Characterization of Locally Isolated Streptomyces Species and Their Potentialities As Sources of Antifungal Agents

Characterization of locally Isolated Streptomyces Species and their Potentialities as Sources of Antifungal Agents

By:

DEENA ABDELFATAH ABDELGADIR MOHAMMED

Post-Graduate Diploma in Applied Microbiology University of Westminster UK

B.Sc. (Honors) Science University of Khartoum

A Thesis Submitted to the University of Khartoum in Fulfillment of the Requirements for the degree of Master of Science in Botany

Department of Botany Faculty of Science University of Khartoum

Jan. 2007 Dedications

To my Beloved Parents To my Ibrahim and Reil And last but not least to Hassan

Table of contents

Contents Page Acknowledgement………………………………...... i Abstract…………………………………………………...... ii Abstract in Arabic ……………………………...... iv Introduction and Literature Review………………...... 1 1 Introduction…………………………………………...... 1 1.1

1.2 Literature Review………………………………………………….. 4 1.2.1 Family Streptomyceteace …………………………………………. 6 1.2.2 Description of the genus Streptomcyes ………..………………….. 6 1.2.3 Isolation of Streptomcyes from soil………………………………... 8 1.2.4 Antibiotic production……………………………………………… 9 1.3 Antibiotics…………………………………………………………. 9 1.3.1 Classification of Antibiotics……..………………………………… 10 1.3.1.1 According to Source……………………………………………….. 10 1.3.1.1. Microorganisms…………………………………………………… 10 1 1.3.1.1. Chemical Synthesis………………………………………………... 10 2 1.3.1.1. Semi-synthesis………………..…………………………………… 10 3

1.3.1.2 According to mode of action………………………………...... 11 1.3.1.2. Inhibition of cell wall synthesis..…………………………………. 11 1 1.3.1.2. Inhibition of protein synthesis…………………………………….. 12 2 1.3.1.2. Inhibition of nucleic acid synthesis………………………………... 12 3 1.3.1.2. Disruption of fungal cell membrane………………………………. 13 4

1.3.2.5 Inhibition of fungal mitosis……………………………………….. 13 1.4 Antibiotics production and recovery………………………………. 14 2. Materials and Methods……………………………………………. 16 2.1 Isolation of Streptomyces species from soil samples..………...... 16 2.2 Preservation and maintenance……………………………………... 17 2.3 Screening for antibiotic production………………………………. 17 2.4 Characterization of Streptomyces isolates………………………… 18 2.4.1 Cultural Characteristics……………………………………………. 18 2.4.2 Microscopic characteristics…………………………………...... 18 2.4.2.1 Gram Staining……………………………………………………… 19 2.4.2.2 Mycelial morphology……………………………………………… 20 2.4.2.3 Acid fast Staining……………………………………………...... 20 2.5 Biochemical Characteristics……………………………………….. 20 2.5.1 Catalase test…………………………………………………...... 20 2.5.2 Oxidase test…………………………………………………...... 20 2.5.3 Aerobiosis…………………………………………………………. 21 2.5.4 Gelatin liquefaction…………………………………………...... 21 2.5.5 Starch hydrolysis test………………………………………………. 21 2.5.6 Utilization of different carbon sources…………………………….. 22 2.5.7 Urease test…………………………………………………………. 22 2.5.8 Organic acid formation……………………………………………. 23 2.5.9 Milk coagulation and peptonization………………………………. 23 2.5.10 Casein hydrolysete…………………………………………...... 24 2.5.11 Blood hemolysis……………………………………………...... 24 2.5.12 Nitrate reductase expression……………………………………… 24 2.5.13 H2S production…………………………………………………….. 25 2.6 Antibiotic production by Streptomyces in submerged cultivation 25 2.6.1 Media preparation ……………………………………………. 25 2.6.2 Streptomyces inoculum and inoculation………………..……. 26 2.6.3 Extraction of the fermentation broth…………………………. 26 3. Results and Discussion………………………………………. 27 3.1 Isolation and screening of Streptomyces………………………… 28 3.2 Characterization of Streptomyces isolates…………………… 35 3.2.1 Cultural characteristics……………………………………….. 35 3.2.2 Microscopical characteristics………………………………… 38 3.2.3 Biochemical characteristics………………………………… 38 Production of antibiotics in submerged culture by Streptomyces 3.3 47 isolates

3.4 General Discussion……………………………... 50 4 References…………………………………….. 52

I wish to express my greatest gratitude to my supervisor Dr. Adil Ali Hussein for his guidance, help and encouragement during the course of this study.

My deep thanks to all members of the Botany department, Faculty of Science, University of Khartoum for their continuous help and encouragement.

My gratitude and warm thanks are extended to my family for their encouragement and support.

I would like to thank my friends and colleagues for help and encouragement.

I am deeply indebted to all those who have assisted me during this study.

Abstract

Fifty isolates of Streptomyces were recovered from soil samples collected from different locations in Sudan. All of the isolates were screened for their abilities to inhibit the growth of certain pathogenic fungi. These included: Cryptococcus humicolus, Penicillium sp., Asperigillus flavus, Asperigillus niger, Candida albicans, and Fusarium oxysporium. The inhibition zone diameters recorded were found to vary among Streptomyces isolates as well as among the tested microorganisms. Sixteen of the Streptomyces isolates succeeded to inhibit the growth of one or more of the tested microorganisms. These were then characterized by different cultural, microscopical and biochemical characteristics. All of the isolates were found to be aerobic, Gram positive, filamentous and non-motile. They were able to express Catalase, oxidase, urease, and nitrate reductase. More, they were also able to haemolyse blood, hydrolyse starch, utilize all of the tested carbohydrates as sole carbon sources. However, a wide range of variation was shown by the isolates in some other biochemical tests. These included : organic acid formation, production of H2S on Kligler Iron Agar (KIA) medium, gelatin liquefaction, and peptonization and coagulation of milk. The isolates were then tested for their abilities to produce inhibitory agents under submerged conditions. The inhibitory agents so produced, were extracted from the fermentation broths using n-butanol. The extracts were found to inhibit the growth of some of the tested pathogenic fungi. The greatest inhibition zone diameter (mm) was shown against Candida albicans by Dee 15. Some of the Streptomyces isolates were found to inhibit the sporulation as well as the vegetative mycelia of the Aspergillus niger such as Dee15 and Dee29. However, none of the Streptomyces isolates have shown any activities against Saccharomyces sp., Penicilium sp. and Fusarium sp.

ii ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ

ﺗﻢ ﻋﺰل ﺧﻤﺴﻴﻦ ﺳﻼﻟﺔ ﻣﻦ ﺑﻜﺘﺮﻳﺎ ال Streptomyces ﻣﻦ ﻋﻴﻨﺎت ﺗﺮﺑﺔ ﺟﻤﻌﺖ ﻣﻦ ﻣﻮاﻗﻊ ﻣﺨﺘﻠﻔﺔ ﻓﻲ اﻟﺴﻮدان . ﺗﻢ اﺧﺘﺒﺎر ﻣﻘﺪرة آﻞ اﻟﺴﻼﻻت اﻟﻤﻌﺰوﻟﺔ ﻋﻠﻰ ﺗﺜﺒﻴﻂ ﻧﻤﻮ ﻓﻄﺮﻳﺎت ﻣﻤﺮﺿﺔ ﻣﻌﻴﻨﺔ . ﺳﺘﺔ ﻋﺸﺮ ﻣﻦ هﺬﻩ اﻟﺴﻼﻻت اﻇﻬﺮت ﻣﻘﺪرات ﻣﺘﺒﺎﻳﻨﺔ ﻋﻠﻰ ﺗﺜﺒﻴﻂ ﻧﻤﻮ واﺣﺪ أو أآﺜﺮ ﻣﻦ اﻟﻔﻄﺮﻳﺎت اﻵﺗﻴﺔ :- Cryptococcus humicolus, Penicillium sp., Asperigillus flavus, Aspergillus niger, Candida albicans, and Fusarium oxysporium اﺧﺘﻠﻒ ﻗﻄﺮ ﻧﻄﺎق اﻟﺘﺜﺒﻴﻂ ﺑﺎﺧﺘﻼف ﺳﻼﻟﺔ ال Streptomyces وأﻧﻮاع اﻟﻔﻄﺮات ﺗﺤﺖ اﻻﺧﺘﺒﺎر . ﻧﺠﺤﺖ ﺳﺘﺔ ﻋﺸﺮ ﺳﻼﻟﺔ ﻣﻦ ال Streptomyces ﻓﻲ ﺗﺜﺒﻴﻂ ﻧﻤﻮ واﺣﺪ أو أآﺜﺮ ﻣﻦ اﻟﻔﻄﺮات ﺗﺤﺖ اﻻﺧﺘﺒﺎر . اﺧﻀﻌﺖ اﻟﺴﻼﻻت اﻟﺘﻲ اﻇﻬﺮت ﻣﻘﺪرة ﻋﻠﻰ اﻟﺘﺜﺒﻴﻂ ﻟﻌﺪة اﺧﺘﺒﺎرات ﻟﻤﻌﺮﻓﺔ ﺻﻔﺎﺗﻬﺎ اﻟﻤﺰرﻋﻴﺔ واﻟﻤﺠﻬﺮﻳﺔ واﻟﻜﻴﻤﻮﺣﻴﻮﻳﺔ . آﻞ اﻟﻌﺰﻻت وﺟﺪت هﻮاﺋﻴﺔ ، ﻣﻮﺟﺒﺔ اﻟﺠﺮام ، ﺧﻴﻄﻴﺔ ، وﻏﻴﺮ ﻣﺘﺤﺮآﺔ . آﻤﺎ اﻧﻪ اﻋﻄﺖ ﻧﺘﺎﺋﺞ اﻳﺠﺎﺑﻴﺔ ﻻﺧﺘﺒﺎرات Nitrate reductase ، Urease ، Oxidase ، Catalase. ﺑﺎﻹﺿﺎﻓﺔ اﻟﻰ أن ﺟﻤﻴﻊ اﻟﻌﺰﻻت آﺎﻧﺖ ﻗﺎدرة ﻋﻠﻰ Blood hemolysis و Starch hydrolysis واﺳﺘﺨﺪام اﻧﻮاع ﻣﺨﺘﻼﻓﺔ ﻣﻦ اﻟﻜﺮﺑﻮهﻴﺪرات .وﺟﺪ ﺑﻌﺾ اﻻﺧﺘﻼف ﻓﻲ ﺑﻌﺾ اﻻﺧﺘﺒﺎرات اﻟﻜﻴﻤﻮﺣﻴﻮﻳﺔ ﻣﺜﻞ اﻧﺘﺎج اﻻﺣﻤﺎض اﻟﻌﻀﻮﻳﺔ واﻧﺘﺎج آﺒﺮﻳﺘﻴﺪ اﻟﻬﻴﺪروﺟﻴﻦ وﺗﺴﻴﻴﻞ اﻟﺠﻼﺗﻴﻦ وﺗﻘﻄﻴﻊ اﻟﻠﺒﻦ . ﺑﻌﺪ ذﻟﻚ ﺗﻤﺖ دراﺳﺔ ﻣﻘﺪرة اﻟﺴﻼﻻت ﻋﻠﻰ اﻧﺘﺎج اﻟﻤﻀﺎدات اﻟﻔﻄﺮﻳﺔ ﻓﻲ ﺑﻴﺌﺔ ﻏﺬاﺋﻴﺔ ﺳﺎﺋﻠﺔ ﻋﻠﻰ ﻃﺮﻳﻘﺔ الSubmerged Culture. ﺑﻌﺪ ﻓﺘﺮة ﺗﺤﻀﻴﻦ ﻟﻤﺪة أﺳﺒﻮع ﺗﻢ اﺳﺘﺨﻼص اﻟﻤﻀﺎدات اﻟﻔﻄﺮﻳﺔ اﻟﻤﻨﺘﺠﺔ ﺑﺎﻟﻤﺬﻳﺐ اﻟﻌﻀﻮي اﻟﺒﻴﻮﺗﺎﻧﻮل -1 . ووﺟﺪ ان اﻟﻤﺴﺘﺨﻠﺼﺎت ﻗﺎدرة ﻋﻠﻰ ﺗﺜﺒﻴﻂ اﻟﻔﻄﺮﻳﺎت اﻟﻤﻤﺮﺿﺔ ﺗﺤﺖ اﻻﺧﺘﺒﺎر . أﻇﻬﺮت ﻣﺴﺘﺨﻠﺼﺎت اﻟﺒﻴﻮﺗﺎﻧﻮل اﻟﺘﻲ ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻴﻬﺎ ﻣﻦ اﻟﺴﻼﻟﺔ Dee15 ﻓﻌﺎﻟﻴﺔ ﻋﺎﻟﻴﺔ ﺿﺪ اﻟﻔﻄﺮ Candida albicans ﺣﻴﺚ ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ أآﺒﺮ ﺗﺜﺒﻴﻂ (45ﻣﻠﻢ ) وﻓﻄﺮ Aspergillus niger (30ﻣﻠﻢ ) آﻤﺎ اﻇﻬﺮت ﺑﻌﺾ اﻟﺴﻼﻻت Dee29 و Dee15 ﻣﻘﺪرة ﻋﻠﻰ ﺗﺜﺒﻴﻂ ﺗﻜﻮﻳﻦ اﻟﺠﺮاﺛﻴﻢ وهﻴﻔﺎت Aspergillus . آﻞ ﻣﺴﺘﺨﻠﺼﺎت ﺳﻼﻻت Streptomyces ﻟﻢ ﺗﻈﻬﺮ أي ﻧﺸﺎط ﺿﺪ ﻓﻄﺮﻳﺎت Penicillum sp., Fusarium sp , وSaccharomyces sp.

