Molecular Profile of Trichophyton mentagrophytes and Microsporum canis based on PCR-RFLP of Internal Transcribed Spacer

Endrawati D, Kusumaningtys E. 2020. Molecular profile of Trichophyton mentagrophytes and Microsporum canis based on PCR-RFLP of internal transcribed spacer. JITV 26(1): 10-21. DOI: http://dx.doi.org/10.14334/jitv.v26i1.2546. Trichophyton mentagrophytes and Microsporum canis are dermatophytes fungi which commonly infect animal and human. Conventional and molecular methods were used for identification of the fungus. The region of internal transcribed spacer (ITS) has a high probability for fungal identification. PCR-RFLP was reported as a useful method to differentiate dermatophytes fungi. The objective of the study was to compare molecular profile of T. mentagrophytes and M. canis based on the result of ITS fragment digestion using Dde I, Hinf I and Mva I. The molds were isolated from skin scrapping of 18 animals which showed dermatophytosis lesion. The isolated molds were grown on agar plate for 14 days of incubation at 37C and then identified based on macro and microscopic morphologies. Amplification of chitin synthase gene was used for confirmation and separation of dermatophytes from other fungi. ITS fragment was amplified and then digested using restriction enzymes Dde I, Hinf I and Mva I. The result showed that digestion products from ITS fragment of T. mentagrophytes and M. canis were different. The fragment 159 bp from Dde I, 374 bp from Hinf I and 89 bp from Mva I were present in T. mentagrophytes but absent in M. canis. Based on these results, specific RFLP profile of digestion ITS region by Dde I, Hinf I and Mva I can be used as a specific marker for species of dermatophytes fungi.


INTRODUCTION
Dermatophytes are the fungus commonly invading stratum corneum of epidermis and keratinized tissues such as skin nails and hair of humans and animals. Cats and dogs are natural hosts which most infected by the fungus (Pasquetti et al. 2017). The fungus is commonly transmitted to human and cause tinea capitis and tinea corporis (Brillowska-Dabrowska et al. 2013). As dermatophytes transmitted from animal to animal, from animal to human and from human to human, identification and differentiation of the related species is important from an epidemiological point of view (Rezaei-Matehkolaei et al. 2012).
In the conventional identification methods, long incubation (7-14) is needed for characteristic traits to appear making the fungi difficult to be identified. Microscopic examination is limited because of the absence of macro or microconidia and the production of hyphae with prominent cross-walls. Identification was difficult because of similarities among colonies of variant Microsporum canis (Rezaei-Matehkolaei et al. 2012). In addition, clinical isolates with similar geographical conditions of nature may show different phenotypes making identification even more complicated (Brillowska-Dabrowska et al. 2013;Katiraee et al. 2016).
A variety of molecular techniques, such as polymerase chain reaction (PCR) need to be considered. Other methods, such as mitochondrial DNA restriction fragment length polymorphism (RFLP) pattern and Chitin synthase 1 nucleotide sequence analysis has reported an as simple, fast and accurate method for identification (Jung et al. 2014). This research used ITS primers (ITS1 and ITS4) because the primers are universal and allow selective amplification of fungal sequences. ITS region is in a ribosomal cistron. The nuclear rRNA cistron has been used for fungal diagnostic and phylogenetics for more than 20 years (Begerow et al. (2010). The eukaryotic rRNA cistron consists of the 18S, 5.8S and 28S rRNA genes transcribed as a unit RNA polymerase I. Posttranscriptional processes split the cistron, removing two internal transcribed spacers. These spacers are the 5.8S which is referred as internal transcribed spacer (ITS) and the 18S nuclear ribosomal small subunit rRNA gene (SSU).
The ITS region has the highest probability of accurate identification for fungi. ITS was referred to a candidate of fungal barcode (Schoch et al. 2012). However, amplification of the internal transcribed spacer (ITS) region representing organism diversity was still unsatisfactory as the sequences of T mentagrophytes, T tonsurans, T rubrum and Microsporum gypseum are very similar (Jung et al. 2014). PCR-RFLP of ITS fragment is a method that combines PCR and enzymatic digestion of the PCR products. The method was reported to be a rapid and accurate technique for fungal identification by generating band patterns on agarose gel electrophoresis, which takes only 5 hours to be carried out (Mohammadi et al. 2015). ITS PCR and RFLP have also been used for differentiation of brewing yeast and brewery wild yeast contaminant (Pham et al. 2011). Mirzahoseini et al. (2009 reported that PCR-RFLP was a reliable tool to identify dermatophytes from a clinical specimen. Application of the Mva I and the Dde I restriction enzyme to the ITS amplicon resulting good, stable and reproducible in the identification of the dermatophytes species (Elavarashi et al. 2013). Previously, it used one or two restriction enzymes to compare molecular profile of dermatophytes fungi. This research used three enzymes to produce fragments of profile from digestion products. It was hoped that application of more enzymes produces more specific molecular profile. In addition, the data would provide information about the most suitable enzyme which used for species identification. Therefore, differentiation among dermatophytes species are more accurate. As dermatophytes fungi infect human and animals such as pets, wild and livestock, the samples were taken from cat and dog which represent pet animals and cattle which represent livestock. This research was conducted to compare molecular profile of Microsporum canis and Trichophyton mentagrophytes based on the result of ITS fragment digestion using Dde I, Hinf I and Mva I. T. mentagrophytes and M. canis produced different digestion product which can be used to distinguished both species

