Abstract: Dermatophytes and Candida are the most common sources of human fungal infections. Identification of dermatophytes and Candida using the traditional methods is sometimes problematic because of atypical microscopic or macroscopic morphology. The aim of this study was to identify and evaluate the genetic relationship among 6 species of dermatophytes and 3 species of yeasts isolated from Egyptian and Libyan patients with skin mycosis using three molecular techniques (RAPD, ISSR and RFLP) and isozyme profiles. Each species was represented by two isolates, one from Egyptian patients and the second from Libyan. Random amplified polymorphic DNA (RAPD) in which four random 10-mer primers and two Inter-Simple Sequence Repeat (ISSR) primers were used to amplify the DNA fragments of target fungi. Restriction Fragment Length Polymorphism (RFLP) in which two universal primers (ITS1 and ITS4) were used to amplify the Internal Transcribed Spacer (ITS) regions of the ribosomal (rRNA) gene in fungal isolates followed by digestion with HinfI and HaeIII endonucleases. Results of RAPD and ISSR markers revealed 78.7% Genetic Similarity (GS) between M. canis and other tested fungi reflecting a relatively longer genetic distance from other isolates of dermatophytes and yeasts. C. krusei and C. tropicalis were closely related showing 93.3% GS. C. albicans showed 90.9% similarity with other species of Candida. E. floccosum was easily separated from all Trichophyton species showing 87.3% similarity. Unique bands were displayed by certain fungi and can be taken as a positive marker for isolate identification and discrimination. RFLP technique revealed differences in the number (1-5) and size (8-378 base pairs) of DNA fragments depending on the fungal isolate and restriction enzyme used. Within each fungal species, different isolates of dermatophytes and Candida from Egypt and Libya showed close relationship. Seven isozyme systems were studied to detect the gene expression and genetic variability among the different isolates of dermatophytes and Candida.
INTRODUCTION
Conventional methods for identification of fungi rely on macro and micro morphological features of the colonies on general and specific culture media and on some biochemical and physiological complementary test. However, conventional identification method is more difficult because its affected by many factors such as temperature, medium and chemotherapy which influence on phenotypic characters (Weitzman and Summerbell, 1995; Faggi et al., 2001; Toshio, 2008; De Baere et al., 2010). Recently, Molecular marker approaches, such as Nested-PCR (Verrier et al., 2013), RAPD-PCR (Kim et al., 2001; Baeza et al., 2006; Leibner-Ciszak et al., 2010; Dobrowolska et al., 2011; Spesso et al., 2013), ISSR-PCR (Cano et al., 2005; Khosravi et al., 2012), PCR-RFLP (Yang et al., 2008; Rezaei-Matehkolaei et al., 2012; Samuel et al., 2013), Real time PCR (Bergmans et al., 2010) and multiplex PCR assay (Arabatzis et al., 2007; Kim et al., 2011) and others have been adapted for detection of dermatophytes from clinical specimens. The random amplified polymorphic DNA (RAPD) approach which used to detect strain polymorphism depends on the application of short arbitrary primers that anneal to multiple genomic sites at appropriate temperature conditions and this technique does not depend on prior knowledge of species-specific sequence (Valerio et al., 2006). RAPD methods have frequently been used for phylogenetic analysis and identification of dermatophytes. For identification Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale. Spesso et al. (2013) used RAPD-PCR technique and they reported that RAPD-PCR is a useful and are liable schema for identification of several indemic strains. For identification of T. mentagrophytes and T. tonsurans, Kim et al. (1999, 2001) proposed a RAPD method, in which T. mentagrophytes was divided into subtypes on the basis of amplification patterns in RAPD using three primers.
The Inter-single sequence- repeat-PCR (ISSR-PCR) technique is highly reproducible and allows detection of interspecific and intraspecific DNA-polymorphisms. This method is based on the Polymerase Chain Reaction (PCR) and uses primers containing microsatellite sequences and degenerate anchors at the 50 end (Zietkiewicz et al., 1994).