iii Chapter One Introduction and Literature Review

1.1 Introduction The history of new drug discovery processes shows that the novel skeletons of antibiotics have, in the majority of cases, come from natural sources (Bevan et al., 1995). This involves the screening of microorganisms as well as plant extracts (Shadomy et al., 1987). Soil is a reservoir of a tremendous species of microorganisms. The majority are native to the soil and are found principally in the upper few inches of the earth crust (Wedberg, 1966). Soil microorganisms are known to produce different types of secondary metabolites that have different biological roles. Of these metabolites, antibiotics predominate due to their therapeutic and other commercial importance (Ounhdouch et al., 2001). An antibiotic is defined as a chemical substance, produced wholly or partially by an organism, which has the capacity in dilute solution to inhibit the growth or kill other microorganisms (O' Grady et al., 1997). Production of antibiotic substances by microorganisms in soil confers an advantage for its producer in competition with other microbes and thus plays a role in the establishment of an equilibrium among the members of the soil micro-flora. Furthermore, most soil microbiologists share the view that many soil antifungal-producing microorganisms are responsible for the control of plant root pathogens (Williams, 1982; Aghighi et al., 2004). Among the different types of antibiotics prevailing in the market, antifungal antibiotics are very few compared to antibacterials, though they are a significant group and have an important role in the control of plant and animal mycotic diseases.

1 The search for new broad-spectrum antifungal agents with greater potency has been progressing slowly (Gupta et al., 2002). The need for new, safe and more effective antifungal agents is a major challenge to the pharmaceutical industry, especially with the increase in the opportunistic infections in the immunocompromised hosts. The reason for the slow progress, compared to antibacterial, is that like mammalian cells, fungi are eukaryotes and therefore any agent that inhibits protein, RNA or DNA biosynthesis in fungi has greater potential for toxicity to the host as well (Georgopapdakou and Tkacz, 1994; Augustine et al., 2005a). The other reason is that, until recently, the incidence of life threatening fungal infections was perceived as being too low hence research in this field receives little support by pharmaceutical companies (Georgopapdakou and Tkacz 1994; Augustine et al., 2005b). Nevertheless, it should be emphasized that fungi cause most of plant diseases and pathogenic fungi are major problem in agriculture (Aghighi et al., 2004). Because of their broad metabolic spectrum, Actinomycetes are considered to be one of the most important organisms existing in soil. They are widely distributed in terrestrial environments from which they are easily isolated. They are known to produce pigments and antibiotics that make them fascinating and a suitable subject for microbiological research (Porter and Wilhem, 1960). Among Actinomycetes members of the genus Streptomyces represent a considerable proportion of the community and have been studied extensively. Many of them have been described, and from time to time a novel Streptomycete is isolated (Meyers et al., 2003). The diversity of Streptomyces spp. in the soil may be influenced by the diversity of cultivated plant species as these bacteria grow profusely in the humus leaf litter layers (Oskay et al., 2004).

2 Recent reports show that Streptomyces still remain an important potential source of new antibiotics (Watve et al., 2001). Screening can possibly reveal Streptomyces species that produces a novel antibiotic or a novel species of Streptomyces can be identified. Thus the specific objectives of this study were: 1. Selective isolation of Streptomyces species from soil samples from seven locations. 2. Screening of these isolates for their antibiotic activities against pathogenic fungi. 3. Characterization of the most potential isolates, by determining their cultural, microscopical and biochemical characteristics. 4. Production of antibiotics by submerged culture. 5. Extraction of the antibiotic from the fermentation broth. 6. Studying the inhibitory effects of the obtained extracts against some pathogenic fungi.

3 1.2 Literature Review Microorganisms are found in the soil, air and water. They represent an important class of the biodiversity. Although some of the microorganisms are pathogenic, there are some which have great importance to humanity in different aspects including, degradation of organic materials, nitrogen fixation, vitamin production, enzyme production and antibiotic production. The first chemotherapeutically effective antibiotic (Penicillin) was discovered by Alexander Fleming in 1929. Since then about 9000 antibiotic substances are known, the majority (66%) is produced by members of the genus Streptomyces which belongs to the eubacterial order Actinomycetales (Champness, 2000; Roubos et al., 2001). Actinomycetes are Gram Positive bacteria; they are widely distributed in terrestrial environments, from which they are easily isolated (Meyers et al., 2003). They produce fine mycelium; its hyphae never exceed 1.5µ (Labeda et al., 1997). Many Actinomycetes produce spores that are different from the endospores of Bacilli not only in the method of formation but also in being mildly resistant to heat. The combination of aerial growth and sporulation usually confer a cottony or powdery texture on the surface of the colony. Colonies which lack aerial mycelium are either glossy or matt (Stanier et al., 1963; Felicitas and Hans, 1992; Alexander, 1967). The basic nutrition of the majority of Actinomycetes is simple and resembles that of a large number of bacteria and the majority of fungi. There is a requirement for an organic source of carbon such as glucose, maltose, starch, sucrose and certain organic acids. Glycerol and proteins may also be used by most species as carbon sources and still some are able to decompose cellulose. Nitrogen can be obtained from either organic or inorganic sources including: nitrates, ammonium salts, organic nitrogenous substances and

4 proteins. Other minerals required include phosphorus, potassium, and sulpher (Felicitas and Hans, 1992). Actinomycetes have considerable economic importance and in nature they contribute to the mineralization of organic residues. Only a few are plant pathogens but a number cause debilitating or even lethal infections of animal and man. They produce 85% of the known antibiotics including substances active against bacteria, fungi, protozoa, rickettsiae, larger viruses and neoplasms. They also generate a great variety of biologically important substances in their environment such as vitamins, pigments, enzymes and antibiotics (Tetsuo et al., 2000). According to Lacey (1973) the order Actinomycetales is divided into four families: a) Mycobacteriaceae, b) Actinomycetaceae, c) Actinoplanaceae and d) Streptomycetaceae. These families can be regarded as representing a series of stages in the morphological transition from unicellularity to the full mycelial state (Alexander, 1967). It has long been known that the Actinomycetes produce inhibitory substances to other microorganisms. Example of antibiotics produced by the members of Streptomycetaceae against gram negative and/or gram positive bacteria include: Streptomycin, Teteracycline, Chloroamphenicol, Oxytetracyline, Oxytetracycline Hydrochloride, Terramycin, Novobiocin, Auromycin and Ertheromycin. Antibiotics such as Cyclohexamide, Nystatin, Mycostatin, Nystan, Mycomycin, are produced by members of Streptomycetaceae and are active against different fungi (Felicitas and Hans, 1992).

1.2.1 Family Streptomycetaceae Decades ago when the members of this family were isolated they were classified morphologically with the fungi rather than with the bacteria (Felicitas and Hans, 1992; Alexander, 1967). Streptomycetes are considered a genus in the family Streptomycetaceae order Actinomycetales and class Schizomycetes. They were defined to be organisms producing an extensively branching non-fragmenting primary or substrate mycelium as well as a more or less abundant secondary or aerial mycelium (Felicitas and Hans, 1992). The mycelium is the only form of the vegetative development and reproduction takes place by the formation, at the tips of the hyphae, of special spherical or oval cells known as conidia (Zhao et al., 2005). The family of Streptomyceteceae is divided into two genera, Streptomyces and Micromonspora (Waksman, 1940).

1.2.2 Description of the genus Streptomyces This is the most recognized genus of the whole order of Actinomycetales because of its wide distribution in nature, especially in soil (Zhao et al., 2005). They have a characteristic earthy smell. Many species have been described, and from time to time, a novel Streptomycete is isolated (Waksman and Henrici, 1943; Felicitas and Hans, 1992; Meyers et al., 2003). Streptomyces species are strict aerobic, Gram positive filamentous bacteria, they produce a well developed mycelia. The hyphae vary greatly in length, some are long with limited branching, and others are short and much branched. The vegetative mycelium does not form cross walls; it does not break up into rod shape and coccus like bodies. They reproduce by means of

6 conidia which are produced in straight or spirally coiled chains, or by bits of mycelium. Conidia are formed in large number as to give a powdery appearance to the colonies on artificial media (Alexander, 1967). According to Prescott et al. (1993) Streptomyces are very flexible nutritionally and can generally hydrolyze one or more of the following: gelatin, casein and starch. In addition, Streptomyces can aerobically degrade resistant substances such as pectin, lignin, keratin and aromatic compounds. The optimum pH is 6.5-8.0 and the optimum temperature is in the range of 25-35˚C, although some species can grow at temperatures within the thermophilic range. The conidiospores, however, are slightly more temperature resistant than the vegetative cells of most bacteria; heating at 65˚C will kill them in 10-30 minutes (Locci, 1990). The growth of Streptomyces colonies on artificial media is smooth or lichnoid, hard and densely textured, raised and adhering to the medium. The colony is usually covered completely or partially in the form of spots or concentric rings by aerial mycelium, which may variously be pigmented depending on the species and the composition of the medium. In liquid media, especially in shaken cultures, growth of Streptomyces is usually in the form of flakes, which gradually fills the container, or in the form of spherical growth of puffballs (pullets). It is the former type of growth that is the most desirable one for antibiotic production. Most of the growth, either in the form of solid medium or in submerged cultures, may undergoes rapid autolysis. The production of antibiotic usually corresponds with the lysis of the culture. These organisms undergo in culture certain qualitative and quantitative variations. For example S. griseus, streptomycin-producing strain has yielded two types of variants, one which produced no aerial mycelium and no streptomycin, whereas the other one produced a red

7 pigmented vegetative mycelium and formed another antibiotic but never streptomycin. The qualitative differences of the variants are best expressed in the amount of the antibiotic produced, which may vary as much as hundred fold for the same species (Waksman and Lechevailer, 1953; Rong and Ming, 1995 and Zhao et al., 2006).

