J Med Microbiol 55 (2006), 429-436; DOI: 10.1099/jmm.0.46236-0
© 2006 Society for General Microbiology
ISSN 0022-2615
Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer
Lilian Cristiane Baeza,
Marcelo Teruyuki Matsumoto,
Ana Marisa Fusco Almeida and
Maria José Soares Mendes-Giannini
Departamento de Análises Clínicas, Faculdade de Ciêlncias Farmacêuticas, UNESP, Rua Expedicionários do Brasil, 1621, Araraquara, SP, CEP 14801-902, Brazil
Correspondence
Maria José Soares Mendes-Giannini
giannini{at}fcfar.unesp.br
Received 8 July 2005
Accepted 30 November 2005
Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T. rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPD products showed a high degree of similarity (>90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60 % similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPD analysis is especially suitable for molecular typing in T. rubrum.
Abbreviations: NTS, nontranscribed spacer; RAPD, randomly amplified polymorphic DNA.
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INTRODUCTION
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Dermatophyte infections are thought to be most common form of human contagious fungal disease. Trichophyton rubrum is the most frequently isolated agent of dermatophytosis worldwide, accounting for approximately 80 % of reported cases of onychomycosis (Evans, 1998).
The identification of T. rubrum at the strain level might help considerably in the treatment and prophylaxis of dermatophytosis. Molecular typing could be particularly useful in solving epidemiological questions, for example revealing infective routes, common sources of infection and areas of dissemination, as well as determining whether the original isolate is responsible for reinfection or a new strain has been acquired.
Molecular biology techniques, such as PCR, RFLP, randomly amplified polymorphic DNA (RAPD) analysis and Southern blotting, have led to dramatic progress in distinguishing among species and strains. In the case of Trichophyton, RAPD succeeded in distinguishing T. rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans (Liu et al., 1996), whereas intraspecific variability was only detected by this method in the T. mentagrophytes group (Kac et al., 1999). Gräser et al. (2000) investigated 96 T. rubrum strains from 4 continents by PCR fingerprinting, amplified fragment length polymorphism and random amplified monomorphic DNA markers. These strains display various colony morphologies, yet none of the methods revealed any polymorphism. RFLP was used for molecular genotyping of T. rubrum from patients with onychomycosis and a total of 5 types were observed among 66 serial isolates of T. rubrum collected from 16 patients. In six patients, more than one type was observed in successive samples. However, the isolation of a different genotype from a nail could not be interpreted as due to infection by a novel strain (Gupta et al., 2001). It is not impossible that individual dermatophytosis patients may be infected by multiple strains, especially patients with a T. rubrum nail infection who have a genetic predisposition making them vulnerable to this organism (Faergemann et al., 2005; Zaias et al., 1996). However, Jackson et al. (2000) described intraspecific variation in T. rubrum strains by the amplification of tandemly repetitive subelements (TRS-1) from the rDNA nontranscribed spacer (NTS) region, 21 TRS-1 PCR types being recognized in 101 clinical isolates. In addition, Kamiya et al. (2004) observed 17 TRS-1 PCR types in 252 clinical isolates. Yazdanparast et al. (2003) also reported two or more T. rubrum strain types from an infectious site when five colonies per culture were selected for typing, the strains being analysed by a PCR-based typing method of varying numbers of repetitive elements in the NTS of rRNA gene repeats, suggesting that in nail infections multiple strains are involved. In contrast, Rad et al. (2005) isolated only a single strain type from patients with tinea pedis and proposed a monotypic aetiology for tinea pedis. Recently, we reported the use of RAPD analysis with six different random primers for T. rubrum molecular typing; two of the six primers (numbered 1 and 6) reflected intraspecific polymorphism, and five patterns were observed among the ten strains tested, with each of the two primers (Baeza & Mendes-Giannini, 2004).
The aim of the present work was to study genotypic variability within T. rubrum by RAPD analysis with the random primers 1 and 6, as well as by subrepeat element analysis of the NTS of rDNA, using the repetitive subelements TRS-1 and TRS-2, to differentiate epidemiologically related and unrelated isolates.
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METHODS
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Dermatophyte isolates.