Cano et al. (2005) used two inter-single-sequence-repeat-PCR (ISSR-PCR) primers (ACA) 5 and (CCA) 5 for typing 24 isolates of M. canis, seventeen isolates tested were from human patients, 5 from cats and 1 from a dog. They concluded that ISSR polymorphism has shown to be a reliable method for M. canis strain identification and probably more discriminatory than RAPDs. Khosravi et al. (2012) using (GACA) 4 ISSR primer to identification of dermatophyte species and they reported that (GACA)4 ISSR-based PCR has utility both as a simple and rapid method for identification of dermatophyte species and for differentiation of T. mentagrophytes variants. Therefore, it is better to use the RAPD and ISSR method in combination with other techniques such as gene-specific PCR-RFLP for fungal identification at the species level.
Rezaei-Matehkolaei et al. (2012) used MvaI restriction enzyme in order to cut the ITS-region and they concluded that this method is a rapid and useful for identification and screening of several pathogenic fungi even closely related species of dermatophytes. In Iran, Mirzahosseini et al. (2009) used RFLP-PCR technique and three restriction enzymes, MvaI, HinfI and HaeIII to identify six dermatophyte isolates. They reported that RFLP-PCR approaches is a rapid identification and reliable for deverntiation of dermatophyte isolates at the genes or species level. Isozyme electrophoretic pattern is a reliable approaches for taxonomic, genetic and population studied (Market and Moller, 1959).
Klaas (1998) reveled that the isozyme pattern is very useful to identify several levels of accession and individuals. Also, they stated that isozyme markers cane be more accurate than for some molecular markers. Siddiquee et al. (2010) used eight enzymes and single protein pattern systems to identify and studying the genetic relationships among 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia. They reported that isozyme patterns and protein profiles were useful for identification of fungi species.
The aim of this study was to identify and study the genetic relationship among 6 species of dermatophytes and 3 species of yeasts isolated from Egyptian and Libyan patients with skin mycosis using three molecular techniques (RAPD, ISSR and RFLP) and isozyme profiles.
MATERIALS AND METHODS
Fungal strains: Eighteen fungal strains were isolated from clinical cases in Assiut (Egypt) and Tripoli (Libya) and used in the present study (Table 1). Conventional morphological methods were employed for identification of these fungi where dermatophytes were cultured on Sabouraud’s Dextrose Agar (SDA) with chloramphenicol and on bromocresol purple milk glucose agar (Ellis et al., 2007). Candida species were allowed to grow on SDA, followed by identification on Chromagar Candida medium (Pfaller et al., 1996). Mycological references including De Hoog et al. (2000) and Ellis et al. (2007) were consulted for identification. All cultures were preserved in the culture collection of the Assiut University Mycological Centre (AUMC).
DNA extraction: Genomic DNA of each fungal strain was extracted using glass bead disruption (Yamada et al., 2002). Briefly, 300 mg of 0.5 mm diameter glass beads, 300 μL of lysis buffer (100 mM Tris-HCl pH 8, 10 mM EDTA, 100 mM NaCl, 1% Sodium Dodecyl Sulfate (SDS), 2% triton X-100) and 300 μL of phenol, chloroform-isoamyl, alcohol (25:24:1) were added to a fresh mycelium or cells in a 1.5 mL tube. Samples were shaken vigorously for 5 min, centrifuged for 10 min at 5000 rpm and the supernatant was transferred to a fresh tube. The supernatant was extracted again with chloroform and DNA was precipitated by adding equal volume of cold isopropanol and 0.1 volume of 3 M sodium acetate (pH 5.2). The solution was incubated for 10 min at -20°C and centrifuged for 15 min at 10000 rpm. The precipitant was washed with cold 70% ethanol and dried in air. DNA was suspended in 80 μL free-nuclease-water and an aliquot was removed to determine nucleic acid purity and concentration by spectrophotometry. DNA was also checked by agarose (0.8%) gel electrophoresis.
| Table 1: | Source and cod number of fungal isolates from Egyptian and
Libyan patient involved in the present study |
| Table 2: | Nucleotide sequences of primers used for PCR-RAPD and PCR-ISSR
analysis |
PCR-amplification for RAPD and ISSR: Four PCR-RAPD primers and two PCR-ISSR primers as shown in Table 2 were used to study the genetic differences and relationships among the 18 isolates of dermatophytes and Candida (Table 2).