1.2.3 Isolation of Streptomyces from Soil Any soil habitat is characterized by a number of properties, which determine the population of microorganisms, quantitatively and qualitatively, present in it. These properties include: 1) vegetation and organic matter, 2) soil type, 3) season and climate, 4) temperature, 5) circulation of water and air, and 6) pH (Felicitas and Hans, 1992). The soil as a reservoir of Streptomycetes does not seem to be exhausted, and there is no doubt that sophisticated selection of microhabitats in soils already screened, as well as of extreme biotopes may lead to the discovery of a novel valuable Streptomycete (Enefiok and Charles, 1978). The procedure for the isolation of microorganisms from its habitat is influenced by the nature of the microorganism and the number of propagules relative to the other microbes within the habitat (Stolp and Starr, 1981). This is rarely the case with Streptomyces; isolation of them usually requires either enrichment and/ or use of selective media. The addition of CaCO3 to air- dried soil samples followed by incubation for 7-9 days at 26ºC compared with untreated control has lead to 100 fold increase of Streptomycete colonies on isolation plates (Taso et al., 1960. Nüesch (1965) observed that drying of the samples or prolonged storage at ambient temperature for mesophytes has resulted in a relative increase of the Streptomycete population. Heat treated soil at 40-50ºC for 2-16 hours reduced the bacterial

8 flora without affecting the colony count of Streptomycetes (Williams et al., 1972). Starch is degraded by most if not all Streptomycetes, the combination of starch with nitrate which is utilized by Streptomycetes as nitrogen source, is favorable for the isolation of these microorganisms (Flaig and Kutzner, 1960). Küster and Williams (1964), in developing a medium for the isolation of these organisms, they stated: “The three best media, allowing good development of Streptomycetes, while suppressing bacterial growth, were those containing starch or glycerol as a carbon source with casein, argnine or nitrate as a nitrogen source.

1.3 Antibiotics An antibiotic was originally defined as a substance, produced by one microorganism. The advent of synthetic methods has, however, resulted in a modification of this definition and an antibiotic now refers to as a substance produced by a microorganism, or to a similar substance (produced wholly or partly through chemical synthesis), which in low concentrations inhibits the growth of other microorganisms (O'Grady et al., 1997; Stephen et al., 2004). They generally fall into two classes, antibacterial antibiotics and antifungal antibiotics, though some of them may act as antibacterial as well as antifungal antibiotics. In contrast to the wide range of antibacterial antibiotics, there are very few antifungal antibiotics that can be used systematically. The lack of toxicity is, as always, of paramount importance, but the differences in structure of some biosynthetic process in fungal cells mean that antibacterial antibiotics are usually inactive against fungi (Arisawa et al., 1996; Stephen et al.,2004).

1.3.1 Classification of Antibiotics Several methods are used to classify antibiotics; the most common ones are as follows: 1.3.1.1 According to sources Generally the major sources from which antibiotics are obtained include: 1.3.1.1.1 Microorganisms: a) Streptomyces species: The majority of antibiotics used today are produced by different species of Streptomyces. Among these are Streptomycin, Oxytetracycline, Chlortetracycline, Tetracycline, Nystatin, Mycostatin and Mycomycin (Stephen et al., 2004) b) Other bacteria: These include antibiotics such as Bacitracin and Polymyxin, which are obtained from Bacillus species, Gentamycin, from Micromonspora and Monobactams from Pseudomonas acidophila and Gluconobacter species. c) Fungi: These include Griseofluvin, Penicillin and Cephalosporin from certain genera of Pencillium and Acermonium (Hugo and Russell., 1998).

1.3.1.1.2 Chemical Synthesis: Certain antibiotics are produced wholely through chemical synthetic processes under specific laboratory conditions, a good example is chloroamphenicol (Stephen et al., 2004).

10 1.3.1.1.3 Semi-Synthesis: Where part of the molecule is produced by microbial activity, such as fermentation process; then the product is modified by a chemical process under laboratory conditions (Hugo and Russell., 1998). 1.3.1.2 According to mode of action The classification of antibiotics has been based on the chemical structure and characterized or proposed mechanism of action. According to Sande and Mandell (1980), the action of an antibiotic may involve:

1.3.1.2.1 Inhibition of cell wall synthesis: This group of antibiotics inhibits the growth of bacteria by inhibiting the synthesis of the cell wall. This include β- lactam antibiotics, they get their common name from the characteristic β- lactam ring, which they share. All β-lactams work by interfering with the synthesis of the cell wall, through the inhibition of the enzyme needed for the synthesis of the peptidoglycan wall (Stephen et al., 2004). They have little effect on resting bacteria but are lethal to dividing bacteria as defective walls cannot protect the organisms from bursting in hypotonic surroundings. β-lactam group has three subdivisions: 1- Penicillins whose official names include or end in “cillin”. 2- Cephalosporins and cephamycins which are recognized by the inclusion of “cef” or “ceph” in their official names. 3- Other β-lactams include: a) Polypeptide Antibiotics (The polypeptide antibiotics comprise a rather diverse group. They include: i) Bacitracin; ii) Polymyxins and iii) Antitubercular antibiotics; b) Miscellaneous antibacterial antibiotics (These antibiotics cannot be considered in any of the other groups mentioned, they include: Chloramphenicol, Fusidic acid and

11 Mupirocin.) and c) Glycopeptide Antibiotics (There are two important Glycopeptide antibiotics,Vancomycin and Tecioplanin.).

1.3.1.2.2 Inhibition of protein synthesis: Most of these antibiotics are produced wholely or partially by Streptomyces species. They inhibit bacterial protein synthesis through different mechanisms: 1) binding to the 30S subunit of the bacterial ribosome and this interfere with the formation of the initiation complex or cause misreading of the mRNA, 2) prevent the transfer of the activated amino acids to the ribosome so protein synthesis is stopped, 3) block the elongation of the peptide chain. Protein synthesis inhibitors include: 1- The Aminoglycosides: The name of each of these ends in “mycin”, indicating that it is derived from Streptomyces. They contain amino sugars in their structure. Examples are; Neomycin, Amikacin, Gentamicin, Kanamycin, Framycetin and Streptomycin. 2- Tetracyclines as the name suggestes are four ringed structures and their names end in “cycline”. They consist of a group of antibiotics obtained as by-products from the metabolic activities of some species of Streptomyces. They include many important compounds such as Tetracyclines and Glycocyclines. 3- Macrolides. Those are characterized by possessing molecular structures that contain large lactone rings linked through glycosidic bonds with aminosugars. The most important members of this group are Erthyromycin. Oleandomycin, Triacetyloleandomycin and Spiramycin. 4- Other drugs which act by inhibiting protein synthesis include: a) Lincosamides, b) Streptogramins, those are Pristinamycin, Virginiamycin, Quinupristin and Dalfopristin.

12 1.3.1.2.3 Inhibition of Nucleic acid synthesis This group include the following: 1. Sulphanoides. Usually their names started with “sulpha”. These drugs contain trimethoprim which combines with the mRNA and hence inhibits the synthesis of nucleic acid precursors. 2. Quinolones, these are structurally related to nalidixic acid; the names of the most recently introduced members of this group end with “oxacin”. They act by preventing DNA replication. 3. Azoles, they contain an azole ring thus their names end in “azole”, e.g. metronidazole. They act by the production of short-lived intermediate compounds which are toxic to DNA (Arisawa et al., 1996). 4. Others include: Rifamycin which, is active against some Gram positive bacteria and some Gram negative bacteria (Stephen et al., 2004).

1.3.1.2.4 Distruption of fungal cell membrane This group includes azoles (imidzoles) and allyamine. They are characterized by possessing a large ring containing a lactone group and a hydrophobic region, examples are Nystatin and Amphotericin. They derive their action from their strong affinity towards sterols, particularly ergosterol. The hydrophobic polyens region binds to the hydrophobic sterol ring system within the fungal membranes. In so doing, the hydroxylated portion of the polyene is pulled into the membrane interior, thus destabilizing the structure and causing leakage of cytoplasmic content (Mahmoud and Louis, 1999).

1.3.1.2.5 Inhibition of fungal mitosis This group include Griseofulvin, Fulvicin, and Grisactin which is metabolic by-product of Penicillum griseofulvum, and is active against the

13 dermatophytes but is ineffective against Candida albicans (Hugo and Russell, 1998). Griseofulvin is insoluble in water. It is effective after oral ingestion and reaches the skin and hair. It is deposited primarily in keratin precursor cells. Ingestion with a heavy meal and reduction in particle size enhances the absorption of griseofulvin. It inhibits fungal mitosis by disrupting the mitotic spindle through interaction with polymerized microtubules (Mahmoud and Louis, 1999).

1.4 Antibiotics production and recovery Large scale manufacturing of the majority of antibiotics is through fermentation, based on the activities of microorganisms, which convert raw materials into antibiotics (Stephen et al., 2004). Fermentation is biological process occurring in the absence of air. However, the term is now commonly applied to any large-scale cultivation of microorganisms, whether aerobic or anaerobic. There are several techniques which are employed in the production of antibiotics from microorganisms. The first method is the surface culture in whish the producing microorganism is grown on the surface of liquid culture medium contained in small vessels (Atlas, 1989). The second method is the deep submerged culture, in which the producer organism is grown in deep aerated fermentation tanks and conditions are adjusted to enable mass growth and production deep within small or large volume media (Prescott et at., 1993). Alternatively, solid-state fermentation technique is preferably employed for antibiotic production using cheap solid substrates. In this respect, Yang and Ling (1989) reported the production of tetracycline by solid-state fermentation using sweet potato whereas Yang and Swie (1996) used corncob as a solid substrate for the production of oxytetracycline.

14 The science of antibiotic fermentation is still imperfectly developed, because it is accomplished with a living microorganism population that is changing, both quantitatively and qualitatively. Throughout the production cycle optimization of the production is difficult, because no two identical batches are ever wholly alike. Dealing with the challenge of this variation is one of the attractions for those who practice in this field (Hugo and Russell 1998; Stephen et al., 2004). Following production by fermentation, antibiotics are recovered, concentrated and purified by a series of down stream processing stages. Extraction of an antibiotic begins with the removal of cells, using several techniques such as filtration, ultrafiltration or centrifugation. Isolation of the antibiotic could be achieved using solvent systems and/ or other available methods including thin layer chromatography, column chromatpgrapy, ion- exchange chromatography, high performance liquid chromatography and precipitation by the addition of an alkali or an acid (El-Nagger, et al., 2001).

15 Chapter Two Materials and Methods

2.1 Isolation of Streptomyces species from soil samples A total of 40 soil samples were collected from different states in Sudan. These were: Khartoum (Geraif West, Blue Nile bank, El-Sunut Forest, Shambat and Tuti), Gedarif State (3 locations in agricultural areas) and Tangasi (Northern State). The samples were collected in sterile plastic bags and then transferred to labeled screw-capped bottles after air- drying for a week. For oven drying one gram of calcium carbonate was added to each 10 gram of the air-dried soil sample, the mixture was then dried in an oven at 50°C for 6-10 hours. For the isolation of Streptomyces one gram of the dried mixture was suspended in 10 ml of sterilized distilled water. Serial of dilution was made by the addition of nine ml of sterile distilled water to one ml of the soil suspension in test tubes. Dilution process was continued until a 10-4 dilution was obtained. One ml from this dilution was transferred and plated on Starch Casein KNO3 Agar (SCKNO3A) medium. The

SCKNO3 agar medium was prepared by dissolving: starch, 10g; KNO3, 2.0g;

KH2PO4, 2.0g; NaCl, 2.0; casein, 0.3; MgSO4.7H2O, 0.05; CaCO3,, 2.0;

FeSO4.7H2O, 0.001g and 18g agar in one liter of distilled water. The medium was then divided equally into four 500 ml Erlenmeyer flasks and was then plugged tightly with cotton and autoclaved for 15 minutes at 121°C and 15 1bs/square inch. The inoculated petri-dishes were incubated at 28° C for 5-7 days. Colonies, with Streptomyces morphological characteristics that appeared in the incubated plates, were repeatedly sub-cultured for purification.