Clinical isolate
Scrapping skin sample was collected from infected cat and dog patients which came to animal hospital around Bogor, Jakarta and Sukabumi, Indonesia. The scrap was inoculated in Sabouraud dextrose agar (SDA) with chloramphenicol 0.05 mg/mL and cycloheximide 0.5 mg/mL (Pal & Dave 2013), to inhibit bacteria and spreading mold. The plates were incubated at 37 o C for 7-14 days. Dermatophytes fungi were purified by picking selected single colony and inoculated in new agar plate.

Conventional identification
The fungus was identified by colonies observation and microscopic direct examination using KOH 10%. The scraping skin was put in object-glass, KOH dropped in surface, press using cover glass. Fixation was done by trough the glass up the flame. Microscopic morphology was examined under microscope. Identification was performed based on mycelia and conidia form.

DNA extraction
DNA extraction was conducted according to White et al. (1990) with some modification. Mycelium of dermatophyte fungi was placed into microtube 1.5 mL. Two grams of mycelia were ground using micro pestle to form small particles. Amount of 500 µL sodium deodecyl sulphate (SDS) was added, then incubated at 65 o C for 30 minutes. The mixture was let until cold, added with 500 µl chloroform isoamyl (CI 24:1) and centrifuged at 10.000 x g for 20 minutes. Supernatant was placed into a new tube and 500 µL phenol-chloroform isoamyl (PCI 25:24:1) was added and centrifuged at 10.000 x g for 10 minutes. The supernatant was placed into new tube and 100 µl Na acetate 2 M (pH 5,2) and 500 µl ethanol 100% were added. The mixture was frozen at -20 o C for 8 hours and then centrifuged at 10,000 x g for 30 minutes. Supernatant was discarded and pellet was dried using vacuum concentrate plus (Eppendorf) for 30 minutes. The dried pellet was added with nuclease free water and 5 µL RNase then incubated at 37 o C for 10 minutes continued with additional incubation at 70 o C for 10 minutes (for RNAse inactivation). Purity and percentage of DNA were measured using NanoDrop spectrophotometer at λ 260/280.

DNA sequencing and analysis
PCR products from amplification of ITS region were sequenced and identified. The PCR product was sent to First Base Laboratories Sdn Bhd All Right Reserved, Selangor, Malaysia for sequencing. DNA sequences were analyzed using Bioedit and Mega-X and aligned with Gene Bank database using BLAST program (www.ncbi.nlm.nih.gov) and clustalw2 (https://www.ebi.ac.uk/Tools/msa/clustalo/). Open Reading Frame was determined using https://www.ncbi.nlm.nih.gov/orffinder/

Polymerase Chain Reaction-Restriction fragment length polymorphism (PCR-RFLP)
Restriction fragment length polymorphism (RFLPs) for PCR products were performed following Mohammadi et al. (2015) using enzymes Dde I, Hinf I and Mva I (Thermo Fisher Scientific Inc). The procedure for enzymes treatment was conducted according to the protocol of each enzyme from the company. Ten µl of ITS PCR product were mixed with 18 µl nuclease-free water (NFW), 2 µl 10x Tango buffer (composed by 33 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg/mL BSA) and 1 µl Dde I (10U/µL) (Thermo Fisher Scientific Inc). The mixture was incubated at 37 o C for 1 hours. The reaction was stopped by incubation in 65 o C water bath for 20 minutes. For Hinf I, ITS PCR product 10 µl, was mixed with 17 µl NFW, 2 µl 10x green buffer and 1 µl Hinf I (Thermo Fisher Scientific Inc) then incubated at 37 o C for 5 minutes. The reaction was stopped by incubation in 65 o C water bath for 20 minutes. For Mva I, ITS PCR product 10 µl, was mixed with 17 µl NFW, 2 µl 10x green buffer and 1 µl Mva I (Thermo Fisher Scientific Inc) then incubated at 37 o C for 5 minutes. The reaction was stopped by incubation in 65 o C water bath for 20 minutes. All digestion products were stored at -20 o C until used. Electrophoresis for PCR digested product was performed using agarose 1.5 % and SYBR TM safe staining, run at voltage 100 Volt. The bands were visualized using UV transilluminator.