Each amplification reaction was performed in a final volume of 25 μL containing 1 μL of genomic DNA, 1.25U of Taq DNA polymerase, 0.3 mM of each four deoxynucleoside triphosphate, 1.5 mM of MgCl2, 0.4 μM of each primer and 2.5 μL of 10X PCR buffer. PCR was carried out with the following program: 1 cycle of 5 min at 95°C, followed by 40 cycles of 1 min at 95°C, 1 min at 34°C and 2 min at 72°C and a final extension step at 72°C for 10 min. Amplified DNA fragments were run onto 1.4% agarose gel electrophoresis in TBE buffer at 80 V for 2.5 h. The products were detected by staining with ethidium bromide and photographed. The molecular sizes of DNA fragments were determined in relation to molecular standards (100-1500 bp). The presence/absence of fragments from dermatophytes and Candida isolates was analyzed using the software package MVSP program of Nei and Li (1979) to estimate the Genetic Similarity (GS).
Restriction Fragment Length Polymorphism (RFLP) Analysis
PCR amplification for rRNA gene: Fungal-specific universal primer pairs
were used to amplify internal transcribed spacer 1 (ITS1)-5.8S -ITS2 regions
of rDNA in the tested fungi. The amplification reaction was performed in a final
volume of 25 μL l containing 1 μL of extracted genomic DNA (about
20 ng), 1.25 U of Taq DNA polymerase, 0.3 mM of each deoxynucleoside
triphosphate mix (dATP, dTTP, dGTP, dCTP), 0.4 μM of each of forward ITS1
(5’-TCC GTA GGT GAA CCT GCG G-3’)
and reverse ITS4 (5’-TCC TCC
GCT TAT TGA TAT GC-3’) primers,
1.5 mM of MgCl2 and 2.5 μL of 10X PCR buffer. PCR was carried
out in a thermal cycler with the following temperature profile: 1 cycle of 5
min at 95°C, followed by 35 cycles of 1 min at 94°C, 1 min at 56°C
and 1 min at 72°C and a final
extension step at 72°C for 10 min. PCR products were analyzed in 2% agarose
gel with 0.5 X Tris-Borate-EDTA buffer, stained with ethidium bromide and visualized
in UV light.
Digestion with restriction enzymes (endonucleases): Ten microliters of each PCR product were separately digested with 10U of HinfI and HaeIII restriction enzymes at 37°C overnight. Restriction fragments were analyzed in 2.5% agarose gel electrophoresis in 0.5 X Tris-Borate-EDTA buffer for about 2.5 h at 80 V and visualized by staining with 0.5 μg mL-1 of ethidium bromide.
Data analysis: Agarose gel photos were scanned by the Gene Profiler 4.03 computer software program that uses automatic lane and peak finding for detecting the presence of banding patterns and calibrating them for size and intensity. A binary data matrix recording the presence (1) or the absence (0) of bands was made. The software package MVSP (Multi-Variant Statistical Package) was used and genetic similarities were computed using the Dice coefficient of similarity of Nei and Li (1979). Cluster analysis was carried out on similarity estimates using the pair-group method with arithmetic average (UPGMA) software.
Isozyme profiles of fungal isolates
Polyacrylamide gel electrophoresis: The electrophoresis was carried out
in vertical polyacrylamide gels, using the slab gel apparatus “ SE 600,
vertical slab gel”, according to Laemmli (1970)
with 7.5% acrylamide.
Preparation of samples: Enzymes were extracted by crushing 1.0 g of fungal hyphae in 1.0 mL extraction buffer (0.1 M Tris-HCl+2 mM EDTA, pH 7.8). To avoid denaturation of the enzymes by overheating, the samples were cooled with ice during crushing and preparation. Then, the samples were centrifuged for 25 min at 10.000 rpm at 4°C. One hundred micro liters of the supernatant was mixed by one hundred microliter of sample application buffer which was composed of 2.5 mM Tris-HCl (pH 6.8), 10.0% glycerol, 4.0% Sodium Dodecyl Sulfate (SDS), 0.02% bromophenol blue and 10% mercaptoethanol. Samples were then loaded directly in the electrophoresis apparatus for isozymes analysis.