16 2.2 Preservation and maintenance of Streptomyces isolates

Streptomyces isolates were maintained in Glycerol Aspargine Agar (GAA) at universal bottles (50 ml). The GAA was prepared by dissolving, agar, 20.0g; glycerol, 10.0g; aspargine, 1.0g; and 1ml of trace elements solution. The trace elements solution is prepared by dissolving g/100 ml of distilled water: FeSO4. 7H2O, 0.1; MnCl2 4H2O, 0.1 and ZnSO4. 7H2O,0.1 in one liter of distilled water in a two liter Erlenmeyer flask (Atlas, 1997). Then slants were prepared by dispensing 6 ml of the GAA medium in test tubes. After plugging with cotton the test tubes were sterilized as described above, left to cool in slanted way and each 4 of them were inoculated with one of the isolates. Each isolate was given a number pre-fixed with Dee. A total of fifty isolates were obtained and were then screened for their ability to produce antibiotics.

2.3 Screening for Antibiotic production: In vitro antibiotic activity of the Streptomyces isolates was tested against some pathogenic fungi. The tested microorganisms include Candida albicans, Aspergillus niger, Aspergillus flavus, Cryptococcus humicolus, Penicillium species and Saccharomyces species. All tested microorganisms were obtained from the National Health Laboratory, Ministry of health, Sudan. Streptomycete Antibiotic Activity medium (Soybean Meal Agar (SAA) was prepared by adding Agar, 15g; D-glucose, 15g; soybean meal,

15g; NaCl 5g; yeast extract, 1g; CaCO3, 1g and glycerol, 2.5 ml to one liter of distilled water. The medium was autoclaved and poured into Petri-dishes as described before. The medium was then inoculated by making a single

17 streak of each Streptomyces isolate in the center of the plate. The plates were then inoculated at 28ºC for 72 hours. Each plate was then inoculated with different pathogenic fungi species at a 90° angle to the Streptomyces streak. The plates were re-incubated for 48, 72, and 96 hours. The inhibition zone made by the Streptomyces isolate against each pathogen indicated the production of an antibiotic.

2.4 Characterization of Streptomcyes Isolates Based on the results of the screening experiment, only sixteen isolates were selected for further studies. Following the direction given by the International Streptomyces Project (ISP) and the selected isolates were characterized by studying some of their cultural, morphological and biochemical characteristics.

2.4.1 Cultural characteristics

The selected isolates were cultured on SCKNO3 agar plates (prepared as in section 2.1). Colony characteristics such as color, shape, elevation, margin, consistency and transparency were determined visually or by placing the Petri- dish, containing the pure individual colonies, under Stereomicroscope .The descriptive terminologies followed were those suggested by Cross, 1989 and Prescott, et al. (1993).

2.4.2 Microscopic characteristics The following microscopic tests were performed on each of the selected isolates.

18 2.4.2.1 Gram staining

Gram staining of 24–hour old SCKNO3 Agar cultures was performed as described by Collins et al. (1995). A loopful of bacterial colony from the slanted stock culture was transferred and mixed with a drop of sterilized distilled water at the center of a clean glass slide .The resulting suspension was spread with sterilized loop to obtain a thin film and was left to air – dry. It was then fixed by passing it over a flame 5- 7 times .The smear was flooded with Crystalviolet-ammonium oxalate complex, prepared by dissolving 2 g of crystal violet in 20 ml of 95% ethanol and 0.8 g of ammonium oxalate in 80 ml of distilled water .The two solutions were mixed and left to stand for 24 hours before they were filtered for one minute. The excess dye, on the smear, was washed off with gently running tap water .The smear was then covered with Gram’s iodine solution (prepared by dissolving 2 g of potassium iodide and 1 g of iodine in 300 ml of distilled water) for 1 minute and then washed under running tap water .The smear was decolorized with 95 % ethanol until no more stain comes out. It was then, washed with tap water and counter stained with safranine dye (prepared by adding 0.25 g of safranine to 10 ml of 95% ethanol then made up to 100 ml with distilled water) for 30 seconds. Excess safranine was washed off with tap water, and the smear blotted dry and finally examined microscopically under oil immersion. Cells which were violet in colour were recorded as Gram –positive, those which were red were recorded as Gram – negative.

19 2.4.2.2 Mycelial morphology The same preparations used for the determination of gram reaction were employed for the determination of the presence or absence of aerial mycelium and conidia and the shape of cells.

2.4.2.3 Acid fast Staining A loopfull from Streptomyces colony was transferred and mixed with a drop of distilled water on the center of a glass slide. The resulting suspension was spread to obtain a thin film and left to dry. It was fixed by passing the slide over the flame 3-5 times. The smear was then flooded with carbol-fuchsin for 5 minutes and heated until steam rised. The wet smear was then washed with tap water and decolorized with acid alcohol for 20 seconds. Washed with tap water and counterstained, for one minute, with methylene blue. Excess methylene blue was washed off with tap water and the smear was dried and examined microscopically under oil immersion lens for acid fast reaction (Collins et al., 1995).

2.4 Biochemical tests 2.5.1 Catalase test:

24 hours old colonies grown on SCKNO3 Agar were examined for catalase production by adding a drop of 3 % hydrogen peroxide to the colony. Appearance of gas bubbles within 10 seconds was considered as a positive result (Prescott et al., 1993).

2.5.2 Oxidase test: Two ml of aqueous solution of methylphenylenediamine dihydrochloride (10 % w/v) were added to a filter paper in sterile Petri –

20 dish. Using a platinum loop, a heavy amount of growth from a 24 – hours old Streptomyces culture, grown on nutrient agar was, added to the filter paper and quickly spread. Development of a purple color within 10 seconds was recorded as a positive reaction for the presence of cytochrome oxidase (Collee et al., 1993).

2.5.3 Aerobiosis The test for aerobiosis was carried out using 10 ml portions of sterilized Thioglycolate Broth (29.8 g in 1000 ml. distilled water) in a set of test tubes. Each of the broth media was inoculated with a loopfull of a bacterial colony. The test tubes were then left undisturbed for 2 days. Aerobiosis was indicated either as aerobic, microaerophilic, anaerobic or facultative anaerobic by observing the growth and specifying whether it occurred at the surface, sub-surface, bottom, or throughout the broth respectively (Collin et al., 1995)

2.5.4 Gelatin liquefaction Nutrient Gelatin (38.4 g) was dissolved in 300 ml of distilled water, and distributed into bijou bottles (4ml each) and autoclaved. Each bottle was then inoculated with each of the Streptomyces isolates and incubated at 30 oC for 7 days. The bottles were placed in the refrigerator for an overnight. Gelatin liquefaction was determined according to the degree of gelatin viscosity (Collins et al., 1995).

2.5.5 Starch hydrolysis Sterilized starch agar plates (prepared by adding 10 ml of 10 % aqueous solution of soluble starch to 100 ml of melted nutrient agar 1) were

21 inoculated each with one of the isolates .The inoculated plates were incubated for 2- 4 days and were then flooded with iodine solution. Hydrolysis is indicated by clear zones around the growth (Collee et al., 1993).

2.5.6 Utilization of different carbon sources The ability of an isolate to utilize a certain carbohydrate as sole carbon source was determined in a modified Starch Casein- KNO3 Agar medium (Atlas, 1997). The medium contained (g/l) KNO3, 2.0 ; K2HPO4

,2.0; NaCI, 2.0; casein, 0.3; MgSO4. 7H2O, 0.05; CaCO3, 0.02; FeSO4,

7H2O, 0.001 and 18, agar. For the test, 10g of each of the carbohydrates under study were added to the medium. The carbohydrates used were: glucose, fructose, rhaminose, lactose, maltose, sucrose, galactose, arabinose, mannitol and salicin. The medium was sterilized, poured in Petri-dishes and incubated for 24 hours before use. Clean uncontaminated plates were inoculated each with one Streptomyces isolate and incubated at 28oC. The plates were examined for the presence of growth after 7, 14 and 21 days (Oskay et al., 2004).

2.5.7 Urease test Urea medium was prepared by mixing peptone, 1.0 g; dextrose, 1.0 g; disodium phosphate, 1.2 g; potassium dihydrogen phosphate, 0.8 g; sodium chloride, 5 g and phenol red, 0.012 g. Of this mixture, 0.9 gram was dissolved in 95 ml of distilled water, in 250 ml conical flasks .The medium was sterilized by autoclaving as described before. After cooling to 55 oC, 5 ml of 40 % filter – sterilized aqueous solution of urea were added. This medium was then divided aseptically into 15 ml lots in sterilized glass vials.

22 Each vial was then heavily inoculated with one of the isolates and incubated at 35 oC. The gradual change of the medium color from yellow to pink was considered as a positive test for urease activity (Collins et al., 1995).

2.5.8 Organic acid formation According to the international Streptomycete Project (ISP) organic acid medium consisted of three solutions: A, B and C. Solution A was prepared by dissolving glucose, 20g; agar, 4g; yeast extract, 1.2g; MgSO4.7H2O, 0.25g; bromo-cresol, 0.012g into 400 ml distilled water. Solution B was prepared by dissolving disodium hydrogen phosphate

(2H2O), 0.534g and KH2PO4, 0.272g into 500 ml of distilled water. Solution C was prepared by dissolving 1.0g calcium carbonate in 100 ml of distilled water. Each of solution A and B were first autoclaved separately and then mixed. Solution C was distributed into test tubes each containing 0.2 ml and autoclaved. Of the mixture of A and B, 1.8 ml was aseptically added to solution C in the test tubes. The content of the tube was inoculated with each of the Streptomyces isolates and incubated at 28°C for 7 days. Acid formation was indicated by the change of the medium color to yellow within the incubation period (Atlas, 1997).

2.5.9 Milk coagulation and peptonization test The solution for milk coagulation and peptonization test was prepared by dissolving 20 g of skimmed milk in 100 ml of distilled water. It was then distributed in test tubes containing 5 ml each. The milk was then autoclaved and inoculated. The appearance of brown color was recorded as positive

23 peptonization reaction whereas clotting of the milk was considered as positive for coagulation reaction (Atlas, 1997).

2.5.10 Casein hydrolysete The solution for casein hydrolysete test was prepared by dissolving 10 g of skimmed milk in 100 ml distilled water. Simultaneously 2g of agar were dissolved in 100 ml of distilled water. The two solutions were then autoclaved and mixed together and were poured in sterile Petri-dishes. The medium was left for 24 hours and then inoculated with isolates and incubated at 28° C for 3 days. The appearance of clear zones around each colony indicated a positive reaction of Casein hydrolystes (Atlas, 1997).

2.5.11 Blood hemolysis Nutrient agar was prepared and Sterilized by autoclaving at 121°C 151bs/inch2 for 15 min. After cooling, 5 ml of human blood were added and mixed with 250 ml of nutrient agar. The blood agar was poured in sterile Petri-dishes, inoculated with Streptomyces isolates and incubated at 28° C for 3 days. Complete blood hemolysis was indicated as alpha hemolysis and partial blood hemolysis was indicated as beta hemolysis (Collins et al., 1995).

