indicate that these bands are composed of only a small number of copies. For example, they may result from the single EcoRI fragments present at either terminus of the junction between the rdna cistrons and the flanking, nonribosomal DNA. Partial digestion by the restriction enzymes is unlikely to be the source of the complexity of the banding profiles, due to checks on reproducibility. We conclude that RFLP analysis of NTS regions of rdna is a valuable molecular marker for subtyping of isolates with the T. mentagrophytes var. interdigitale genotype, and it may contribute to the solution of epidemiological and clinical problems of dermatophytoses. ACKNOWLEDGMENT This work was supported by a grant-in-aid for scientific research (grant C-2, no. 10670811) from the Japanese Ministry of Education, Science and Culture. REFERENCES 1. Beltrame, M., Y. Henry, and D. 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