Staining of gel and detection of enzymes: The gels were stained for seven enzyme systems. The staining protocols were according to Guikema and Sherman (1980) for Peroxidase (E.C.1.11.1.7), for Esterase (E.C.3.1.1.2), Glutamate-oxaloacetate-transaminase (E.C.2.6.1.1), Malate dehydrogenase (E.C1.1.1.37) Acid phosphatase (E.C.3.1.3.2) staining protocols were according to Tanksley and Orton (1986), staining protocol for Urease (E.C.3.5.1.5) was according to Fishbein (1969) and Protease (PROT E.C.3.4.2.3) staining gel was according to Sarath et al. (1999).
Data collection and analysis: Stained gels were placed in a light box to determine their isozyme Banding Patterns (BP). The number of bands was recorded and their relative mobility (Rf) was obtained using the formula:
Multivariate analysis of the isozyme profiles was done and clustering was based on the results of Unweighted Pair Group Method Using Averages (UPGMA) cluster analysis performed on the (Nei and Li, 1979) similarity coefficient matrices. Dendrogram presenting the genetic relationships of the different isolates were constructed using the Numerical Taxonomy and Multivariate Analysis System (NTSYS).
RESULTS
Genotypic relationship among dermatophytes and Candida species
RAPD and ISSR analysis: A total of 58 DNA bands ranging from 119 bp (HB15)
to 863 bp (OPW15) were generated by the six primers. The number of bands per
one primer ranged from 12 bands in case of OPA06 RAPD primer to 8 bands with
844A ISSR primer. C. krusei displayed the highest number of total DNA
fragments (43 bands/six primers), whereas T. rubrum yielded the lowest
(35 bands) as shown in Fig. 1 and Table 3.
These variations are mainly due to variations in primer structure and number
of annealing sites in the genomic DNA of fungal strains (Kernodle
et al., 1993).
| Fig. 1(a-f): | Agarose gel electrophoresis of fungal DNA fragments amplified with the RAPD and ISSR primers, (a)844A (b) OPA09 (c)OPA06, (d) OPW15 (e) HB 15 and (f) OPA13 |
| Table 3: | No. of DNA fragments amplified by the 6 primers of RAPD and
ISSR analysis of fungal isolates |
Polymorphic bands were detected with all tested primers in all isolates of dermatophytes and Candida. Out of 58 DNA-bands, 26 were conserved among all tested isolates while 32 (55.2%) were polymorphic (Table 3). The monomorphic bands are constant bands and cannot be used to study the diversity, while polymorphic bands revealed differences and could be used to establish a systematic relationship among the fungal genotypes (Hadrys et al., 1992). The presence of a unique band for a given isolates is taken as a positive marker while the absence of a unique band is referred as a negative marker. Such bands could be used as DNA markers for isolate identification and discrimination. The 844A ISSR primer showed one specific band for T. mentagrophytes at molecular weight of 377 bp and three unique bands for M. canis, two of which are expressed as positive bands (261 and 221 bp) and the 3rd was negative band (287 bp). One DNA fragment at molecular weight 212 bp generated by OPA09 primer could be used as positive marker for C. krusei. The OPA13 RAPD primer produced specific bands for M. canis and E. floccosum with molecular weights of 485 and 552 bp, respectively. Cluster dendrogram (Fig. 2) based on similarity matrix obtained with unweight pair group method using arithmetic means (UPGMA) showed that the genetic similarity among the 18 isolates of dermatophytes and Candida was high, ranging from 78.7 to 93.3%. The dendrogram also showed that M. canis isolates were separated in a single branch with 78.7% genetic similarity, reflecting a relatively longer genetic distance from other isolates. C. krusei and C. tropicalis are closely related (93.3% genetic similarity). Candida albicans showed 90.9% similarity with both C. krusei and C. tropicalis. E. floccosum was easily separated from all Trichophyton species showing 87.3% similarity.