2.5.12 Nitrate reductase Streptomycete medium was prepared by adding glycerol, 5g; agar, 4g;

NaCl, 2g; KNO3,1g; Na2HPO4.7H2O, 0.534g; KH2PO4, 0.272g;

MgSO4.7H2O, 0.5g and 1 ml of trace element solution to one liter of distilled water. The trace element solution was prepared by dissolving (mg/100ml)

FeSO4.7H2O, 0.1; MnCl2.4H2O, 0.1 and ZnSO4.7H2O, 0.1 in distilled water.

24 One ml of the Streptomycete medium was distributed each into a test tube and then autoclaved as described before. After 24 hours the tubes were inoculated with the Streptomyces isolates and incubated for 5 days, after which Griess-Iiosvay reagent was added to the culture. The change in color to pink indicated a positive reaction for the nitrate reductase (Atlas, 1997).

2.5.13 H2S production Production of Hydrogen sulphide was tested using Kligler Iron Agar (KIA) medium, which was prepared by dissolving 8.62g of KIA medium in 150 ml of distilled water. The medium was then divided into tubes (3ml each) and autoclaved. The tubes were allowed to solidify in a slanting way and were then heavily inoculated with the Streptomyces isolates. Appearance of a black color on the medium indicated a positive H2S production activity of the isolate (Collins et al., 1995).

2.6 Antibiotic production by Streptomyces in submerged cultivation Streptomyces isolates, which expressed activity against any of the test microorganisms, were inoculated in submerged culture to test their abilities to produce inhibitory substances in liquid medium. Mineral Yeast Extract (MYE) broth was used as a medium for production under submerged conditions.

2.6.1 Media preparation

MYE medium was prepared by dissolving (g/liter) NaCl, 0.8; NH4

CL, 1.0; KCL, 0.1; KH2 PO4, 0.1; MgSO4 .7H2 O, 0.2; CaCl2. 2H2O, 0.04; glucose, 2.0 and yeast extract, 3 in distilled water. The final pH value of the medium was adjusted to 7.3 with 0.1 N HCL or 0.1 N NaOH using a pH

25 meter. The medium was divided in 500 ml Erlenmeyer flasks each containing 100 ml of MYE medium.

2.6.2 Streptomyces inoculum and inoculation Streptomyces isolates were incubated at 28ºC for 14 days on a slant containing (gL-1) soluble starch 10; yeast extract 1; beef extract 1; tryptone 2; hydrated ferrous sulphate 0.1; and agar 20 at pH 7.3. Streptomyces spores were harvested with a tween 80 solution (5ml, 0.5% v/v). the spore concentration was diluted with sterilized water to give approximately suspension 107 to 108 spores per ml (Yang and Wang, 1999). Two ml of this Streptomyces spore suspension were used to inoculate 25ml of sterilized Mineral Yeast Extract broth in 500 ml cotton plugged Erlenmeyer flasks. The flasks were incubated at 28ºC on a rotary shaker operating at 180 rpm for 168 hours (Sahin and Ugur, 2003).

2.6.3 Extraction of the fermentation broth After eight days of incubation, the fermentation broth was collected and centrifuged at 10,000 rpm for 20 minutes and the supernatant was separated from the mycelial cake. The supernatant was then extracted with n- butanol (Augstine et al., 2005a). One volume of broth was extracted in two and half volume of n- butanol (1:2.5 v/v supernatant: n-butanol). The mycelium cake was extracted with methanol overnight, methanol was evaporated and the watery residue was then extracted twice using n-butanol as previously described (Pullen et al., 2002). The n-butanol extracts of the culture filtrate and mycelium cake were combined in one glass vial.

26 2.6.4 Testing of extracts for antifungal activity:- The cup-plate agar diffusion method (Kavanagh, 1972) was adopted, with some minor modifications, to assess the antifungal activity of the prepared extracts. One ml of the standardized fungal stock suspension was thoroughly mixed with 100ml of sterile molten Mueller-Hinton agar which was maintained at 45◦C. Twenty ml aliquots of the inoculated Mueller- Hinton agar were distributed into sterile Petri-dishes. The agar was left to set, and in each of these plates, two cups (10cm in diameter) were cut using a sterile cork borer (No.4) and the agar discs were removed. Alternate cups were filled with 0.1 ml samples of each of the extracts using standard Finn pipette adjustable volume digital pipette, and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position, at 28◦C for 72 hours. Four replicates were carried out for each extract against each of the test organisms. Simultaneously, controls, involving the addition of the solvent instead of the extracts, were included. After incubation, the diameter of the resultant growth inhibition zones were measured and recorded.

27 Chapter three Results and Discussion

Actinomycetes have been recognized as potential producers of metabolites such as antibiotics, growth promoting substances for plants and animals, immunomodifiers, enzyme inhibitors and many other compounds of use for man. They have provided about two thirds of the naturally occurring antibiotics discovered; including many of those which are important in medicine, such as aminoglycosides, anthracyclines, chloramphenicol, tetracyclines (Gupta et al., 2002). Fungi are eukaryotic and have machinery for protein and nucleic acid synthesis similar to that of higher animals. It is therefore, very difficult to find compounds that selectively inhibit fungal metabolism without toxicity to humans. Fungal infections have been gaining prime importance because of the morbidity of the hospitalized patients (Beck-Sague and Jarvis, 1993; Gupta et al., 2002). Although synthetic drugs contribute a major proportion of the antifungal used, natural antifungals have their own place in the antimycotic market (Augustine et al., 2005a).The genus Streptomyces is a genus of bacteria marked by several distinguishing features. They are the source of the majority of antibiotics applied in human, veterinary medicine, agriculture as pesticides, and in food industry as in the case of several important enzymes.

28 3.1 Isolation and screening of Streptomyces: Species of Streptomyces were reported to occur predominantly in soil. In the present study 50 isolates were recovered from samples of soil collected from different locations in Sudan. Each isolate was given a number prefixed with the abbreviation Dee (Table 1). Fifteen isolates were recovered from soils collected from EL-Gedarif; thirty were recovered from different locations in Khartoum State, 11 from the Bank of the Blue Nile (opposite to the University of Khartoum), 5 from Tuti Island, 4 from White Nile (ELSunt Forest), 5 isolates from Shambat, 5 isolates from Al-Geraif west; and 5 isolates from Tangsi, Northern State. All the isolates were found to be Streptomyces depending on their distinct morphological and microscopical characteristics (Plate 1). Isolation was done on Starch Casein Potassium Nitrate Agar which is a semi-selective medium for Streptomyces (O’Grady et al., 1997). Moreover, isolation was enhanced by the addition of Calcium Carbonate to the soil samples (to reduce the number of fungal spores) and heating at 40˚C for 6-10 hours in order to decrease the number of non- filamentous bacteria (Felicitas and Hans and Hans, 1992). The obtained isolates were screened for their abilities to produce inhibitory metabolites against certain pathogenic fungi. Screening was performed by the streak method (plate 2) the inhibition zone was measured in millimeter for each isolate; results shown in Table 2, indicated that only 30% (16 isolates) of the tested isolates have potent in vitro antifungal activities against one or two of the tested fungi. Of these isolates, 60% have strong activities with growth inhibition zones ranging between 12- 50 mm. A very high inhibitory activity against Candida albicans was expressed by isolate Dee 16 (50 mm); Dee 26 (45 mm); Dee 2, Dee 18 and Dee 33 (each

29 Table 1: Soil origin of Streptomyces presumptive Isolates

ISOLATE SOURCE OF SOIL SAMPLE STATE Dee1 Dee2 Dee3 Dee4 Dee5 Dee6 Dee7 Dee8 GEDARIF GADARIF STATE Dee9 Dee10 Dee11 Dee12 Dee13 Dee14 Dee15 Dee16 Dee17 Dee18 Dee19 Dee20 Dee21 BLUE NILE BANK Dee22 Dee23 Dee24 Dee25 Dee26 Dee27 Dee29 ELSUNT FOREST Dee30 Dee31 KHARTOUM STATE Dee32 Dee33 TUTI ISLAND Dee34 Dee35 Dee36 Dee37 Dee38 SHAMBAT Dee39 Dee40 Dee41 Dee42 Dee43 GERAIF WEST Dee44 Dee45 Dee46 Dee47 Dee48 TANGASI NORTHERN STATE Dee49 Dee50

DEE3

DEE12

DEE17

plate 1a: morphological features of different Streptomyces isolates grown on Starch Casein potassium Nitrate Agar

Plate 1b continued: filamentous mycelia of different Streptomyces isolates

A- Dee20

B-Dee 28

Plate 2 (A& B) showing the inhibition of Streptomyces isolates Dee 20 and Dee28 to the growth of a. Aspergillus niger b. Candida albians c. Fusarium oxysporium

33 Table 2 Inhibition zones diameters (mm) shown by different Streptomyces isolates on the growth of seven microorganisms

34 40 mm); Dee10 (34 mm); Dee 4 (31 mm) and Dee 3 (30 mm). The highest inhibition zones against Aspergillus niger were recorded by Dee 18 (30 mm); Dee 26 (28 mm) and Dee 17 (22mm). On the other hand, the highest activities against Cryptococcus humicolus (24 mm) and Aspergillus flavus (23 mm) were expressed by Dee 15. Most of the isolates showed narrow inhibitory spectra since they inhibited the growth of one or two of the test pathogens. However, none of the isolates have shown activity against Saccharomyces sp., Pencillium sp. and Fusarium oxysporium. Since the inhibition process is a function of both Streptomyces isolate and the pathogen, inhibition in the growth could be affected by the sensitivity of the tested microorganism, which might not be sensitive to the inhibitory substance. Low concentrations of the inhibitory substance could give negative results as well, but it may also develop resistance against the antifungal produced by Streptomyces isolate. Arikan et al. (1999) reported that several antifungal agents has higher Minimum Inhibitory Concentrations (MICs) with Fusarium spp. when compared with other Asperigllus spp., they due that to the slow growth of this species. The 16 isolates which proved to have good potential to inhibit the growth of fungi were selected and characterized. Arikan et al., (1999) reported that Fusarium spp. requires higher concentrations of antifungal agents when compared with Aspergillus spp. They due this to the slow growth rate of Fusarium spp. which lead an increase in the Minimum Inhibitory concentrations needed. The 16 isolates which proved to have good potential to inhibit the growth of fungi were selected and characterized.

3.2 Characterization of Streptomyces isolates

35 Characterization of the obtained Streptomyces isolates was carried out according to the directions given by the International Streptomyces Project (Felicitas and Hans, 1992; Gottlieb and Shirling, 1967 and Cross 1989) as follows:-

3.2.1 Cultural characteristics Results of cultural characteristics are shown in table 3. After three days of incubation all of the isolate colonies were opaque and dry. Two different edge shapes were observed; eleven of the isolates expressed a filamentous margin while five expressed a smooth margin. The colonies shape varies from filamentous (11 isolates) to small round (5 isolates), all the isolates had smooth leathery surfaces, which later were developed to give a powdery appearance. After 5-7 days, scattered very fine droplets that have watery appearance were sometimes observed on the top of the mycelium. Most of the isolates where white at the beginning of the growth (2-3 days) but later they produced a variety of pigments that colored the vegetative mycelia and spores (plate 3). The colors observed were: white (2 isolates); black (one isolate); red (one isolate); gray (3 isolates); gray with white margin (2 isolates); black and white (one isolate); cream (one isolate) and greenish blue (one isolate). All the isolates that expressed a filamentous shape were found to have flat elevation while the round-shaped colonies were raised. The cultural characteristics were confirmed by the growth on Starch Casein Agar medium and Glycerol Arginine Agar medium. The results obtained are in agreement with the description of Rong and Ming (1995); Felicitas and Hans (1992) and Lacey (1973). Based on the results obtained all the isolates were therefore, considered as typical Streptomycetes.