Isolation of dermatophytes fungi
Dermatophytes fungi were isolated from cat, dog and cattle which came to animal hospital, pet clinics and animal husbandry around Jakarta, Bogor and Sukabumi city. There was a total of 18 patients which showed clinical signs of dermatophytosis such as itchy, red, scaly, circular rush and some hair loss as showed in Figure 1. The fungi infect certain organs or even around the body. Table 1 provides information regarding the animal and the organ which had suspected dermatophytosis in this study. The patients were dominated by cats. It may due to less dog population compare to cat. Besides cats and dogs, dermatophytes such as Microsporum canis and Trichophyton mentagrophytes were also infected calves (Pal & Dave 2013). In this research, only one from 100 examined cattle were infected. Intensive husbandry with good sanitation reduced the possibility to be infected by the dermatophytes fungi. As shown in table 1, there was no organ or breeds preference. Age ≤ 12 months more frequently infected by the fungi. Aneke et al. (2018) reported that in dogs and cats, male and young individuals develop more frequently clinical lesions. Ilhan et al. (2016) found no significant association between genders in cats. The most likely risk factor for dermatophytes infection were seasons and age of animals. Winter and spring are the   Table 1 was only performed on the animal which showed the clinical sign of dermatophytoses, but the fungi had not been yet identified. In some cases, the sign leads to dermatophytoses, but the dermatophytes fungi failed to be isolated in culture and not detected in native preparation or molecular identification. The scrapping of infected skins was then observed under a microscope using KOH 10% and some were inoculated in agar medium. Colonies and microscopic of dermatophytes fungi are shown in Fig 2. Identification was performed based on the macro and microscopic morphology and confirmed by molecular identification.
As shown in Figure 2, colony of Microsporum canis is coarsely fluffy, furrier on top and darker in the underside of the growth medium than that of Trichopyton mentagrophytes. The dark yellow pigment was absent in some strains of M canis due to failure to develop macroconidia and retardation of colony growth. Macroconidia divided into compartments which are separated by coss-wall. Microconidia M. canis also resemble other dermatophytes therefore it is not useful for diagnostic or identification Spora of Trichophyton mentagrophytes was more abundant therefore easily recognized. On the contrary for Microsporum canis, even with prolonged 14 days incubation, the conidia were still hardly present. As consequence, molecular identification is a necessity.
In this research, internal transcribed spacer and chitin synthase were amplified for fungal identification and characterization. According to Cafarchia et al. (2013), the first and second internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA and the part the chitin cynthase gene (pchs1) have shown promise as markers for specific identification of dermatophytes.

Internal Transcribed Spacer (ITS)
ITS primers were used for amplification DNA region specific for fungi. The ITS bands were detected in fungi such as Candida sp., Fusarium sp. and dermatophytes but absent in bacteria (Elavarashi et al. 2013). Therefore, the ITS amplification products can be used as a fungal marker. This research use primers ITS 1 and ITS 4 to amplify both of marker specific and conserved sequence. These primer pairs are universal primers and are commonly used for fungal molecular diagnostic and identification (Ferrer et al. 2001;Aala 2012). The length sizes were various among genus and species. Amplification of the region using primer ITS 1 and ITS 4 in some dermatophytes from the previous research indicated that the region was conserved among dermatophytes fungi. PCR product using ITS1 and ITS4 primers is shown in Figure 3. PCR products were then sequenced for identification.
As shown in Figure 3 some of the fungi sequenced identified as dermatophytes fungi. One isolate was identified as Trichophyton mentagrophytes (Tm) and 7 isolates were Microsporum canis (Mc). However, not all fungi isolated from skin sent for sequencing. Mostly the fungi which had been confirmed as dermatophytes as the chitin synthase amplicon was detected (Figure 3 and 4), or the fungi which genus identified from macro and microscopic morphology.
PCR product using ITS 1 and ITS 4 primer was reported producing 690-720 bp for T. mentagrophytes and M. canis (Abdel-Fatah et al. 2013). ITS amplicon of Microsporum genus was also reported varied in size from ~851 bp in Microsporum gypseum to ~922 bp in Microsporum canis and ITS region of M. canis being ~50 bp longer than that of other dermatophytes (Cafarchia et al. 2013).
In this research, both T. mentagrophytes and M. canis produced 686-739 bp. Using the same primers pairs, the M. canis amplicon shorter than that reported by Zhang et al.    Table 2) such as Aspergillus niger (600bp), Aspergillus bridgeri (600 bp), yeast (around 500bp) and Chaetomium pachypodiodes (around 500 bp), were shorter than that of dermatophytes fungi. A similar result was reported by (Elavarashi et al. 2013) who revealed that ITS 1 and ITS 4 pairs primer produced around 550-600 bp PCR products in Candida sp and Fusarium sp. This result showed that PCR products of ITS 1 and ITS 4 pair primers can be used to distinguish dermatophytes and non-dermatophytes fungi.