RFLP analysis: Two consecutive PCR steps were done. Firstly, the ITS region of rDNA gene was amplified using the universal primers ITS1 and ITS4. Amplicons produced by T. mentagrophytes, T. verrucosum and T. rubrum were about 680 bp in length. Those from T. violaceum, M. canis and E. floccosum, were larger, 690, 720 and 780 bp, respectively.
| Fig. 2: | Dendrogram developed from PCR- RAPD and PCR-ISSR primers using UPGMA analysis. The scale is based on Nei and Li coefficients of similarity |
| Table 4: | RFLP analysis of ITS region of rDNA after digestion by HinfI
and HaeIII endonucleases |
The ITS region amplified from C. krusei, C. tropicalis and C. albicans, were 510, 524 and 550 bp, respectively (Fig. 3a and Table 4). Secondly, PCR products were subjected to RFLP analysis by digestion with the restriction enzymes HinfI and HaeIII in order to generate species-specific patterns for fungal identification. Digestion with HinfI endonuclease produced different fragment patterns varying in number and size. T. mentagrophytes, T. verrucosum and E. floccosum created five fragments, T. rubrum, T. violaceum and C. tropicalis showed four fragments and three fragments for C. krusei and C. albicans. Restriction fragments showed marked variations in size ranging from 8 to 378 bp. It was interestingly to note that in case of M. canis, HinfI was not able to produce any obvious cutting pattern (Table 4 and Fig. 3b). The endonuclease HaeIII was able to digest the ITS region of rDNA gene producing 2 – 3 fragments of 40 to 450 bp depending on the fungal isolate. Great similarity was observed among fragments of C. albicans and C. tropicalis (420 and 90 bp for each). C. krusei yielded three fragments of different sizes (370, 90 and 40 bp). Similarly, dermatophytes produced three fragments one of which (100 bp) was shared by all isolates of Trichophyton and Microsporum but not by Epidermophyton. However, these dermatophytes showed marked variations in the size of other fragments as seen in Fig. 3c and Table 4.
Isozyme profiles of dermatophytes and Candida isolates
Esterase (EST, E.C.3.1.1.2): Three loci of isozyme activity (EST1, EST2
and EST3) were observed for esterase (Fig. 4a). EST1 locus
was polymorphic and produced one band shown in T. mentagrophytes, T. verrucosum,
T. rubrum and T. violaceum isolates while, it not detected in other
fungi. EST isozyme was dimeric as confirmed by the presence of three bands in
the heterozygous genotypes. All tested fungi were heterozygous for EST2 and
EST3 loci. The tested fungi displayed three bands of EST3 at Rf values of 0.69,
0.73 and 0.76. Although at EST2 loci (Rf = 0.38, 0.43 and 0.48), a number of
isolates including E. floccosum, C. albicans, C. tropicalis and C.
krusei, showed only two out of the three bands (Table 5
and Fig. 4a). The missing of such bands in the heterozygous
state may be due to the least expression of such isozyme band or lower activity.
Peroxidase (PRX, E.C.1.11.1.7): Six peroxidase isozyme loci (Prx-1 to PRX-6) were detected in the tested fungi. PRX-3, 4, 5 and 6 isozyme bands were monomorphic, which all fungi displayed these bands. While, PRX-1 and PRX-2 were polymorphic in which PRX-1 was detected in all fungi, except C. albicans, C. krusei and C. tropicalis. Prx-2 showed lower polymorphism (2/18) than PRX-2 (16/18) which was detected in all tested isolates, except E. floccosum (Table 5, Fig. 4b).
| Fig. 3(a-c): | Agarose gel electrophoresis of fungal isolates (a) ITS-PCR product, (b) ITS-PCR after digestion with Hinf1 endonuclease, (c) ITS-PCR after digestion with HaeIII endonuclease |
| Fig. 4(a-e): | Electrophoretic isozyme patterns detected in all tested fungal isolates (a) esterase (EST), (b) peroxidase (PRX), (c) Protease (PROT), (d) Urease (URA) and (e) glutamate oxaloacetate transaminase (GOT) |
| Table 5: | The electrophoretic banding patterns of the EST, PER, URA,
PROT, MDH, GOT and ACP isozymes in fungal isolates |
.+:Present, -:Absent |
|
Protease (PROT, E.C.3.4.2.3): Fungal protease was shown to be controlled by two loci PROT1 and PROT2 (Table 5, Fig. 4c). PROT1 was monomorphic and showed one band in all isolates at Rf value of 0.62. On the other hand, PROT2 locus showed heterozygosity in all tested fungi, except C. albicans, C. tropicalis and C. krusei were homozygous for PROT2a allele which produced one band at this locus.
