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Isolation and Identification of Streptomyces Spp. from Desert And International Journal of Environmental Research and Public Health Communication Isolation and Identification of Streptomyces spp. from Desert and Savanna Soils in Sudan Mohamed E. Hamid 1,2,* , Adil Mahgoub 2,3, Abdulrhman J. O. Babiker 4, Hussein A. E. Babiker 5, Mohammed A. I. Holie 3, Mogahid M. Elhassan 6 and Martin R. P. Joseph 1 1 Department of Clinical Microbiology and Parasitology, College of Medicine, King Khalid University, P.O. Box 641, Abha 61314, Saudi Arabia; [email protected] 2 Department of Preventive Medicine, Faculty of Veterinary Medicine, University of Khartoum, Khartoum North 13314, Sudan; [email protected] 3 Department of Microbiology, College of Medical Laboratory Science, Alzeim Alazhari University, Khartoum North 12217, Sudan; [email protected] 4 Al-Amal National Hospital, Khartoum North 23622, Sudan; [email protected] 5 Department of Clinical Science, College of Veterinary Medicine, King Faisal University, P.O. Box 400, Al-Ahsa 31982, Saudi Arabia; [email protected] 6 Department of Clinical Laboratory Science, College of Applied Medical Science, Taibah University, Al-Madinah 13215, Saudi Arabia; [email protected] * Correspondence: [email protected]; Tel.: +966-5-0977-3687 Received: 20 September 2020; Accepted: 12 November 2020; Published: 25 November 2020 Abstract: The purpose of this study was to investigate streptomycete populations in desert and savanna ecozones in Sudan and to identify species based on 16S rRNA gene sequences. A total of 49 different Streptomyces phenotypes (22 from sites representing the desert and semi-desert ecozone; 27 representing the savanna ecozone) have been included in the study. The isolates were characterized phenotypically and confirmed using 16S rRNA gene sequence analysis. The two ecozones showed both similarities and uniqueness in the types of isolates. The shared species were in cluster 1 (Streptomyces (S.) werraensis), cluster 2 (Streptomyces sp.), cluster 3 (S. griseomycini-like), and cluster 7 (S. rochei). The desert ecozone revealed unique species in cluster 9 (Streptomyces sp.) and cluster 10 (S. griseomycini). Whereas, the savanna ecozone revealed unique species in cluster 4 (Streptomyces sp.), cluster 5 (S. albogriseolus/ S. griseoincarnatus), cluster 6 (S. djakartensis), and cluster 8 (Streptomyces sp.). Streptomycetes are widely distributed in both desert and the savanna ecozones and many of these require full descriptions. Extending knowledge on Streptomyces communities and their dynamics in different ecological zones and their potential antibiotic production is needed. Keywords: actinomycetes; ecosystem; biodiversity; phenotypic identification; 16S rRNA gene 1. Introduction The phylum Actinobacteria (order Actinomycetales) hosts diverse high G+C, Gram-positive bacteria including members of the genus Streptomyces (S.) Streptomycetes are Gram-positive spore-forming bacteria which include more than 900 species (https://www.ncbi.nlm.nih.gov/Taxonomy/ Browser/wwwtax.cgi?id=1883). Streptomyces represents the largest taxonomic group within the Bacteria Domain which is ubiquitously distributed in both aquatic and terrestrial ecosystems [1,2]. They are found in a wide range of habitats but are particularly abundant in soil, representing around 1 to 20% of the total viable count [2,3] and are known to prefer the characteristic earthy smell (geosmin) [4]. Streptomycetes are chemoheteroorganotrophs and are distinguished by their tough, leathery colonies and filamentous growth. They have an important ecological role in the turnover of organic material and Int. J. Environ. Res. Public Health 2020, 17, 8749; doi:10.3390/ijerph17238749 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 8749 2 of 10 are capable of using complex organic materials such as carbon and energy sources in the breakdown of theseInt. products J. Environ. inRes. the Public soil Health [5]. 2020, 17, x 2 of 10 The microbial communities including streptomycetes are affected by biotic and abiotic factors, notablyecological vegetation, role in the type turnover of soil, of organic and climate material [6 and] Actinomycetes’ are capable of using diversity, complex and organic particularly materials that of streptomycetes,such as carbon and is widelyenergy sources documented in the br foreakdown various of these reasons products including in the soil the [5]. continued generation of environmentalThe microbial isolates communities for pharmaceutical, including streptomycetes biodegradative, are affected and by biotic biotechnological and abiotic factors, screening. Actinomycetesnotably vegetation, isolated type from of soil soil, and and related climate substrates [6] Actinomycetes’ show primary diversity, biodegradative and particularly