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Isolation of Keratinophilic Fungi from the Soil of Greater Tunb, Abu-Musa

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Isolation of Keratinophilic Fungi from the Soil of Greater Tunb, Abu-Musa Current Medical Mycology 2017, 3(2): 13-19 Isolation of keratinophilic fungi from the soil of Greater Tunb, Abu-Musa, and Sirri islands in the Persian Gulf, Iran Nosratabadi M1, Kordbacheh P1, Kachuei R2*, Safara M1, Rezaie S3, Afshari MA4 1 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 3 Division of Molecular Biology, Department of Medical Mycology and Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4 Arya Tina Gene Biopharmaceutical Company, Tehran, Iran Article Info A B S T R A C T Article type: Background and Purpose: Keratinophilic fungi are among the important groups of Original article fungi living in the soil. This study aimed to isolate and identify keratinophilic fungi from the soil of three Iranian islands, namely Greater Tunb, Abu Musa, and Sirri, located in the Persian Gulf using morphological and molecular (polymerase chain reaction) methods. Materials and Methods: In this study, a total of 60 soil samples were collected from the Article History: three islands of Greater Tunb, Abu Musa, and Sirri. The samples were analyzed for the Received: 09 September 2017 presence of the keratinophilic fungi using a hair baiting technique. Furthermore, the Revised: 30 September 2017 identification of keratinophilic fungi was accomplished through the employment of Accepted: 23 October 2017 molecular and sequencing techniques. Results: A total of 130 fungal isolates, including 11 genera with 24 species, were collected. Accordingly, Chrysosporium tropicum (24;18.5%), C. keratinophilum (17; 13.1%), Chrysosporium species (15; 11.5%), Aspergillus species ( 8;6.1%), Aspergillus * Corresponding author: flavus (8; 6.1%), Penicillium species (8;6.1%), Alternaria spp ( 6; 4.6%), Phoma Reza Kachuei species (5; 3.8%), Aphanoascus verrucosus (4;3.1%), Fusarium chlamydosporum (4; Molecular Biology Research Center, 3.1%), Aspergillus trreus (4;3.1%), Acremonium species (4; 3.1%), and other fungi( 23; Baqiyatallah University of Medical 17.8 %) isolates were identified . All isolates of keratinophilic fungi were isolated from Sciences, Tehran, Iran. the soils with the pH range of 7-9. Email: [email protected] Conclusion: The results of this study contributed towards a better conceptualization of the incidence pattern of keratinophilic fungi in the regions of Iran. Given that no study has investigated this issue, the findings of the present study can be beneficial for the management of public health surveillance, physicians, and epidemiologists. Keywords: Abu Musa, Greater Tunb, Keratinophilic fungi, PCR, Sirri, Soil How to cite this paper Nosratabadi M, Kordbacheh P, Kachuei R, Safara M, Rezaie S, Afshari MA. Isolation of keratinophilic fungi from the soil of Greater Tunb, Abu-Musa, and Sirri islands in the Persian Gulf, Iran. Curr Med Mycol. 2017; 3(2): 13-19. DOI: 10.29252/cmm.3.2.13 Introduction ungi are a group of microorganisms with a as saprophytes, which are termed as geophilic wide distribution in soil. These organisms play dermatophytes [6]. On the other hand, the non- F an important role in the soil ecosystem and dermatophyte fungi, such as Aspergillus flavus, soil-borne fungal diseases [1]. A number of soil fungi Fusarium Oxysporum, and Chrysosporium species are known as potential pathogens for humans and have the ability to colonize around hair and are isolated animals [2]. Keratinophilic fungi are among important from the cutaneous lesions of humans and animals as groups of fungi living in the soil that colonize in opportunistic agents [7, 8]. various keratinous substrates, produce keratinases, and Keratinophilic fungi have a variable distribution in decompose them into components with lower the environment depending on such natural factors as molecular weight [3, 4]. keratin sources, soil pH, temperature, humidity, The ability of these microorganisms to invade and environmental light, and climate [9, 10]. Vanbreu- colonize on the keratinous tissues is closely associated seghem was the first one detecting the existence of with their ability to use keratin [5]. Dermatophytes are keratinophilic fungi in the soil in 1952 [11]. In the a group of keratinophilic fungi that are often recent years, many studies have been carried out in anthropophilic or zoophilic in their natural habitat. different parts of the world to study the distribution of However, some of these fungi occur in the soil these fungi in soil [12-14]. Accordingly, several studies Copyright© 2017, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY) License (http://creativecommons.org/) which permits unrestricted use, distribution and reproduction in any medium, provided appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Isolation of keratinophilic fungi from the soil Nosratabadi M et al. have been conducted in Iran targeting this domain technique [11]. After mixing soil samples, 70-100 g of [15-17]. However, there are no data regarding soil was transferred to sterile large deep glass plates. keratinophilic fungi in the soil of Greater Tunb, Abu Subsequently, 1 g of a mixture of sterile human child Musa, and Sirri islands. girls’ hairs and horse was added and distributed evenly In this study, the identification and chara- on the whole surface of the soil. Then, almost 20 cc of cterization of the isolated keratinophilic species were sterile distilled water was added and kept for 6-8 weeks performed using the molecular techniques along with at ambient temperature (i.e., 25°C). traditional methods. To this aim, the internal The isolation was carried out by direct transfer of transcribed spacer (ITS) region of ribosomal DNA mycelium from the baits to sabouraud dextrose agar was amplified and the polymerase chain reaction medium (Merck, Germany) with chloramphenicol (PCR) products were sequenced. This region is the (0.1 mg/mL; Sigma-Aldrich, USA) as well as most widely sequenced DNA region in the molecular sabouraud dextrose agar medium with cycloheximide ecology of fungi and has been recommended as the (1 mg/mL; Sigma-Aldrich, USA) and chloramphenicol universal fungal barcode sequence. It has typically (0.1 mg/mL). They were then incubated at room been most useful for molecular systematics at the temperature for a period of two weeks. The species level and even within species due to its higher identification of keratinophilic fungi was carried out degree of variation than that of other genic regions of according to standard procedures [20, 21]. In addition, rDNA [18]. the unknown fungal isolates were identified through With this background in mind, the present study performing DNA sequence analysis. was carried out with the aim of isolating keratinophilic fungi from the soil of the Iranian islands of Greater Molecular identification Tunb, Abu Musa, and Sirri. The findings of this study DNA extraction can be helpful since no study has investigated this issue DNA was extracted following Lee et al. [22] with in the given regions. slight modifications. To this aim, the harvested mycelial mass was flash-frozen in liquid nitrogen and Materials and Methods ground to a fine powder in a porcelain mortar. The Geographical characteristics of the studied islands mycelial powder was suspended in DNA extraction Greater Tunb, Abu Musa, and Sirri islands are buffer containing 1 mM NaCl, 10 mM Tris (pH 8.0), 1 located at the Persian Gulf in the most southern part of mM ethylenediaminetetraacetic acid, 1% SDS, and 1% Iran. These three islands are considered as part of Triton X-l00, and then the DNA was extracted. The Hormozgan province. The Greater Tunb (10.3 km2 quality and quantity of the extracted DNA were wide) has a longitude and latitude of 55˚ 28-55˚ 34 and evaluated using electrophoresis and Nano- Drop, 26˚ 34-26˚ 30 respectively. Abu Musa Island (12 km2 respectively. wide) has a longitude and latitude of 54˚ 26-55˚ 19 and 25˚ 51-26˚ 19, respectively. Furthermore, Sirri Island is Polymerase chain reaction situated 76 km from Bandar-e Lengeh and 50 km west The ITS1-5.8S-ITS2 rDNA was amplified using of Abu Musa Island. This island is almost 5.6 km long ITS1 and ITS4 as forward and reverse primers with a width of about 3 km. It covers an area of following White et al. [23]. The amplification was 17.3 km². All three islands have a warm and humid performed in a total volume of 25 μL in each tube climate [19]. containing 12.5 µL master mix (Ampliqon, Denmark) (buffer, dNTP, Taq DNA polymerase, 2 mM MgCl2), Sample collection 0.5 μL of the template DNA, 1.5 µL of each This descriptive study was conducted in the second primer (Cinaclone, Iran) ITS1, namely 5'- half of 2011 in three Iranian islands of Greater Tunb, TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'- Abu-Musa, and Sirri. A total of 60 soil samples (i.e., 20 TCCTCCGCTTATTGATATGC-3' (20 pmol final samples from each island) were collected. The samples concentration of each primer), and 9 휇L distilled water. were collected from the superficial layer of soil with the The PCR reaction was carried out using a thermal maximum depth of 10 cm and weight of 300-500 g. cycler (Biometra, Germany) with initial denaturation During the sampling, necessary accuracy was at 94°C for 5 min, 35 cycles with denaturation at considered to provide samples from different locations 94°C for 30 sec, annealiation at 56°C for 45 sec, and from places not directly exposed to sunlight. The extension at 72°C for 45 sec, and a final extension samples were placed in sterile polyethylene bags, step of 7 min at 72°C.
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