36 Table 3: cultural characteristics of Streptomyces isolates

Isolate Shape Chromogensis Edge Opacity Elevation Surface consistency

Dee2 Filamentous Green Filamentous opaque Flat Powdery Dry

Dee3 Filamentous White and Black Filamentous opaque Flat Powdery Dry

Gray with White Dee4 Filamentous Filamentous opaque Flat Powdery Dry margin

Dee6 Filamentous Cream Filamentous opaque Flat Powdery Dry

Dee10 Round Greenish blue Smooth opaque raised Powdery Dry

Dee12 Filamentous Black Filamentous opaque Flat Powdery Dry

Dee15 Round Red Smooth opaque Raised Powdery Dry

Dee16 Round Gray Smooth opaque Flat Powdery Dry

Gray with White Dee17 Filamentous Filamentous opaque Flat Powdery Dry margin

Dee18 Filamentous Gray Filamentous opaque Flat Powdery Dry

Dee20 Filamentous Gray Filamentous opaque Flat Powdery Dry

Green with Dee26 Round Smooth opaque raised Powdery Dry White margin

Dee30 Filamentous White Filamentous opaque Flat Powdery Dry

Green with Dee33 Filamentous Filamentous opaque Flat Powdery Dry White margin

Dee41 Filamentous Green Filamentous opaque Flat Powdery Dry

Dee45 Round White Smooth opaque raised Powdery Dry

DEE3 DEE6

DEE6 DEE15

DEE2

DEE12

DEE10 DEE33

DEE26 DEE16

Plate 2: morphological characteristics of some Streptomyces isolates

38 3.2.2 Microscopical Characteristics Results of microscopical examination are shown in Table 4 and plate 4. All of the tested isolates were Gram positive, Acid fast negative and filamentous. Acid fast staining differentiate between Streptomyces and Micromonospora, the latter is Acid fast positive (Felicitas and Hans, 1992). The vegetative hyphae were branched and were non-fragmented. The non- fragmentation of the mycelium is generally accepted as a diagnostic characteristic of the Streptomyces (Goodfellow et al. 1998). At maturity, the aerial mycelia of all isolates could be differentiated into straight or long spiral chains of cylindrical immotile spores with smooth, warty or hairy appearance. Similar results were reported by Wendisch and Kutzner (1991).

3.2.3 Biochemical characteristics Streptomyces is one of the best recognized genera of the whole order of Actinomycetales because of its wide distribution in nature, especially in soils. Many bacterial genera including Streptomcyes are not only morphologically and microscopically identical but yielded colonies which are not clearly distinguishable. The biochemical activities of such pure cultures frequently allow genera and species characterization and identification (Lacey, 1973; Gillies, 1984). Moreover, the selection of a range of biochemical tests to be used depends upon the diversity and nature of the group of bacteria understudy. Biochemical characterization of the isolates recovered in this study involved 12 diagnostic characters that are recommended by International Streptomycete Project (ISP), and were successfully utilized by various

39 Table 4 Microsopical characteristics of 16 Streptomyces isolates

Isolate Gram Acid-fast Motility Aerial Conidia Stain Stain mycelium

Dee2 + ve - ve - ve Present present

Dee3 + ve - ve - ve Present present

Dee4 + ve - ve - ve Present present

Dee6 + ve - ve - ve Present present

Dee10 + ve - ve - ve Present present

Dee12 + ve - ve - ve Present present

Dee15 + ve - ve - ve Present present

Dee16 + ve - ve - ve Present present

Dee17 + ve - ve - ve Present present

Dee18 + ve - ve - ve Present present

Dee20 + ve - ve - ve Present present

Dee26 + ve - ve - ve Present present

Dee30 + ve - ve - ve Present present

Dee33 + ve - ve - ve Present present

Dee41 + ve - ve - ve Present present

Dee45 + ve - ve - ve Present present

b Plate 4: Microscopical characteristics of Streptomyces isolate showing the mycelia and spiral chains of conidia a- gram stain positive b- acid fast stain negative

41 investigators in the field (Anderson and Wellington, 2001; Oskay et al., 2004).

All isolates were found to be aerobic, catalase, oxidase, urease, protease and nitrate reductase have expressed positive result (Table 5). These results are inconsistent with the results obtained by Oskay et al. (2004) and Anderson and Willington (2001). Starch hydrolysis was observed by twelve of the Streptomyces isolates while the left four isolates do not have the ability to hydrolyze starch. Although it is generally considered that amylase and protease are mainly fungal and eubacterial products, the results of starch hydrolysis and protease expression (plate 4) expressed by the Streptomyces isolates have indicated the ability of Streptomyces to produce amylase and protease. Yang and Wang (1999) reported the production of amylase and protease enzymes by different species of Streptomyces. Only Dee 2, 3, 10, 15, 30 and 45 had the ability to produce Hydrogen Sulfide in Kligler Iron Agar medium (plate 4) while the rest of the isolates express negative results with the KIA medium. Moreover, gelatin liquefaction showed similar variation since only eight have the capacity to liquefy gelatin whereas the other eight did not. Out of the sixteen isolates eleven have the capacity to peptonize milk and eight were able to coagulate it. All the isolates were able to hemolyse blood either complete hemolysis (β hemolysis) or partially (γ hemolysis) and only ten isolates can produce melanin. Less variation was obtained with the organic acid formation test since thirteen isolates had the ability to produce organic acid and only three could not. As for the utilization of different carbohydrates as sole carbon source, all of the isolates were able to utilize all of the carbohydrates under

42 table 5 biochemical characteristics

Protease: clear zone indicates positive results

Starch hydrolysis: clear zone indicates positive results

Plate 4: results of biochemical tests

Negative positive

A: nitrate reduction

Negative positive

(B) Organic acid formation

Plate 4: biochemical tests (continued)

A-Blood haemolysis: clear zones indicates positive results

Negative positive B-Urease test

Plate 4 (continued)

46 test (Table 6). This is not surprising, because it is generally reported that Streptomyces spp. possess several metabolic pathways that are supported by large-sized genomes (Ōmura et al., 2001). This provides insights into the intrinsic abilities of Streptomyces to utilize different carbohydrate sources.

3.3 Production of antibiotics in submerged culture by Streptomyces isolates Isolates that showed activity against any of the tested microorganisms were grown in 500 ml flasks containing 100 ml of liquid Mineral Yeast Extract Medium (MYE). Fermentation broths were extracted with n-butanol. The extracts were bioassayed for their antimicrobial activities. The inhibition zones, exhibited against the tested fungi are shown in Table 7 and plate 5. Table 7 shows that the n-butanol extracts of all selected Streptomcyes isolates have shown activity against one or more of the tested organisms. However, the activities were strong against the following fungi: Candida albicans, Aspergillus niger, Aspergillus flavus and Cryptococcus humicolus. The greatest zone of inhibition (45mm) was exhibited by Dee15 against C. Albicans. This was followed by Dee 12 (30mm), Dee 10 and Dee 16 (20mm) and Dee 18 (15mm). For Aspergillus niger the greatest inhibition zone was 24 mm which was expressed by Dee 15, 20 mm by Dee12 and 15mm by Dee 33. The greatest inhibition zone exhibited against Aspergillus flavus was 14 mm by the isolate Dee 15. From table 7, most of the extracts have higher activities against yeast (Candida) and Aspergillus than the other tested fungi. Similar results were observed by Augustine et al. (2005a)

47 Table 6: Carbohhydrate utilization by different Streptomyces isolates

Isolate Arabinose Rhaminose Glucose Galactose Fructose Sucrose Maltose Lactose Manitol

Dee2 + + + + + + + + +

Dee3 + + + + + + + + +

Dee4 + + + + + + + + +

Dee6 + + + + + + + + +

Dee10 + + + + + + + + +

Dee12 + + + + + + + + +

Dee15 + + + + + + + + +

Dee16 + + + + + + + + +

Dee17 + + + + + + + + +

Dee18 + + + + + + + + +

Dee20 + + + + + + + + +

Dee26 + + + + + + + + +

Dee30 + + + + + + + + +

Dee33 + + + + + + + + +

Dee41 + + + + + + + + +

Dee45 + + + + + + + + +

+=positive

48 Table 7: Diameter of growth inhibition zone in (mm) shown by n- butanol extracts of isolates against some tested microorganisms

Isolate C. humicolus Pencillium sp. A.flavus A.niger C. albicans F.oxysporium Saccharomyces

Dee2 00 00 05 10 05 00 00

Dee3 00 00 00 05 10 00 00

Dee4 00 00 03 06 10 00 00

Dee6 10 00 00 12 04 00 00

Dee10 12 00 12 10 20 00 00

Dee12 00 00 10 20 30 00 00

Dee15 24 00 14 24 45 00 00

Dee16 00 00 05 12 20 00 00

Dee17 00 00 00 06 10 00 00

Dee18 00 00 00 05 15 00 00

Dee20 05 00 10 15 10 00 00

Dee26 00 00 06 05 05 00 00

Dee30 10 00 05 12 05 00 00

Dee33 00 00 11 14 12 00 00

Dee41 00 00 00 00 14 00 00

Dee45 00 00 00 00 05 00 00

e c e c

(a) Aspergillus niger (b) Candida albicans

Plate 5: shows the effect of n-butanol extract of Dee 15 (e) against A. niger and C. albicans, compared with the control (c) (n-butanol)

50 Candida albicans, Aspergillus niger, Cryptococcus humicolus, Penicillium and Fusarium oxysporium. Augustine et al. (2005b) reported that Fusarium oxysporium was found to be resistant against Streptomyces rochei AK 39 extract, this result is in agreement with the results obtained from this study where all of the isolates extracts were not active against Fusarium oxysporium. The inhibition zones for Aspergillus flavus and Cryptococcus humicolus were less than those for C. albicans. The highest inhibition zone for C. humicolus was obtained by Dee 15 (24 mm), Dee 12 (20 mm), Dee 20 (15 mm) and Dee 33 (14 mm). The highest inhibition zones for A. flavus were exhibited by Dee15 (14 mm), Dee 10 (12 mm) and Dee 33 (11 mm). Fig. 1 shows that the most potential isolate was Dee 15 which has a profound activity against most of the pathogenic fungi tested, followed by Dee 12 and Dee 33. However, all of the isolates extracts have not shown any activity against Penicillium sp, Saccaromyces sp and Fusarium sp.