activity that of [5 ]. streptomycetes, is widely documented for various reasons including the continued generation of Exploring new habitats around the globe is increasingly becoming the focus for discovering new environmental isolates for pharmaceutical, biodegradative, and biotechnological screening. taxa of Actinobacteria, namely streptomycetes, for potential antibacterial, antitumoral, and antifungal Actinomycetes isolated from soil and related substrates show primary biodegradative activity [5]. activitiesExploring [7,8]. The new habitat habitats of Sudanaround hasthe globe remained is incr virtuallyeasingly becoming untouched the focus except for fordiscovering very few new studies. Sometaxa areas of Actinobacteria, in Sudan were namely found streptomycetes, to be rich sources for potential of microbial antibacterial, biodiversity, antitumoral, holding and antifungal withinthem immenseactivities novelty [7,8]. and The potentiality habitat of Sudan of identifying has remained new isolatesvirtually for untouched the production except for of life-savingvery few studies. drugs such as amphotericinSome areas in A. Sudan Marine were habitats found of to Sudan be rich were source founds of microbial to be a promising biodiversity, source holding for Streptomyceswithin them spp. diversityimmense and novelty a potential and potentiality source of of productionidentifying new of secondaryisolates for the metabolites production [of9]. life-saving Two actinomycin drugs D-producingsuch as amphotericin streptomycetes A. Marine from habitats Sudanese of Sudan soil have were been found characterized to be a promising and weresource found for Streptomyces to be diff erent fromspp. previous diversity actinomycin-producing and a potential source of species producti [10on]. of The secondary finding metabolites of distinct [9] phylogenetic Two actinomycin lineages D- and producing streptomycetes from Sudanese soil have been characterized and were found to be different the variation in the spatial distribution of clones suggests that selection pressures may vary over the from previous actinomycin-producing species [10]. The finding of distinct phylogenetic lineages and soil landscape [11]. the variation in the spatial distribution of clones suggests that selection pressures may vary over the Thissoil landscape study aimed [11]. to isolate and identify streptomycetes from desert and savanna ecozones in Sudan andThis to findstudy out aimed the to species isolate diversity and identify in the strept twoomycetes ecozones from using desert 16S and rRNA savanna gene ecozones analysis. in Sudan and to find out the species diversity in the two ecozones using 16S rRNA gene analysis. 2. Materials and Methods 2. Materials and Methods 2.1. Study Samples and Geographical Locations 2.1. Study Samples and Geographical Locations Soil samples were collected from different sites in two ecozones: (1) a desert and semi-desert ecozone,Soil and samples (2) a low were rainfall collected woodland from different savanna sites ecozone in two ecozones: (Figure 1(1)), accordinga desert and to semi-desert the ecological classificationecozone, byand Harrison (2) a low and rainfall Jackson woodland [12]. The savanna soil samples ecozone were(Figure collected 1), according during to the the dry ecological season from the surfaceclassification as well by as Harrison from 5–10 and Jackson cm deep. [12]. From The soil five samples sections, were 10 collected g within during each sitethe dry were season collected from with the surface as well as from 5–10 cm deep. From five sections, 10 g within each site were collected with a sterilized spatula. Soils were then merged, sieved, and thoroughly mixed. The composite sample a sterilized spatula. Soils were then merged, sieved, and thoroughly mixed. The composite sample was was transferred into sterile plastic bags, labeled, transported to the laboratory, and stored at 4 C transferred into sterile plastic bags, labeled, transported to the laboratory, and stored at 4 °C awaiting ◦ awaitinganalysis. analysis. FigureFigure 1. Map 1. Map of Sudan of Sudan showing showing soil soil collection collection sites and and ecological ecological zones zones (modified (modified from from the World the World ConservationConservation Union Union (IUCN) (IUCN) (http: (http://www.catsg.or//www.catsg.orgg/cheetah/04_country-information/North-African-/cheetah/04_country-information/North-African- regionsregions/sudan/sudan-sat2.jpg)./sudan/sudan-sat2.jpg). Int. J. Environ. Res. Public Health 2020, 17, 8749 3 of 10 2.2. Pretreatment of Soil Samples The Higher Nitrogen Content (HNC) medium [13] was used as a pretreatment method to assist the extraction and isolation of streptomycetes from soil. HNC medium was prepared and stored at 4 ◦C. After collection,
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