Urease (URA, E.C.3.5.1.5): Urease electrophoretic pattern showed five different enzymatic bands URA1-URA6 in the tested isolates (Fig. 4d, Table 5). URA1 and URA4 were monomorphic bands which expressed in all isolates while the other bands were polymorphic. URA2 and URA3 were detected in all isolates except, T. rubrum, C. albicans, C. tropicalis and C. krusei. The URA5 enzymatic band was only expressed in T. mentagrophytes (L), T. verrucosum (L) and T. violaceum (L). It is worthy to mention that the similarity in urease isozyme profile among T. rubrum and all Candida isolates is in harmony with the biochemical test for urease production by these fungal species which appeared to be urease negative as shown in Table 5.
Glutamate-oxaloacetate-transaminase (GOT, E.C.2.6.1.1): Two bands (GOT1, GOT2) were detected in the zymogram of Glutamate oxaloacetate transaminase. GOT1 was monomorphic which appeared in all tested isolates. Meanwhile, GOT2 was detected in all isolates, except C. albicans, C. tropicalis and C. krusei (Table 5 and Fig. 4e).
Malate dehydrogenase (MDH, E.C. 1.1.1.37): The electrophoretic analysis of MDH reveled two enzymatic bands, one of them (MDH1) was monomorphic which appeared in all tested isolates. The other band (MDH2) was polymorphic and detected in all isolates, except C. albicans, C. tropicalis and C. krusei (Fig. 5a, Table 5).
Acid phosphatase (ACP, E.C. 3.1.3.2): No differences were observed among the tested isolates of dermatophytes and Candida in the isozyme pattern of ACP in which only one band was detected for such enzyme (Fig. 5b, Table 5).
Cluster analyses of isozyme profiles: Cluster analysis based on the combined data of all tested isozymes was more valuable for determining relationships among the isolates than if each isozyme was to be analyzed separately. Genetic distances were calculated on the basis of similarity coefficient matrix among all different isolates involved in the cluster analysis.
The most noticeable results are that the dendrogram separated the tested fungi according their taxonomic order rather than geographical origin, i.e., all species of the same genera were clustered together. All isolates fell into two major clusters, labeled as A and B (Fig. 6). Cluster-A was further subdivided into four sub-clusters comprising 12 isolates of dermatophytes and Cluster-B contained the 6 isolates of Candida. Cluster-B is distinctly away from the cluster-A with a similarity of GS = 0.791 (Fig. 6). These results suggested that the genetic differences between the genera and species of dermatophytes and Candida were higher than that between the isolates of the same species. Close relationship between the isolates of the same specie, supporting the theory of a common lineage. Also RAPD approach showed considerable potential for identifying and discriminating dermatophytes and Candida spp.
| Fig. 5(a-b): | Electrophoretic isozyme patterns detected in all tested fungal isolates (a) malate dehydrogenase (MDH) and (b) acid phosphatase (ACP) |
| Fig. 6: | Dendrogram based on the UPGMA analysis of genetic similarity derived from the simple matching coefficient of Nei and Li, based on isozyme data, showing the relationships among 12 isolates of dermatophytes and 6 Candida |
DISCUSSION
Identification of dermatophytes at the species level is essential because of the therapeutic and epidemiological importance. Conventional methods on the basis of phenotype variations and molecular methods on the basis of molecular differences were used to identification of dermatophytes species. Due to some limitations in traditional methods such as the high degree of phenotypic similarity between these relative species identification, time-consuming many isolates reveal unusual characteristics (Weitzman and Summerbell, 1995; Graser et al., 2008; Shehata et al., 2008; De Baere et al., 2010). To overcome these problems, in the recent years, molecular marker approaches relying on genetic makeup are regarded as useful in the exact and rapid recognition of dermatophytes (Mochizuki et al., 2003; Kanbe et al., 2003; Liu et al., 2000; Graser et al., 1998).
