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Research Article Antidermatophytes from Bioactive Secondary Metabolites of Local Streptomyces Spp

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Research Article Antidermatophytes from Bioactive Secondary Metabolites of Local Streptomyces Spp Journal of Innovations in Pharmaceuticals and Biological Sciences JIPBS www.jipbs.com ISSN: 2349-2759 Research article Antidermatophytes from bioactive secondary metabolites of local Streptomyces spp. Ahmed A. Hamed1, Mohamed S. Abdel-Aziz1, Mohamed Fadel1*, Mohamed F. Ghali2 1Microbial Chemistry Department, National Research Centre, 33 El- Bohouth St. Dokki- Giza- Egypt- P. O. 12622. 2Botany Department, Faculty of Science, Zagazig University, Egypt. Abstract Dermatophytes were considered as pathogenic fungi that invade human skin, nails and hairs and cause dermatomycosis. One hundred and eleven were isolated from both marine and terrestrial Egyptian habitats were screened for their ability to produce bioactive secondary metabolites with antidermatophytic this has been done by cultivating them on ISP2 liquid media for 10 days. The antdermatophytic activity was tested for the produced extracts by well-diffusion methods. Extracts from isolates A1, A3, A10, G5 and P4 exhibited potent antidermatophytic activity against trichophyton mentagrophytes (RCMB 09285), Microsporumcanis (RCMB 07321) and Microsporumgypseum (RCMB 07336). These potent isolates were identified by studying their morphological, physiological and biochemical characteristics and microscopical studies. Key words: Antidermatophytes, Bioactive secondary metabolites, Streptomyces. *Corresponding Author: Mohamed Fadel, Microbial Chemistry Department, National Research Centre, 33 El- Bohouth St. Dokki- Giza- Egypt- P. O. 12622. 1. Introduction Natural products (secondary metabolites) Actinomycetes, the cultivable group of considered the most important source of Gram-positive, filamentous bacteria from potential drug leads to be used diverse ecological niches, they are in drug discovery [1], furthermore there is saprophytes and are responsible for the an urgent need to discover new antibiotic degradation of complex biopolymers [5]. to counter and reverse the spread of Actinomycetes considered as one of the antibiotic resistant pathogens [2] and to most important suppliers of antibiotics. combat life-threatening diseases [3]. Even Further, they can produce an array of though there are a considerable progress secondary metabolites, many of which within the fields of chemical synthesis and have antibacterial or antifungal engineered biosynthesis of antimicrobial properties. In fact, most antibiotics compounds, nature still remains the most developed from human pharmaceutical important source for new antibiotics [4]. use are produced by actionmycetes, and ©JIPBS, All rights reserved Mohamed Fadel et al., JIPBS, Vol 3 (2), 155-165, 2016 many being derived from Streptomyces sp. Collection of marine and soil samples [6]. Streptomyces is the most predominant Marine Samples were collected from genera representing about 57% of the different locations of the Red sea (Ghardga), total soil actinobacteria population], and Marine. Ain Sokhna Sediment and Ras Sedr is widely distributed in terrestrial and sediment. Terrestrial samples are collected aquatic habitats [7]. This member of the from Mansoura governorate, National order Actinomycetales with complex life research, Dokki, Giza NRC and Pyramids cycle involving three stages of zone, Giza soil. All the samples were kept in differentiation. It is thought that the sterile tubes in refrigerator at 4oC for morphological and physiological further analysis. differentiation and onset of secondary metabolites production result from Isolation of Streptomyces spp. common elements of regulation [8]. Streptomyces spp. were isolated by serial Dermatomycosis is a skin mycotic disease dilution technique [14] in which Sample of skin caused by Dermatophytes. were added to a sterile saline solution Pathogenic fungi, which invade the (0.85% NaCl) in a percent of 1:10(w/v or keratinized and cutaneous areas of the v/v) starch casein (SCA) of the following body (nail, hair and skin). Most of them constituents (g/l): Soluble Starch (10); belong to the Hyphomycetes but several KNO3 (2.0); Casein (0.3) MgSO4.7H2O are now known to have perfect states (0.05); FeSO4.7H2O (0.01); CaCO3 (0.02); (teleomorphs) in the family agar, 20.0 and distilled water (1000 ml) in Gynmoascaceae of the order Eurotiales case of soil isolation and filtered aged sea [9,10]. The dermatophytes are water in case of marine isolation the pH was represented by three genera: adjusted to 7.2. Proper antibacterial Microsporum, Trichophyton, and (Penicillin G) and antifungal (Nystatin) Epidermophyton on the basis of agents were added. After sterilization at morphology of their macro and 121oC for 20 min. the media were poured microconidia. The teleomorphs of the into Petri dishes and left over night to Microsporum and Trichophyton spp. are prevent moisture film [15]. 