The 67 strains of T. rubrum and details of their origins are listed in Table 1
. All isolates were recovered from humans with clinical lesions. The majority of the patients (68 %) were aged between 10 and 39 years; of the patients with tinea pedis, 75 % were in this age range, as were 62·9 % of the patients with onychomycosis, while 22·3 % of all the patients were >50 years old. There were epidemiological associations between the isolates 1 to 13, collected from patients who lived together in a public institution (an orphanage), and seven pairs of isolates were derived from two different sites on single patients. The remaining strains were epidemiologically unrelated.
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Table 1. Human clinical isolates and PCR types for the 67 strains of T. rubrum analysed
Patients indicated by p (tinea pedis), o (onychomycosis), cr (tinea cruris) and co (tinea corporis) had two different sites of infection. U, epidemiologically unrelated; R, epidemiologically related.
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The clinical samples were isolated in the Clinical Mycology Laboratory of the School of Pharmacy, UNESP, Sao Paulo State University, Araraquara, Brazil, and identified to species level using standard mycological procedures (Summerbell & Kane, 1997).
Isolation of fungal DNA.
All isolates were grown on Sabouraud's dextrose agar (Difco) supplemented with chloramphenicol, 0·05 % (w/v), and incubated at 25 °C for 10 days. Liquid nitrogen was added to 1–2 g of mycelium in a mortar, and the cells were ground finely with a pestle. Genomic DNA was extracted as described by Del Sal et al. (1989) with a few modifications. In brief, the mycelial powder was transferred to an Eppendorf tube and 500 µl lysis buffer (10 mM Tris/HCl pH 8·0, 1 mM EDTA, 1 % SDS, 100 µg proteinase K ml–1) was added. The mixture was incubated for 1 h at 37 °C, 200 µl 5 mol l–1 NaCl was added and the contents incubated for 10 min at 65 °C. After that, 100 µl 10 % (w/v) cetyltrimethylammonium bromide (CTAB; Sigma) was added and incubation continued for a further 20 min at 65 °C. The solution was treated with RNase A (Roche) at a final concentration of 50 µg ml–1 for 1 h at 37 °C, and then extracted with an equal volume of phenol/chloroform/isoamyl alcohol [25 : 24 : 1 (v/v)], and centrifuged at 12 000 g for 15 min at 4 °C. This procedure was repeated three times. The DNA was precipitated with two volumes of ice-cold 2-propanol at –20 °C for 20 min, washed twice in 500 µl 70 % ethanol, air dried, and resuspended in 100 µl TE buffer (40 mM Tris/HCl pH 8·0, 2 mM EDTA). DNA concentration was estimated by measuring the absorbance at 260 nm.
RAPD.
The following decamer oligonucleotides of arbitrary sequence were used as single primers in the RAPD experiments: primer 1, 5'-d[GGTGCGGGAA]-3', and primer 6, 5'-d[CCCGTCAGCA]-3' from the Ready-To-Go RAPD analysis beads kit (Amersham Pharmacia Biotech). Amplification reactions were performed in 25 µl volumes containing 50 ng template DNA and a lyophilized mixture of reaction buffer (30 mM KCl, 3 mM MgCl2, 10 mM Tris pH 8·3), 0·4 mM (each) dNTP, 2·5 µg BSA, 25 pmol primer and thermostable polymerases (Ampli Taq DNA polymerase and Stoffel fragment). The samples were overlaid with sterile light mineral oil (Sigma) and amplification was performed in a thermal cycler (Perkin-Elmer; 9700) as follows: an initial denaturation of 5 min at 95 °C was followed by 45 cycles consisting of denaturation for 1 min at 95 °C, annealing for 1 min at 36 °C and extension for 2 min at 72 °C, and then a final extension for 10 min at 72 °C. Amplification products were separated by electrophoresis in 2 % agarose gels, visualized by staining with ethidium bromide and photographed under UV.
RAPD profiles were analysed by GelCompar software (version 2.0; Applied Maths). The similarity coefficient (SAB) between band patterns for a given pair of isolates A and B was computed by the formula SAB=2E/(2E+a+b), where E is the number of common bands in the patterns of A and B, a is the number of bands in pattern A with no correlates in pattern B, and b is the number of bands in pattern B with no correlates in pattern A. Dendrograms based on SAB values were generated by the unweighted pair group method with arithmetic means (UPGMA), implemented in the GelCompar software.
PCR assay for strain characterization.