3.4 General Discussion Streptomcyes is the largest antibiotic producing genus in the microbial world discovered so far. The number of antimicrobial compounds reported from the species per year increased almost exponentially for about two decades to reach its peak in 1970 and with a substantial decline in the late 1980's and 1990's. Recently, Watve et al. (2001) presented a mathematical model which estimated the total number of antimicrobial compounds that this genus is capable of producing to be in the order of a 100,000-a tiny fraction of this has been unearthed so far. This means that if the screening efforts are maintained, novel antibiotics are expected to be discovered

Fig1: Variation of inhibition zones exhibited by four different Streptomycs isolates

40 Dee15 35 Dee12 Dee10 30 Dee33 inhibition zones 25 diameter (mm) 20

0 C. albicans A.niger A.flavus C. humicolus Tested pathogenic fungi

52 regularly. It should be emphasized that, the search for a metabolite of pharmaceutical interest requires a large number of isolates (Sahin and Ugur, 2003). The search will be more promising if diverse actinomycetes are sampled and screened (Watve et al., 2001). In this study, soils were specifically collected from different locations in Sudan. Most of the soil samples collected were from different agricultural locations. This was based on the assumption that actinomycetes diversity may be influenced by the diversity of cultivated plant species as these bacteria grow profusely in the humus and leaf litter layers (Oskay et al., 2004). Although our screening efforts have been limited to few sampling sites, yet they possess many actinomycetes. Fifty isolates were obtained, 16 of them produced inhibitory substances. With reference to the results of screening experiments obtained morphological and physiological characterization studies were carried out. Based on these characteristics, all of these isolates were classified in the order, Actinomycetales; family Streptomycetaceae and genus Streptomcyes. When grown in submerged culture, sixteen of the isolates produced inhibitory substances. The isolates showed varying degrees of retarding effect against one or more of the tested microorganisms. The absence or the presence of mild activity by many of our isolates may be due to the low concentration of the crude preparation used. A shortcoming of our study was to limit crude preparation concentrations to the level that allowed better diffusion of the bioactive compounds. If higher concentrations of the crude preparations could be used, stronger inhibition might have been noted. Nevertheless, the antifungal activities shown by the isolates highlighted them as candidates for further in vitro and in vivo investigations before they

53 are to be recommended for the control of pathogenic fungi (Aghighi et al., 2004). The results of this study also demonstrated that the isolation of Streptomyces species from diverse geographical locations in Sudan may present significant capacity for antifungal production. Therefore, we recommend a long term screening programme in order to discover a novel antibiotic. This should be accompanied by a systematic approach to evaluate and optimize production under different fermentation conditions using locally available substrates. Parameters such as temperature, pH, sugar and nitrogen concentration in the fermentation media should be carefully studied and adjusted under our conditions. According to Desai et al. (2002) the pH value affects the production of antibiotics at shake flasks fed fermentation. It is worth mentioning that, in similar investigations, antibiotics production is usually enhanced by the addition of Sodium Alginate in very low concentrations. Production may also be elicitated by the addition of γ- butyrolcatone to the fermentation media (Eriko et al., 2000). Production of antibiotic was also improved by the addition of phosphate, at a sub-optimal level, to the fermentation media (Raytadpadar and Paul, 2001). Most of the antibiotics, in use today, are produced by microorganisms specifically by Streptomyces species that are isolated from soils. Sudan soils have different physical and chemical properties according to the location and environment, which play an important role in the diversity of the soil populations. However, Sudan soils have not been fully explored. This study shows the great potentiality of Streptomyces species that are isolated and yet to be isolated to produce novel antimicrobial agents.

54 Chapter four References Aghighi S. G. H.; Shahidi Bonjar and I. Sadoun, (2004). First report of Antifungal properties of a New Strain of Streptomyces plicatus (strain 101) Against Four Iranian Phytopathogenic Isolates of Verticillum dahliae, A New Horizon in Biocontrol Agents. Biotechnology 3: 90-97.

Alexander, M. (1967). Introduction to Soil Microbiology. John Wiley and Sons, New York, London, Sydney. 472 pp.

Anderson, A. S. and Wellington, E. M. H. (2001). The taxonomy of Streptomyces and related genera. Int. J. Syst. Evol. Microbiol. 51: 797-814.

Arikan, S.; Lozano-Chiu, M.; Paetzick, V.; Nangia, S. and Rex, H. J., (1999). Microdilution Susceptibility Testing of Amphotericin B, Itraconazole and Voriconazole against Clinical Isolates of Aspergillus and Fusarium species. Journal of Clinical Microbiology 37: 3946-3951

Arisawa, A.; Kawamura, N.; Okumura, K.; Tsunekawa, H.; Yashioka, T. and Okamoto, R. (1996). Direct fermentative production of acyltylosins by genetically engineered strains of Streptomyces faradiae. J. Antibiotics 49: 349-354.

Atlas, R. M. (1989). Microbiology Fundementals and Applications. 2nd ed. MacMillan publishing Company, New York 806 pp.

55 Atlas, R. M. (1997). Handbook of Microbiological Media; 2nd ed., CRC Press, Boca Raton, New York, London, Tokyo, 1706 pp.

Augustine, S. K.; Bhavsar, S. P. and Kapadnis, B. P. (2005a). A non-polene antifungal antibiotic from Streptomcyes albidoflavous PU23. J. Bioscience 30: 201-211.

Augustine, S. K.; Bhavsar S. P. and Kapadnis B. P. (2005b). Production of a growth dependent metabolite active against dermatophytes by Streptomyces rochei AK 39. Indian J Med Res 121: 164-170.

Beck-Sague, C. M. and Jarvis, W. R. (1993). Secular trends in the Epidemiology of nosocomial fungal infections in the United States 1980- 1990. J. Infect. Dis. 167: 1247-1252.

Bevan, P.; Ryder H. and Shaw I. (1995). Identifying small molecule lead compound: The Screening Approach to Drug Discovery; Trends Biotechnology, 113: 115-121.

Champness, W. (2000). Actinomycetes Development, Antibiotic Production and Phylogeny: questions and challenges. In: Burn Y. V.; Skimkets, L.J. (eds). Prokaryotic Development. American Society for Microbiology, Washington D.C., 11.

Collee, J. G.; Fraster, A. G.; Marion, B. P. and Simmons, A. (1996). Mackie and McCartney. Practical Medical Microbiology. 14th ed., Longman, Singapore, 978 pp.

Collins, C. H.; Lyne, P. M. and Grande, J. M. (1995). Collins and Lyne’s, Microbiological Methods. 7th ed., Butterworth- Heinemann Ltd, 493 pp.

Cross, T. (1989). Growth Examination of Actinomycetes: Some Guidelines. In: Bergey’s Manual of Systematic Bacteriology. Williams & Wilkins Company, Baltimore, 4: 2340-2343.

Desai, R. P.; Leaf, T.; Woo, E. and Licari, P. (2002). Enhancement production of hetrologous macrolide aglycones by fed-batch cultivation of Streptomyces coelicolor. J Ind. Microbiol. Biotechnol. 28 (5): 29301

El-Naggar, M. Y.; Hassan, M. A. and Said, W. Y. (2001). Isolation and Characterization of and Antimicrobial Substance Produced by Streptomyces violatus. Egyptian J. Biol. 3: 11-21.

Enefiok JN. and Charles H. (1978). Detection of Antibiotic-Producing Streptomyces Inhabiting Forest Soils. Antimicrob Agents and Chemo14: 51- 59.

Eriko, T.; Nihiras, T.; Haras, Y.; Jones, J.J.;Gershater, C. J. L.; Yamadas, Y. and Bibb, M. (2000). Purification and structural determination of SCB1, a butyrolactone that elicit Antibiotic Production in Streptomyces coelicolor A3(2). J. Biol. Chem. 275: 1110-1116.

Felicitas KW. and Hans JK. (1992). The Family of Streptomycetaceae, Chapter 41, The Prokaryotes: a handbook on the biology of the bacteria:

57 ecophisiology, isolation, identification and application. 2nd edition , Springer-Verlag: 921-980.

Flaig, W, and Kutzner, H. J. (1960).Bitrag zur Őkologie der Gattung Streptomyces Waksman et Henrici. Arch. Mikrobiol. 35: 207-228.

Georgopapdakou, N. H. and Tkacz, J. S. (1994). Human Mycosis: Drugs and targets for emerging pathogens; Science 264: 371-373.

Gillies, R. R. (1984). Gillies and Dodds Bacteriology. 5th edtion, Churchill Livingstone, 224 pp.

Goodfellow, M. and Williams, S. T. (1983). Ecology of Actinomycetes. Annu. Rev. Microbiol.37: 189-216.

Goodfellow, M.; Kim, S. B.; Falconer, C.; Williams, E. and. (1998). Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately thermophilic carboxydotrophic species from soil. Int. J. Bacteriol. 48: 59.

Gottlieb, D. and Shirling, E. B. (1967). Co-operative description of type cultures of Streptomyces. I. The International Streptomyces Project. Int. J. Syst. Bacteriol. 17: 315-322.

58 Gupta, M.; Kulkarni, P. and Ganguli, B. N. (2002). Antifungal Antibiotics; Appl. Microbiol. Biotech., 58: 46-57.

Gupte, M. D. and Kulkarni, P. R. (2002). A study of antifungal antibiotic production by Streptomyces chattanoogensis MTCC 3423 using full factorial design. Lett. Appl. Microbiol. 35: 22-26.

Hugo, W. B. and Russell, A. D. (1998). Pharmaceutical Microbiology. 2nd edition, Blackwell Scientific publications, 481pp.

Kavanagh, F. (1972). Analytical Microbiology. Kavanagh(Ed)vol.2, Academic press, New York and London, 11 pp.

Kűster, E. and Williams, S. T. (1964). Selection of the media for the isolation of Streptomycetes. Nature 20: 928-929.

Labeda, D.P.; Lechevalier, M. P. and Testa, R. T. (1997). Streptomyces stramineus sp. nov., A New Species of the Verticillate Streptomycetes. Int. J. Syst. Bacteriol. 4: 747-753.

Lacey, J. (1973). Actinomycetales: characteristics and practical importance. Edited by G. Skyes and F. Skinner. The Society for Applied Bacteriology Symposium Series. No. 2. Academic press, London, New York.

Locci, R. (1990). Streptomyces and related genera. In: Bergey’s Manual of Systematic Bacteriology, vol. 4. Williams & Wilkins Company, Baltimore, 2451-2508.

59 Mahmoud A. Ghannoum1,2,* and Louis B. Rice3 (1999). Antifungal Agents: Mode of Action, Mechanisms of Resistance, and Correlation of These Mechanisms with Bacterial Resistance. Clinc. Microbiol. Rev 12: 501-517.

Meyers, P. R.; Donovan, S. P.; Clifford, O.J.; Motsamai, P. and Thembelani, K. (2003). Streptomcyes speibonae sp. nov. a novel streptomycete with blue substrate mycelium isolated from South Africa soil. Int.J. Syst. Evaluti. Microbiol. 53: 801-805.

Nűesch, J. (1965) Isoierung and Selectionirung von Actinomyceten. In: Symposium (Anreicherungskulter and Ma\utantenauslese) , Hyg. At. Suppl. 1: 234-252.

O’Grady, F.; Lambert, R. G. and Greenwood, D. (1997). Antibiotic and Chemotherapty. 7th edition, Churchill Livingstone, New York, 987 pp.

Ōmura, S.; Ikeda, H.; Ishikawa, J.; Hanamoto, A.; Takahashi, C.; Shinose, M.; Takahashi, Y.; Horikawa, H.; Nakazawa, H.; Osonone, T.; Kikuchi, H.; Shiba, T.; Sakaki, Y. and Hottori, M. (2001). Genome sequence of an industrial microorganism Streptomyces avermitilis: Deducing the ability of producing secondary metabolites. Proc. Natl. Acad. Soc. 98: 12215-12220.

Oskay, M.; Tamer, A. Ű and Azeri, C. (2004). Antibacterial activity of some Actinomycetes isolated from farming soils of Turkey. Afri. J. Biotechnol. 3: 441-446.

60 Ounhdouch, Y.; Barakate, M; Finanse, C. (2001). Actinomyctes Habitats: Isolation and Screening for Antifungal activities. Eur. J. Soil biology. 37: 69-74.

Porter, J. N. and Wilhelm, J. J. (1960). The effect on Streptomyces population of adding various supplements to the soil samples. Development in Industrial Microbiology, vol.2 Society for Industrial Microbiology, H. Stillwater, Oklahoma. USA.

Prescott, L. M.; Harely, J. P. and Klein, D. A. (1993). Microbiolgy. 2nd edition. WM. C. Brown publishers. 912 pp.

Pullen, C.; Schimtz, P.; Groth, I.; Schlegel, B.; Mollmann, U.; Gollmick, F.; Grafe, V. and Leistner, E. (2002). New and bioactive compounds from Streptomyces strains residing in the wood of celastraceae. Planta 216: 162- 167.