Chitin synthase
Amplification of chitin synthase region was aimed to confirm that the isolate was dermatophytes fungi. The existence of chitin synthase band indicated a dermatophytes fungi. Saprophytic fungi isolated from skin scrapping did not produce this band. PCR for chitin synthase 1 gene was powerful to identify the presence of dermatophytes fungi from clinical isolate such as skin scraping and hair. Sharma et al. (2017) found 10 samples that were negative on the fungal culture but were positive for dermatophytes by PCR of chitin synthase indicating that PCR was more sensitive than culture. Putty et al. (2018) reported that amplification of chitin synthase I gene resulting in 288 bp product size. They added that amplification of the gene was able to be considered as a rapid test for dermatophytosis to decided appropriate antifungal therapy. In this research, the same primer pairs produced longer PCR products, around 400 bp. According to Emam & Abd El-salam (2016) PCR products may be varied among the dermatophyte genus and amplicon size 288 bp was  The result showed that both Trichophyton tonsurans and T. mentagrophytes produced 366 bp, almost similar to the PCR product in this research. Based on the result, amplification of the chitin synthase using primer CHS 1 was powerfull to differenciate dermatophytes and non dermatophytes but did not able to distinguish among genera within dermatophytes. This result also indicates that primer CHS 1 can be used for determination of dermatophytes fungi from clinical samples such as skin scraping from the animals suspected suffer from dermatophytosis.

Identification of dermatophytes fungi
Sequence analysis of PCR product of ITS genes showed that they were confirmed as Trichophyton mentagrophytes and Microsporum canis. The similarity percentage of both fungi with sequence database in GeneBank is more than 99% (Table 2).
Seven dermatophytes fungi were identified based on the characteristic colonies, microscopic morphologies and their nucleotide sequences of ITS PCR product. The fungi were identified as M. canis and T. mentagrophytes. Sequencing result of ITS 1 to ITS 4 regions of Mc1-7 showed that they had similarity almost 100% with ITS regions of M. canis from GeneBank. Conventional identification using macro dan microscopic morphology of T. mentagrophytes was also confirmed by sequencing result of the ITS region. Identification of dermatophytes and non-dermatophytes fungi isolated from cats, dogs and cattle suspected dermatophytosis as displayed in Table 3.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP)
For further molecular profile, PCR products of ITS fragment were then digested using restriction enzymes Dde I, Hinf I and Mva I. The enzymes cut the ITS sequence in their cleavage site. ITS sequence affects the cleavage site position resulting difference in fragment size of digestion products. The digestion product was presented at Figure 5. T. mentagrophytes (Tm) and M. canis (Mc) were digested by Dde I produced different fragments. Fragment 159 bp in Tm and 201 bp in Mc was able to differentiate both genera. Microsporum canis (Mc) 1-5 isolated from Bogor has a different pattern with Mc 6-7 which was isolated from Jakarta. This different pattern may represent different strains circulating between both regions, although it needs further examination to prove it.

CONCLUSION
Molecular profil from PCR_RFLP using Dde I, Hinf I and Mva I was different between Trichophyton mentagrophytes and Microsporum canis. Digestion product 159 bp from Dde I, 374 bp from Hinf I and 89 bp from Mva I were present in T. mentagrophytes but absent in M. canis. Based on these differences, it is possible that specific RFLP profile of digestion ITS region using Dde I, Hinf I and Mva I are used as a specific marker to differentiate among species, especially between T. mentagrophytes and M. canis local isolate from Indonesia.

AUTHOR CONTRIBUTIONS
Endrawati D and Kusumaningtyas E had full access to all data in this study and contributed equally to this work.