Following the method for strain characterization of T. rubrum described by Jackson et al. (2000), two primer pairs (flanking sequences from the NTS located between the 18S and 25S regions) were used to amplify two novel tandemly repetitive subelements: TRS-1, containing a 27 bp palindromic sequence, and TRS-2. Primers TrNTSF-2 (5'-ACCGTATTAAGCTAGCGCTGC-3') and TrNTSR-4 (5'-TGCCACTTCGATTAGGAGGC-3') were used to amplify TRS-1, and primers TrNTSR-1 (5'-CTCAGTCGAACCGTGAGGC-3') and TrNTSC-1 (5'-CGAGACCACGTGATACATGCG-3') were used to amplify TRS-2.
These amplification reactions were carried out in reaction buffer (50 mM KCl, 10 mM Tris/HCl pH 9·0, 0·1 % Triton X-100) with 2·0 mM magnesium chloride, containing each dNTP at 0·25 mM (dATP, dCTP, dGTP and dTTP), 50 pmol each primer, 1·25 U Taq polymerase (BioTools) and approximately 20 ng template DNA. The final reaction volume was made up to 50 µl with pure water. The amplification of TRS-1 was carried out in a thermal cycler with an initial denaturation for 2 min at 94 °C, followed by 30 cycles consisting of denaturation for 0·5 min at 94 °C, annealing for 0·5 min at 58 °C and extension for 3 min at 72 °C, and then a final extension for 10 min at 72 °C. The conditions for amplification of TRS-2 were: initial denaturation for 1 min at 94 °C, followed by 30 cycles consisting of denaturation for 0·5 min at 94 °C, annealing for 0·5 min at 55 °C and extension for 2 min at 72 °C, with a final extension step for 10 min at 72 °C. Amplification products were separated by electrophoresis in 2 % agarose gels, visualized by staining with ethidium bromide and photographed in UV light.
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RESULTS
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RAPD analysis
Two random primers were used to amplify DNA polymorphisms within T. rubrum. RAPD profiles were assigned according to major bands. All visible and well-defined bands were identified visually and confirmed by software. Dendrograms were used to determine the relatedness of isolates on the basis of these RAPD bands. The analysis with primer 1 yielded 12 profiles among the 67 isolates. The mean SAB value of the isolates was 0·737±0·173. Three groups were formed, I, II and III, with 60 % similarity. The group I isolates had 80 % similarity and were distributed into two subgroups, IA and IB, which included 45 (67·2 %) of the isolates. The ten isolates strongly related epidemiologically had >95 % similarity. Nine of these had similarity coefficients of 100 %, being genotypically identical with an SAB value of 1·00, while one, isolated from patient 2, had a value of 0·923. Among the seven patients represented by two samples each that were isolated from different body sites, three patients (1023, 1033 and 1042) had identical profiles at both sites. The strains from patient 2264 were included in two different groups, IB and III, the similarity between the isolates being 60 %; strains from patient 2291 were in the subgroups IA and IB, with 80 % similarity, and strains from patient 2480 were in two groups, II and III, the similarity value being 60 % (Fig. 1).
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Fig. 1. Dendrogram of the RAPD patterns obtained from T. rubrum isolates with random primer 1. M, molecular mass marker (Gibco; 100 bp).
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The 11 RAPD profiles obtained with primer 6 were clustered in three main groups (IA, IB and III), with a similarity coefficient of about 60 %. The mean SAB value for the isolates was 0·732±0·184. The similarity coefficient of group I strains (IA and IB) was 85 %. Isolates from the ten epidemiologically related patients were divided into two clusters with high overall homogeneity (92 %); eight of these isolates showed SAB values of 1·00, representing identical strains, while the SAB value of isolates 6 and 8 and the others was found to be 0·933. To assess the genetic similarity of the seven patients each represented by two samples isolated from different body sites, SAB values between each pair of isolates obtained with the two primers were computed and listed in Table 2. A total of 6 (3 pairs) of the 14 isolates from these patients showed differences between the paired isolates, and were distributed as follows: strains of patient 2264 were included in 2 different clusters within the same group, IA, the similarity coefficient between these isolates being 90 %; strains from patient 2291 were in the groups 1B and III, with 60 % similarity; strains from patient 2480 were in 2 subgroups, IA and 1B, the similarity value being 80 % (Fig. 2).