Raytapadar, S. and Paul, AK. (2001). Production of an antifungal antibiotic by AStreptomyces aburaviensis 1DA-28. Microbiol Res. 155: 315-323.

Rong-Yang W. and Ming-Ho C. (1995). Identification of the Streptomyces Strain KS3-5. Bot. Bull. Acad. Sin. 36: 201-205.

Roubos J. A.; Perben K.; Ruud, G. M. L.;, Henk B. V. and Joseph J. H. (2001). A quantitative approach to characterizing cell lysis caused by mechanical agitation of Streptomcyes clavuligerus. Biotechnol Prog. 17: 339-347.

61 Sahin, N. and Ugur, A. (2003). Investigation of the antimicrobial activity of Streptomyces isolates. Turk. J. Biol. 27: 79-84.

Sande, M. A. and Mandell, G. L. (1980). Antimicrobial agents, general consedration. In: The Pharmacological Basis of Theraputics.

Shadomy HJ, Wood-Helie S, Shadomy S, Dismukes WE, Chau RY. (1987). Biochemical serogrouping of clinical isolates of Cryptococcus neoformans. Diagn. Microbio.l Infect Dis.7:159.

Stanier, R. Y.; Michael, D. and Adelberg, E. A. (1963). General Microbiology. London, Macmillan publishers, 753p.

Stephen, P. Denyer; Norman, A. Hodges and Sean, P. Gorman (2004). Hugo and Russell's Pharmaceutical Microbiology. 7th edition, 481pp, Blackwell Science Ltd.

Stolp, H. and Starr, M. P. (1981). Principles of isolation, cultivation and conservation of bacteria. In:The prokaryotes,. Springer Verlag, Berlin p135- 175.

Tetsuo, N.; Koichiro, M.; Masato I.; Toru M. and Ryoichi K. (2000). Mechanism of the Incidental Production of a Melanin-Like Pigment during 6-Demethylchlortetracycline Production in Streptomyces aureofaciens. Appl Evviron Micro. 66:1400-1404

62 Tsao, P. H.; Leben, C. and Keitt, G. W. (1960). An enrichment method for isolating Actinomycete that produce diffusible antifungal antibiotics. Phytopathology 50: 88-89.

Waksman, S. A. and Henrici, A. T. (1943). The nomenclature and classification of the Actinomyces. J. Bacteriol. 46: p337-341.

Waksman, A. S. and Lechevailer, A. H. (1953). Guide to the Classification of the Actinomycetes and their Antibiotics. The Williams and Wilkans Company. Baltimore, ,246pp.

Watve, M. G.; Tickoo, R.; Jog, M. M.; Bhole, B. D. (2001). How many antibiotics produced by the genus Streptomyces?. Arch. Microbiol. 176: 386- 390.

Wedberg, S. E. (1966). Introduction to Microbiology. Reinhold publishing Corporation, 426pp.

Wendisch,F. K. and Kutzner, H. J. (1991). The Family Streptomyceteaceae in Prokaryotes. A. Ballows (eds), Springer Verlag , vol1, 2nd edition, 922- 995.

Williams,S. T.; Shameemullah, M. ; Watson, E. T. and Mayfield, C. I. (1972). Studies on the ecology of the Actinomycetes in soil. The influence of the moisture tension on the growth and survival. Soil Biol. Biochem. 4: 215-225.

63 Williams, S. T. (1982). Are Antibiotics produced in Soil?. Pedobiologia, 23: 427-435.

Yang, S.S. and Ling, M.Y. (1989). Tetracycline Production with sweet potato residue by solid state fermentation. Biotech. Bioeng. 33: 1021-1028.

Yang, S.S. and Sewi, W.J. (1996). Oxytetracycline production by Streptomyces rimosus in solid fermentation of corncorb. Microbiol. Biotech. 12: 43-46.

Yang, S.S. and Wang, J.Y. (1999). Protease and Amylase production of Streptomyces rimosus in submerged culture and solid state cultivation. Bot. Bull. Acad. Sin. 40: 256-259.

Zhao H.; Rachel, L. P.; David, I. E.; Gareth, W. G. and Royston, G. (2006). The rapid differentiation of Streptomyces isolates using Fourier transform infrared spectroscopy. Vibrational Stpecrascopy 40: 213-218.

64 Table 1: Streptomyces presumptive Isolates ISOLATE SOURCE OF SOIL SAMPLE DEE1 DEE2 DEE3 DEE4 DEE5 DEE6 DEE7 DEE7 GADARIF GADARIF STATE DEE8 DEE9 DEE10 DEE11 DEE12 DEE13 DEE14 DEE15 DEE16 DEE17 DEE18 DEE19 DEE20 BLUE NILE RIVER DEE21 BANK DEE22 DEE23 DEE 24 DEE25 DEE26 DEE27 DEE28 NILE RIVER BANK DEE29 DEE30 KHARTOUM STATE DEE31 DEE32 DEE33 TWTI DEE34 DEE35 DEE36 DEE37 DEE38 SHAMBAT DEE39 DEE40 DEE41 DEE42 DEE 43 GERIF WEST DEE44 DEE 45 DEE 46 DEE 47 DEE 48 TANGASI NORTHERN STATE DEE 49 DEE 50

TABLE 2: Inhibition zones diameters (mm) shown by different Streptomyces isolates on the growth of seven microorganisms Isolate Penicillium Cryptococcus A.flavus Candida Aspergillus Fusarium Saccharomyces albicans niger oxysporium Dee1 ------Dee2 - 13.0 - 40.0 - - - Dee3 - - - 30.0 - - - Dee4 - - - 31.0 10.0 - - Dee5 ------Dee6 - - 12.0 - - - - Dee7 ------Dee7 ------Dee8 ------Dee9 ------Dee10 - - - 34.0 - - - Dee11 ------Dee12 - - 12.0 - - - - Dee13 ------Dee14 ------Dee15 - 24.0 23.0 - - - - Dee16 - - - 50.0 - - - Dee17 - - - 15.0 22.0 - - Dee18 - - - 40.0 30.0 - - Dee19 ------Dee20 - - - 28.0 14.0 - - Dee21 ------Dee22 ------Dee23 ------Dee24 ------Dee25 ------Dee26 - - - 45.0 - - - Dee27 ------Dee28 ------Dee29 ------Dee30 - - - - 14.0 - - Dee31 ------Dee32 ------Dee33 - - - 40.0 - - - Dee34 ------Dee35 ------Dee36 ------Dee37 ------Dee38 ------Dee39 ------Dee40 ------Dee41 - - - - 35.0 - - Dee42 ------Dee 43 ------Dee44 ------Dee 45 - - - - 33.0 - - Dee 46 ------Dee 47 ------Dee 48 ------Dee 49 ------Dee 50 ------

Table 6:Carbohhydrate utilization by different Streptomyces isolates:

Isolate Arabinose Rhaminose Glucose Galactose Fructose Sucrose Maltose Lactose Manitol

Dee2 + + + + + + + + +

Dee3 + + + + + + + + +

Dee4 + + + + + + + + +

Dee6 + + + + + + + + +

Dee10 + + + + + + + + +

Dee12 + + + + + + + + +

Dee15 + + + + + + + + +

Dee16 + + + + + + + + +

Dee17 + + + + + + + + +

Dee18 + + + + + + + + +

Dee20 + + + + + + + + +

Dee26 + + + + + + + + +

Dee30 + + + + + + + + +

Dee33 + + + + + + + + +

Dee41 + + + + + + + + +

Dee45 + + + + + + + + +

+= positive

Table 3: cultural characteristics of Streptomyces isolates:

Isolate Shape Chromogensis Edge Opacity Elevartion Surface consistancey

Dee2 Filamentous White Filamentous opaque Flat Powdery Dry

White with Dee3 Filamentous Filamentous opaque Flat Powdery Dry Gray Margin Gray with Dee4 Filamentous Filamentous opaque Flat Powdery Dry White margin

Dee6 Filamentous Cream Filamentous opaque Flat Powdery Dry

Dee10 Round Greenish blue Smooth opaque raised Powdery Dry

Dee12 Filamentous Black Filamentous opaque Flat Powdery Dry

Dee15 Round Red Smooth opaque Raised Powdery Dry

Dee16 Filamentous Gray Filamentous opaque Flat Powdery Dry

Gray with Dee17 Filamentous Filamentous opaque Flat Powdery Dry White margin

Dee18 Filamentous Gray Filamentous opaque Flat Powdery Dry

Dee20 Filamentous Gray Filamentous opaque Flat Powdery Dry

Dee26 Round White Smooth opaque raised Powdery Dry

Dee30 Filamentous White Filamentous opaque Flat Powdery Dry

Dee33 Filamentous White Filamentous opaque Flat Powdery Dry

Dee41 Filamentous White Filamentous opaque Flat Powdery Dry

Dee45 Round White Smooth opaque raised Powdery Dry

Table 4: Microsopical characteristics of Streptomyces isolates:

Isolate Gram Acid-fast Motility Aerial Conidia Stain mycelium

Dee2 + ve - ve - ve Present present

Dee3 + ve - ve - ve Present present

Dee4 + ve - ve - ve Present present

Dee6 + ve - ve - ve Present present

Dee10 + ve - ve - ve Present present

Dee12 + ve - ve - ve Present present

Dee15 + ve - ve - ve Present present

Dee16 + ve - ve - ve Present present

Dee17 + ve - ve - ve Present present

Dee18 + ve - ve - ve Present present

Dee20 + ve - ve - ve Present present

Dee26 + ve - ve - ve Present present

Dee30 + ve - ve - ve Present present

Dee33 + ve - ve - ve Present present

Dee41 + ve - ve - ve Present present

Dee45 + ve - ve - ve Present present

Table 5: Biochemical characteristics of Streptomyces isolates:

Gelati Blood Starch Test Nitrate Organic acid H S Melanin ne Peptonizati Coagulatio Catalase Oxidase Urease Protease Aerobiosis 2 hemolys hydrolys reductase formation formation formation liquef on of milk n of milk /Isolate is is action Dee2 +ve +ve +ve +ve +ve +ve +ve +ve +ve -ve + β -ve -ve -ve Dee3 +ve +ve +ve +ve +ve +ve +ve +ve +ve -ve + β -ve +ve +ve Dee4 +ve +ve +ve +ve +ve +ve +ve -ve +ve -ve + β +ve -ve +ve Dee6 +ve +ve +ve +ve +ve +ve +ve -ve -ve +ve + β +ve +ve +ve Dee10 +ve +ve +ve +ve +ve +ve - ve +ve -ve +ve + + α +ve +ve +ve Dee12 +ve +ve +ve +ve +ve +ve -ve -ve -ve +ve + β +ve +ve +ve Dee15 +ve +ve +ve +ve +ve +ve +ve +ve -ve +ve + + α +ve -ve +ve Dee16 +ve +ve +ve +ve +ve +ve +ve -ve +ve +ve + β +ve -ve -ve Dee17 +ve +ve +ve +ve +ve +ve +ve -ve +ve -ve + + α +ve -ve +ve Dee18 +ve +ve +ve +ve +ve +ve +ve -ve +ve -ve + β +ve -ve +ve Dee20 +ve +ve +ve +ve +ve +ve +ve -ve -ve -ve + β -ve -ve +ve Dee26 +ve +ve +ve +ve +ve +ve +ve -ve -ve +ve + β -ve -ve +ve Dee30 +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve + + α +ve +ve +ve Dee33 +ve +ve +ve +ve +ve +ve +ve --ve +ve +ve + + α +ve +ve +ve Dee41 +ve +ve +ve +ve +ve +ve +ve -ve +ve -ve + β +ve +ve -ve Dee45 +ve +ve +ve +ve +ve +ve -ve +ve +ve -ve + β -ve +ve -ve