100 µl from called nannizia and arthroderma each dilution were used to inoculate a Petri respectively, however an asexual form of dish of 10 cm diameter containing about 20 Epidermophyton floccosunt is ml medium and the inoculums was spread unknown[11]. As the dermatophytes have on the dish top using a sterilized glass rod. developed resistance to antimycotic drugs The inoculated plates were incubated at [12], there is an urgent need to discover 28oC for 7-14 days and noticing any growth. safe and cost-effective antidermatophytes The isolation of streptomycetes based on drugs [13]. This study is undertaken with their special morphological characteristics the aim of isolating, cultivating and (deep sitting colonies, sporulation, screening of Streptomyces spp. Extracts characteristic color, etc.) The plates that for their ability to work as showed countable single colonies were antidermatophytic agents. The potent selected and purified by a streak plate Streptomyces isolates were further technique. identified by studying the morphological, cultural physiological as well as the Fermentation and extraction biochemical characteristic. Streptomyces spp. were cultivated on ISP2 medium (g/l): Glucose (4.0), Yeast extract 2. Materials and Methods (4.0), Malt extract (10.0). The pH was 156 Mohamed Fadel et al., JIPBS, Vol 3 (2), 155-165, 2016 adjusted to 6.8. One liter volume of Streptomyces species according to Shirling Erlenmeyer flasks each containing 200 ml of and Gottleib [18-20]. Alternatively, the ISP2 medium were inoculated with 5 ml strains were rather identified using the spore suspension. The inoculated flasks Bergey’s Manual of Determinative were incubated on a rotary shaker with 150 Bacteriology [21] and Bergey’s Manual of rpm rotation at 30ºC for 10 days. The Systematic Bacteriology [22]. Streptomyces cells were removed by centrifugation at 5000 rpm for 20 min, and 3. Results and discussion the culture supernatants were extracted using ethyl acetate Table (1), the ethyl Results acetate phase was evaporated till dryness Isolation of actinomycete One hundred and and used for further studies. eleven of actinomycetes was isolated from different marine and terrestrial habitats Antidermatophytic activity including Ghrgada marine sea, Ras Sedr The antidermatophytic activity of ethyl sediment, Ain Sokhna. Results in Table (1) acetate extracts were tested using the agar indicate Highest number of strains were well diffusion method [16], 100 µl of each isolated from Hurgada sea water (20 %) extract culture was placed in a well made followed by Ras sedr sediments (16 %) and with a sterile cork borer on Sabouraud soil isolates from Mansoura (17 %). dextrose agar plates (pH 5.6) seeded with the test fungal cultures Trichophyton Table 1. Distribution and percent of mentagrophytes (RCMB 09285), streptomyces spp. isolated from different Microsporum canis (RCMB 07321) and marine and terrestrial localities. Microsporum gypseum (RCMB 07336). The Isolates Percentage plates were incubated at 28oC and observed Location account incidence (%) for antibiosis after 3-4 days [17] Results are Marine sea water shown in Table (2). Amphotericin B was 22 20 % Hurgada used as a control. Twenty-one isolates were Marine algae 13 12 % primarily selected as they exhibited Ras Sedr 18 16 % interesting antidermatophytic activities. sediments Ten isolates (A1, A3, A8, A10, A12, G5, P2, Ain Sokhna 13 12 % P4, P7, P9) exhibited antidermatophytic sediment activity by a clear zone of inhibition against Marine algae 2 5 4 % all the test dermatophytes. While nine Soil from 19 17 % isolates (B13, B17, F5, F7, F12, F20, S1, Mansoura Soil from NRC S5, S14) exhibited antidermatophytic 10 9 % activity by a clear zone of inhibition against garden Microsporumcanis (RCMB 07321) and Pyramids soil 11 10 % Microsporumgypseum (RCMB 07336) and Total isolates 111 100 % two isolates(H3, H10) exhibited antidermatophytic activity by a clear zone Antidermatophytic activity test: Result in of inhibition against (Trichophyton Table 2 revealed that twenty-one isolates mentagrophytes (RCMB 09285) and exhibited antidermatophytic activities. Ten Microsporumcanis (RCMB 07321). extracts from. (A1, A3, A8, A10, A12, G5, P2, Taxonomic identification: The diagnostic P4, P7, P9) showed antidermatophytic properties of the strain were compared activity against all test dermatophytes: with those reported for identification of Trichophyton mentagrophytes (RCMB 157 Mohamed Fadel et al., JIPBS, Vol 3 (2), 155-165, 2016 09285), Microsporum canis (RCMB 07321) antifungal activity against V. ceratosperma and Microsporum gypseum (RCMB [29]. Taxonomic identification of the most 07336).While the most potent streptomyces potent actinomycetes strain (P4) has been spp. extracts were (A1, A3, A10, G5 and restricted
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