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Table 2. Genetic similarity between strains from patients with two samples isolated from different body sites
p, Tinea pedis; o, onychomycosis; cr, tinea cruris; co, tinea corporis.
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Fig. 2. Dendrogram of the RAPD patterns obtained from T. rubrum isolates with random primer 6. M, molecular mass marker (Gibco; 100 bp).
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Strain typing of T. rubrum by specific amplification of the TRS-1 and TRS-2 repeat regions
Three TRS-1 types (1, 2 and 3) were observed among the isolates studied (Fig. 3a). One pattern, PCR type 1, was the most common, representing 60 (89·5 %) out of 67 strains; it was followed by PCR type 2 (4 out of 67 strains; 6·0 %) and PCR type 3 (3 out of 67 strains; 4·5 %). Only one PCR type (1) was recognized among all 10 epidemiologically related isolates taken from patients living together in a public institution (an orphanage). The strains isolated from two different body sites on the same patient had identical profiles (Table 1
).
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Fig. 3. (a) Amplification of the TRS-1 subrepeat element produced3 PCR types from 67 clinical isolates of T. rubrum. Lanes: M, molecular mass marker (Promega; 100 bp); 1, strain 02; 2, strain 1029; 3, strain 2044; 4, strain 2060; 5, strain 2291; 6, strain 1033; 7, strain 2232; 8, strain 2249; 9, strain 2413; 10, strain 2455. (b) Amplification of the TRS-2 subrepeat element produced 2 PCR types from 67 clinical isolates of T. rubrum. Lanes: M, molecular mass marker (Promega; 100 bp); 1, strain 1044; 2, strain 2044; 3, strain 2236; 4, strain 02; 5, strain 1021; 6, strain 2230; 7, strain 2255; 8, strain 2292; 9, strain 2412; 10, strain 2418.
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In the NTS region containing TRS-2, two PCR types were found (I and II) (Fig. 3b). A single 502 bp product representing two complete copies of TRS-2 (PCR type I) was obtained from the majority of strains tested (57 out of 67 strains; 85 %). The smaller fragment, with a length equivalent to one 77 bp TRS-2 repeat unit, was found in 15 % of strains, all of them having a single copy of TRS-2 (PCR type II). The distribution of TRS-2 types recovered from the patients who lived together showed that one isolate (patient 5) was PCR type II and the rest were PCR type I. The strains isolated from different sites on the same patient had identical patterns (PCR type I) (Table 1).
Reproducibility and stability of the methods
To evaluate the stability of the methods, each sample of genomic DNA was amplified in duplicate in repeated PCRs at different times. To test for reproducibility, the DNAs from 14 isolates were extracted from 2 independent cultures. All amplifications were done with rigorously standardized concentrations of reagents, the same thermal cycler and the same cycling conditions. RAPD profiles showed identical band patterns (data not shown) and reproducibility was 100 % (Fig. 4). In the typing by specific amplification of the TRS-1 and TRS-2 repeat regions, reagents from different sources were used, Taq polymerase (Invitrogen) and MasterMix (Eppendorf), giving identical results (data not shown).
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Fig. 4. (a) Comparison of RAPD patterns obtained using primer 1 from clinical isolates of T. rubrum amplified in repeated PCRs at different times. Lanes: M, molecular mass marker (Gibco; 100 bp); 1, strain 2292; 2, strain 2379; 3, strain 2396; 4, strain 2413; 5, strain 2455. (b) Comparison of RAPD patterns obtained using primer 1 from clinical isolates of T. rubrum amplified from genomic DNA extracted from two independent cultures. Lanes: M, molecular mass marker (Gibco; 100 bp); 6,strain 2060; 7, strain 2264co.
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DISCUSSION
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Molecular typing of dermatophytes could provide new insights into the epidemiology, population biology and pathogenicity of these fungi. Techniques for the detection of intraspecific variation have long been sought, in order to allow population genetics studies of Trichophyton species, especially of T. rubrum, the predominant species in most isolates.
However, anthropophilic (human-specific) dermatophyte species are products of recent evolutionary adaptive radiation events (Harmsen et al., 1995; Summerbell et al., 1997) and also have a restricted ecological niche, resulting in species with unusually high degrees of genetic uniformity (Summerbell et al., 1999). Studies by Gräser et al. (2000) have further highlighted the uniformity in the T. rubrum complex by comparing the morphological and physiological features with the results of sequencing of the internal transcribed spacer (ITS) region of the ribosomal operon, PCR fingerprinting and amplified fragment length polymorphism analysis.