During the present work three molecular marker systems based on PCR were applied to study the genetic relationships among 18 selected isolates of dermatophytes (12 isolates) and yeasts (6 isolates). Four PCR-RAPD primers and two PCR-ISSR primers were used to amplify randomly the DNA fragments of target fungi. After gel electrophoresis, the total number of bands per fungal isolate was ranging from 35 to 43 bands depending on the primer structure and number of annealing sites in the fungal genome. Polymorphic bands were detected with the different primers used to amplify the DNA of the fungal isolates. These bands accounted for 55.2% of total bands and were analyzed to establish a relationship between the genotypes of fungal isolates. Establishment of a dendrogram revealed that M. canis was separated in a single branch (0.787 GS) reflecting a relatively longer genetic distance from other isolates of dermatophytes and yeasts. It was also observed that both C. krusei and C. tropicalis are closely related showing 0.933 genetic similarities. C. albicans showed 0.909 similarities with other species of Candida. E. floccosum was easily separated from all Trichophyton species showing 0.873 similarities. It is to be noted that the monomorphic bands which accounted for 44.8% of total bands are constant bands and cannot be used to study the diversity while, polymorphic bands revealed differences and could be used to examine and establish systematic relationships among the genotypes (Hadrys et al., 1992). The presence of a unique band for a given genotype is taken as a positive marker while, the absence of a unique band referred as negative marker. Such bands could be used as DNA markers for isolate identification and discrimination. It is also important to observe that the results of RAPD-PCR came in complete harmony with the conventional methods employed for identification and characterization of these fungal species.
Randomly amplified polymorphic DNA (RAPD) and ISSR-PCR methods have frequently been used for phylogenetic analysis and identification of dermatophytes (Kim et al., 1999, 2001; Cano et al., 2005; Leibner-Ciszak et al., 2010). Spesso et al. (2013) demonstrated that the detection of intra-species polymorphisms in isolates of M. canis by RAPD-PCR may be applied in future molecular epidemiological studies in order to identify endemic strains, the route of infection in an outbreak and the coexistence of different strains in a single infection. In contrast to that reported and using the same method, Leibner-Ciszak et al. (2010) were not able to detect genetic variations in 13 clinical isolates of M. canis. These discrepancies could have been due to different conditions in the PCR reaction or that the strains of M. canis used by these authors had no clonal diversity.
RAPD-PCR as well as phenotype were also used by De Pinho Resende et al. (2004) to identify 242 yeasts isolated from hospitalized patients. There was both agreement and consistency between phenotypic and genotypic analysis using RAPD, demonstrating that is possible to apply this method for the identification of Candida species. While, Rocha et al. (2008) concluded that the RAPD proposed might be used to confirm Candida species identified by microbiological methods.
On the other hand, the Internal Transcribed Spacer (ITS) regions of the fungal ribosomal DNA (rDNA) were frequently used for species identification because it is faster, accurate species determination, specific and are less feasible to be affected by exterior effects such as temperature changes and chemotherapy (Girgis et al., 2006; Kong et al., 2008). Ellis et al. (2007), Aala et al. (2012) and Samuel et al. (2013) used both conventional and molecular methods to identified dermatophytes species. They revealed that the conventional methods are generally prolonged and may be indecisive. However, molecular studies based on Internal Transcribed Spacer (ITS) sequencing provide a very accurate result.
In this study PCR-RFLP in which two universal primers (ITS1 and ITS4) were used to amplify the ITS region of the rDNA gene in all fungal isolates studied, followed by digestion of the PCR product with restriction enzymes (HinfI and HaeIII endonucleases). Digestion with HinfI created 5 fragments for T. mentagrophytes, T. verrucosum and E. floccosum, 4 fragments for T. rubrum, T. violaceum and C. tropicalis, 3 fragments for C. krusei and C. albicans. Application of HaeIII endonuclease resulted in the cutting of 2-3 fragments of varying size (90-450 bp) depending on the fungal isolate. The cutting pattern was similar for C. albicans and C. tropicalis (420 and 90 bp) showing differences from C. krusei (370, 90 and 40 bp). It is worthy to mention that the ITS region in M. canis was not digested by HinfI but the same region produced 3 fragments when HaeIII endonuclease was used. Therefore, PCR-RFLP technique can be considered as a powerful technique for discrimination and identification of fungal species especially when the proper restriction endonucleases are selected. These results also suggested that dermatophytes and Candida species isolated from different geographical regions (Assiut and Tripoli) are closely related, supporting the theory of a common lineage. Also, RAPD and ISSR approaches showed considerable potential for identifying and discriminating dermatophytes and Candida spp.
