East African Scholars Journal of Biotechnology and Genetics Abbreviated Key Title: East African Scholars J Biotechnol Genet ISSN: 2663-189X (Print) & ISSN : 2663-7286 (Online) Published By East African Scholars Publisher, Kenya Volume-1 | Issue-.4 | July-Aug -2019 | Research Article Isolation, characterization and antibacterial activity of a Rare Actinomycete: Saccharopolyspora sp. In Iraq Prof. Dr. Mohsen Hashim Risan 1, Rusul Alaa Jafar1 and Shams Ahmed Subhi2 1College of Biotechnology, Al-Nahrain University, Iraq 2Al-Turath University College, Iraq *Corresponding Author Prof. Dr. Mohsen Hashim Risan Abstract: The aimed of the study is isolating and identifying the rare actinomycetes species in Iraq, that are capable of producing antimicrobial agent. Thirty-three colonies were found. Colonies having characteristic features such as powdery appearance with convex, concave or flat surface and color ranging from buff, brown, pink, red, white, yellow and gray were selected. Sub-cultured on ISP2 were supplemented with 12 % (w/v) NaCl for growth, and incubation of plates for 10-12 days at 28°C, only five isolates demonstrated cultural characteristics similar to that of genus Saccharopolyspora sp. Five isolates were selected and purified by pure culture techniques. All isolates were given a number as AW12, AW 4, MK 7, AW 9 and AW 2. The isolates were identified as Saccharopolyspora sp. based on their morphological, physiological and biochemical characteristics. All Saccharopolyspora isolates were screened for their antibacterial activity on malt extract yeast extract agar medium (ISP2) using scross-streak technique. The highest activities were shown by AW12 isolate against Staphylococcus aureus with inhibition zone diameters 17 mm. Keywords: rare actinomycetes, Saccharopolyspora, antibacterial, Iraq. INTRODUCTION pseudonocardiaceae belong to rare actinomycetes. This Actinomycetes are filamentous bacteria, family currently contains seven genera, (Ventura et al., 2007). The soil actinomycetes produce Actinopolyspora, Amycolata, Amycolatopsis, a volatile compound called geosmin, which literally Kibdelosporangium, Pseudonocardia, translates to “earth smell” of healthy soil (Gust et al., Saccharomonospora and Saccharopolyspora. It 2003; Sprusansky et al., 2005). Actinomycetes are includes several cultures with industrial interest as Gram-positive bacteria. Although Streptomyces species antibiotic producers (erythromycin, vancomycin, still promise to remain fruitful sources of new rifamycin) or in bioconversion processes (Okazaki et antibiotics (Amin et al., 2016; Risan et al., 2016; Risan al., 1983). All of pseudonocardiaceae members are et al., 2017; Risan et al., 2018; Al-Rubaye et al., aerobic and catalase-positive, but exhibit a wide range 2018a, b). The Recent tendency has shown more of different physiologies (autotrophic, thermophilic, frequent detection of a number of new antibiotics from halophilic, etc.) (Embley 1992). micro-organisms other than Streptomyces as rare Pseudonocardiaceae family includes genus Actinomycetes. Rare actinomycetes once isolated from Saccharopolyspora (Lazzarini et al., 2000; Tiwari and soils are much easier to die than Streptomyces species. Gupta, 2013). The genus Saccharopolyspora was first Rare actinomycetes, show much slower growth than described by Lacey and Goodfellow (1975). The genus Streptomyces species. Some require vitamin and biotin Saccharopolyspora includes species: for growth and most of them have more complex Saccharopolyspora erythraea (Labeda, 1987), nutritional requirements (Lechevalier and Lechevalier Saccharopolyspora gregorii and Saccharopolyspora 1981). The characteristics of rare actinomycetes are hordei (Goodfellow et al., 1989), Saccharopolyspora slower growth, more complex nutritional requirements, rectivirgula and Saccharopolyspora taberi (Korn- poorer sporulation and less stable as for preservation Wendisch et al., 1989), Saccharopolyspora spinosa (Kroppenstedt and Goodfellow 2006). The family (Mertz and Yao, 1990), Saccharopolyspora hirsuta Quick Response Code Journal homepage: Copyright @ 2019: This is an open-access http://www.easpublisher.com/easjbg/ article distributed under the terms of the Creative Commons Attribution license which Article History permits unrestricted use, distribution, and Received: 15.06.2019 reproduction in any medium for non Accepted: 25.06.2019 commercial use (NonCommercial, or CC-BY- Published: 10.07.2019 NC) provided the original author and source are credited. Published By East African Scholars Publisher, Kenya 49 Mohsen Hashim Risan et al., East African Scholars J Biotechnol Genet; Vol-1, Iss-4 (July-Aug, 2019): 49-60 (Lacey and Goodfellow, 1975), Saccharopolyspora were obtained from Central Public Health Labrotory in spinosporotrichia (Zhou et al., 1998), Baghdad city. They were maintained on Mueller- Saccharopolyspora flava and Hinton (MH), plates at 4ºC. Stock cultures were kept in Saccharopolyspora thermophila (Lu et al., 2001), 20% glycerol at -70ºC. Saccharopolyspora antimicrobica (Yuan et al., 2008), Saccharopolyspora cebuensis (Pimentel-Elardo et al., Isolation of Actinomycetes 2008), Saccharopolyspora shandongensis (Zhang et al., Each soil sample was air dried at room 2008), Saccharopolyspora endophytica (Qin et al., temperature and sieved through a 2 mm pore size sieve 2008), Saccharopolyspora halophila (Tang et al., to get rid of large debris. The sieved soil was used for 2009a), Saccharopolyspora jiangxiensis (Zhang et al., the isolation purpose. One g was suspended in 99 ml 2009), Saccharopolyspora qujiaojingensis (Tang et sterile distilled water and incubated at 28°C with al., 2009b), Saccharopolyspora rosea (Yassin, 2009), shaking at 180 rpm for 1 h. (Oskay et al., 2004), 0.1 ml Saccharopolyspora tripterygii (Li et al., 2009) and of each solution, at dilutions of 10-4-10-5, then spread Saccharopolyspora patthalungensis (Duangmal et al., on Glycerol yeast extract agar medium (GYEA) (2g 2010). Yeast extract, 10ml Glycerol, 20 agar and 1 L water) supplemented with tetracycline 50 μg/ml and 50 μg/ml Members of this genus are aerobic, Gram- nystatin, to prevent growth of other bacteria and fungi, positive, non-acid-fast organisms with substrate hyphae respectively. (The pH value of the medium was that either fragment into rod-shaped elements, do not adjusted to 7-7.2 and then sterilized in an autoclave at fragment or are transformed partially into chains of 121°C for 15 minutes), then incubated at 28°C for 8-12 spores (Korn-Wendisch et al., 1989). The combination days still actinomycetes colonies appear. (El-Nakeeb of a green to bluish green color of the aerial and and Lechevalier, 1963; Kuster and Williams, 1964; substrate mycelia and monosporic morphology appears Qasim and Risan, 2017). After the incubation period, to be a good indication that an isolate belongs to the the plates were examined for typical actinomycetes genus Saccharomonospora. The genus encompasses colonies, which had regular round, small, opaque, 27 recognized species of which 17 were identified in compact, frequently pigmented with white, brown, the last 10 years. Most of these species were isolated gray-pink, or other colors, the colonies were examined from soil samples (Cheng et al., 2013), saline lakes under a light microscope to observe their morphology (Tang et al., 2009) and endophytic associations (Qin et and distinguished from fungi colonies. The isolated al., 2008). Furthermore, some members of actinomycetes were re-cultured and stored at 4°C for Saccharopolyspora have been recovered from further study (Venkateswarlu et al., 2000; Gesheva et symbiotic associations with marine sponges. So the aim al., 2005). of the study is isolating and identifying the rare actinomycetes species in Iraq, that are capable of Purification And Identification Of Actinomycetes producing antimicrobial agent. spp After incubation, plates were examined for the MATERIALS AND METHODS appearance of actinomycetes colonies. Colonies of Sample Collection actinomycetes were recognized by their microscopic Soil samples were collected from two characteristics (optical microscopy, aerial, substrate provinces in 2018, Baghdad city (Al-Jadriya) and Wasit mycelium colour and Gram’s stain). The stock cultures province (Al - Noamaniya). The samples were taken at were maintained and transferred on International a depth of 5-7 centimeters below the surface, where Streptomyces project Medium slants (ISP-2 ) (4g Yeast most of the microbial activity takes place, and thus extract, 10g Malt extract, 4g Dextrose, 20 agar and 1 L where most of the bacterial population is concentrated. water) to obtain pure colonies used for identification Soil samples (100 g sample of each soil type) were (Nonoh et al., 2010 ; Whitman et al., 2012). Once in collected by using clean, dry and sterile four months and stored at 4°C. Polyethylene bags along with sterile spatula, marking pen, rubber band and other accessories. The site Identification by Cultural Characterizations: selection was done by taking care of the point where The identification of Saccharopolyspora widely varying characteristics as possible with regard to isolates were based largely upon the morphological the organic matter, moisture content, and particle size observations. It includes some basic observations, and colour of soil and to avoid contamination as far as Gram’s stain, produced Substrate mycelia (Branched or possible. Samples were stored in boxes and transported Fragments), spores on substrate mycelia, colors of the to the laboratory