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