The identification results are in agreement with established and recent taxonomical insights into the dermatophytes; for example, highly related species also had closely related and sometimes difficult-to-discriminate ITS2-RFLP patterns. Analysis of ITS region based on RFLP-PCR was used to identify and discriminate between 57 T. rubrum clinical isolates (Hryncewicz-Gwoozdz et al., 2011) and between different species or varieties of Trichophyton, Microsporum and Epidermophyton (Graser et al., 1999, 2000a, b; Mirzahosseini et al., 2009). They reported that PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum isolates and other species of dermatophytes, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing. While, De Baere et al. (2010) reported that ITS2-RFLP analysis proved to be most useful for identification of species of the genera Arthroderma, Chrysosporium and Epidermophyton but could not distinguish between several Trichophyton species. Recently, Rezaei-Matehkolaei et al. (2012) used PCR-RFLP assay to find the exact differentiating restriction profiles for each dermatophyte species. They reported that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.
Identification of Candida species and isolates using PCR-RFLP for amplification the ITS region has been applied by Pinto et al. (2004), Mirhendi et al. (2005, 2006) and Saltanatpouri et al. (2010).
Isozymes analysis is more reliable than traditional methods since the expression of isozymes loci are co-dominant, in addition this technique provides a fairly rapid and inexpensive alternative tool for taxonomic studies and for identifying fungi species (Ryan and Scowcroft, 1987; Bonde et al., 1993; Bragaloni et al., 1997). Klaas (1998) reported that the Isozyme markers can correctly identify several levels of taxa, accessions and individuals, since the assumption of homology can be more accurate than for some genomic DNA markers.
In this study analysis of isozyme profiles were based on the data from all seven isozymes pattern. The combined data of the present work give more complete information than if each isozyme was to be analyzed separately. All isolates fell into two major clusters. First cluster (A) was further subdivided into four sub-clusters comprising 12 isolates of dermatophytes and second cluster (B) of 6 isolates of Candida. Cluster-B is distinctly away from the cluster-A with a similarity of GS = 0.791. However, two disadvantages were observed. One is inability to differentiate the 3 different species of Candida from each other. The second is the confusion resulted from the grouping of 3 different Trichophyton species in the same sub cluster. These are T. mentagrophytes, T. violaceum and T. verrucosum which can be easily identified with the conventional culturing and microscopic examination. It can be suggested try more different isozymes to design a set of isozymes suitable for successful clustering and identification of the different medically important yeasts and dermatophytes. It is also very important to compare the results of isozyme profiles with those of conventional and molecular based identification methods in order to achieve correct identification and characterization of the pathogenic fungi.
In Trichoderma, the first characterization was done by Zamir and Chet (1985) who reported that the 23 geographically diverse isolates of T. harzianum were grouped into 5 types according to their isozyme profiles. The results indicated that enzyme electrophoresis was useful for distinguishing Trichoderma at the intraspecies level. Rosa et al. (2000) used Multilocus Enzyme Electrophoresis (MLEE) and numerical taxonomic methods to establish the degrees of relatedness among five Candida species commonly isolated from humans oral cavities. Of twenty enzymic systems assayed, five showed no enzymic activity (aspartate dehydrogenase, mannitol dehydrogenase, sorbitol dehydrogenase, glucosyl transferase and a-amylase). The obtained data revealed that some of these enzymes are capable of distinguishing strains of different species but most of them could not organize all strains in their respective species-specific clusters. Numerical classification based on MLEE polymorphism must be regarded for surveys involving just one Candida species.
Isozyme and protein electrophoresis data from mycelia extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated by Siddiquee et al. (2010). The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. They suggested that the T. harzianum isolates had high levels of genetic variation compared with the other isolates of Trichoderma species.
Generally, the utilization of genetic markers such as isozymes, RAPD, ISSR and PCR-RFLP together with morphological analysis to study dermatophytes and Candida spp. help alleviate confusion in the identification of isolates at the species level. It is to be emphasized that data obtained from molecular analysis (RAPD, ISSR and RFLP) came in harmony with identifications based on conventional morphological examination of all fungal species studied.