In order to improve epidemiological monitoring, it is important to follow standardized methods. Here we describe the application of RAPD analysis with two random primers, as well as subrepeat element analysis of the NTS region of rDNA. RAPD analysis has demonstrated enormous success in fungal infection studies (Soll, 2000). However, the main problem is the reproducibility, not only among laboratories, but also within a laboratory over time. Artifactual variation can occur as a result of small differences in the primer : template concentration ratio, the temperatures during amplification and the concentration of magnesium in the reaction mixture (Ellsworth et al., 1993). In contrast to most of the results in the literature, in this study we showed identical band patterns and a reproducibility of 100 %. These results were obtained from a lyophilized mixture, with standardized concentrations of the reagents and the same thermal cycler for all the RAPD reactions.
The intraspecific polymorphism achieved in our study contrasts with what has been demonstrated in most of the available literature on the use of RAPD. Zhong et al. (1997) examined 30 isolates of T. rubrum by RAPD analysis and found that 22 strains were indistinguishable and 8 showed very minor differences, while Liu et al. (1996), using arbitrarily primed PCR, reported no differences between 8 strains of T. rubrum. The sequences employed in our study probably hybridize with less-conserved regions of the genomic DNA and generate a larger degree of polymorphism.
Epidemiologically related T. rubrum strains showed profiles that differed only in one band. Dendrogram analysis, irrespective of the primer, generated similarity coefficients >90 %, demonstrating that these isolates are highly related. These isolates came from an orphanage, where the use of common bathrooms, towels and shoes and recreational area may have provided ideal conditions for the proliferation of a single or small number of particularly anthropophilic or pathogenic T. rubrum clones. The SAB value for these isolates was >0·90. According to Chong et al. (2003), an SAB value of 0·80–0·99 represents highly similar (but not identical) strains, suggesting that these infections were caused by a single strain that underwent microevolution.
Interestingly, of the seven patients that yielded two samples from separate infected sites, three showed different profiles at the two sites, with each primer. These lesions may be due to infection by two different strains. Yazdanparast et al. (2003) detected two or more T. rubrum strains involved in nail infection by analysis of the variation in numbers of repetitive elements in the NTS region of the rDNA. By contrast, no variation was observed in RAPD analysis of pairs of T. mentagrophytes strains from foot skin and nail lesions (Kac et al., 1999).
T. rubrum has subrepeat elements in the rDNA and it has been demonstrated that variations in the copy number of these elements are useful for strain identification (Jackson et al., 2000). In our study, three TRS-1 PCR types (1, 2 and 3) were found and 60 strains (89·5 %) were classified as PCR type 1, a higher proportion than that (75 %) reported for the most common type seen by Jackson et al. (2000), and the total number of types was considerably lower than in their work (21 PCR patterns, of which only 6 types were straightforward). One explanation for this pattern assumes that multiplication of the number of copies of TRS-1 by unequal crossover is a rare event (Jackson et al., 2000). The other class (TRS-2) was less variable, with just two different types, and 85 % of all strains examined showed two complete copies of TRS-2 in the amplification product.
One reason for this limited range of common PCR types may relate to the presumed asexual nature of T. rubrum, as genomic diversity at the rDNA locus may be restricted by the absence of sexual recombination. Also, the strains used in this study were isolated from a limited area in Brazil and may have resulted from the rapid proliferation of a limited number of comparatively undifferentiated clones, reflected in the restricted number of prevalent PCR types reported here. This distribution pattern may also involve strain types that may possess enhanced infectivity, invasiveness or other virulence characteristics.
Although intraspecies strain variability was observed by analysis of the NTS of rDNA, this typing system may not be very useful in relapse and reinfection studies, since it has a low discrimination index. On the other hand, our results from RAPD analysis demonstrate the substantial molecular diversity present in the genome of T. rubrum, and interstrain variations may subsequently be identified. This technique provides a valuable tool for strain typing and epidemiology of disease caused by these organisms.
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ACKNOWLEDGEMENTS
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We are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CNPq, PADC-FCF-UNESP and FUNDUNESP, who provided the financial support for this project.
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REFERENCES
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INTRODUCTION
METHODS
