biodiversity and antibiotic activity of actinomycetes isolated from cat ba island, vietnam = đa dạng sinh học và hoạt tính kháng sinh của các chủng xạ khuẩn phân lập ở đảo cát bà, việt nam

CONTENTS Acknowledgements i Abbreviations ii List of figures iii List of tables iv Abstract 1 Tóm tắt 3 Foreword 5 Chapter 1. Introduction 6 1.1 Antibiotic 6 1.1.1 General introduction 6 1.1.2 History of the development of antibiotics 7 1.1.3 Classification of antibiotics 9 1.1.4 Anti-tumor antibiotics 13 1.1.5 The need of developing new antibiotics 14 1.2 Actinomycetes 14 1.2.1 General characteristics 14 1.2.2 Actinomycetes and secondary metabolites 16 1.3 Objectives of the study 16 Chapter 2. Materials and methods 18 2.1 Work flow 18 2.2 Methods 19 2.2.1 Isolation of actinomycetes 19 2.2.2 Targeted microorganisms 21 2.2.3 Screening antibiotic producing actinomycetes 21 2.2.4 Ethyl-acetate extraction 22 2.2.5 Chromatography analyses of antibiotics 22 2.2.6 Screening for cytotoxicity 24 2.2.7 Taxonomical identification of actinomycete isolates 25 Chapter 3. Results and discussion 28 3.1 Biodiversity of the actinomycete isolated from Catba island 28 3.2 Screening for antibiotic producing actinomycetes 29 3.3 Chromatography analyses of crude extracts of the selected strains 31 3.3.1 Thin Layer Chromatography (TLC) 31 3.3.2 High – Performance Liquid Chromatography (HPLC) 32 3.4 Primary study on cytotoxicity activity 33 3.4.1 pH-dependent color change by the selected strains 33 3.4.2 Cytotoxicity assay against human cell lines 33 3.5 Identification of the actinomycete isolates 34 3.5.1 Morphology-based identification for the Streptomyces isolates 35 3.5.2 16S rDNA sequence-based identification for the non-Streptomyces isolates 37 Conclusion and Suggestion 38 Bibliography 40 Appendix 1 Culture media 46 Appendix 2 The 16S rDNA sequence………………………………………… …….46 iii LIST OF FIGURES Figure Title Page Figure 1 Discovery of important antibiotics and other natural products over the years .7 Figure 2 Evolution of penicillin G 8 Figure 3 Chemical structures of daunorumycin and idarumycin .13 Figure 4 Catba national park 19 Figure 5 Evaluation of antimicrobial activity of the actinomycete isolates 30 Figure 6 Analyses of crude extracts of the selected actinomycete strains via TLC 31 Figure 7 HPLC analysis of crude extract from actinomycetes 32 Figure 8 Color change by strains A1018 and A1073 depending on pH in the medium 33 Figure 9 Colony morphology of the representative Streptomycete strains… 35 Figure 10 Spore-bearing aerial hyphae of the representative Streptomyces strains… 36 Figure 11 Neighbor-joining tree of 16S rDNA partial sequences showing phylogenetic positions of the 7 actinomycete strains in the relationship to type strains of the genus Nonomuraea 37 iv LIST OF TABLES Chapter 1 Table 1.1 Grouping of antibiotics bases on their chemical structures 10 Chapter 2 Table 2.1 Condition for HPLC analyses of the standards 23 Chapter 3 Table 3.1 Taxonomical grouping of the actinomycete isolates 28 Table 3.2 Antimicrobial activity of the 17 selected actinomycete strains 30 Table 3.3 Toxicity assay against three human cell lines 34 1 ABSTRACT In this study, a total of 424 actinomycete strains isolated from soil and litter samples on Catba island (Haiphong, Vietnam) were subjected to a screening for the inhibitory activities against microorganisms, including bacteria (Micrococcus luteus, and Escherichia coli) and eukaria (Candida albicans and Fusarium oxysporium). Through two screening steps, 17 strains were selected for their inhibitory activity against one or more target microorganisms. Crude extracts in ethyl acetate from culturing media of the selected strains were analyzed via thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), in which chloramphenicol, kitasamycin, erythromycin and raw extract of anthracycline were used as standards. The obtained results showed that antibiotic substances produced by the selected strains could not be able to classify in any group of the analyzed standards, except the strain A396 which appeared to produce chloramphenicol-like antibiotic. Besides that, several human tumor cell lines have also been used for testing the inhibitory effects. The results showed that among the 17 selected strains, 3 strains (A1018, A1022 and A1073) exhibited cytotoxicity against all three cell lines including hepatocellular carcinoma, lung cancer, and rhabdosarcoma. Taxonomical studies based on the morphology and 16S rDNA gene sequencing indicated that the collection of actinomycetes isolated from Catba island contained mainly Streptomyces species (about 70%) and the group of rare actinomycetes (non-Streptomyces) which made of 30% of the collection was dominated by Micromonospora, Nonomureae and Nocardia genera. Of the 17 selected strains with highest antimicrobial activity, ten strains affiliated to the genus Streptomyces (as based on morphology) and seven strains to the genus Nonomuraea (as based on 16S rDNA sequence analyses). The strains 2 selected in this study could serve as valuable sources for discovering new antibiotic substances in Vietnam. 3 TÓM TẮT Tên luận văn: Đa dạng sinh học và hoạt tính kháng sinh của các chủng xạ khuẩn phân lập ở ñảo Cát Bà, Việt nam Người hướng dẫn: TS. Nguyễn Huỳnh Minh Quyên TS. Nguyễn Quỳnh Uyển Viện Vi sinh vật và Công nghệ Sinh học, Đại học Quốc gia Hà nội Ngành: Công nghệ Sinh học Chuyên ngành: Công nghệ Sinh học Mã số: 60 42 80 Tổng số 424 chủng xạ khuẩn phân lập từ mẫu ñất và lá mục thu thập ở ñảo Cát bà (Hải Phòng, Việt Nam) ñược sử dụng ñể sàng lọc hoạt tính kháng ñối với cả vi khuẩn (Micrococcus luteus và Escherichia coli) và vi sinh vật nhân thực (Candida albicans và Fusarium oxysporium). Qua hai bước sàng lọc, 17 chủng ñã ñược chọn lọc dựa trên hoạt tính ức chế ñối với một hoặc nhiều vi sinh vật kiểm ñịnh. Chiết xuất thô trong ethyl acetate của các chủng lựa chọn ñược phân tích bằng sắc ký bản mỏng và sắc ký lỏng hiệu năng cao, trong ñó chloramphenicol, kitasamycin, erythromycin và chiết xuất thô của chủng vi sinh vật sinh anthracyclin ñược sử dụng làm chất chuẩn. Kết quả thu ñược cho thấy các chất kháng sinh do các chủng lựa chọn tạo ra không nằm trong nhóm của các chất kháng sinh sử dụng làm chất chuẩn. Trường hợp ngoại lệ duy nhất là chủng A396 tạo chất kháng sinh cùng nhóm với chloramphenicol. Bên cạnh ñó, việc thử nghiệm khả năng ức chế một số dòng tế bào ung thư cũng ñược thực hiện. Kết quả cho thấy rằng, 3 trong số 17 chủng ñược chọn (A1018, A1022 và A1073) thể hiện tính ñộc ñối với cả 3 dòng tế bào gồm ung thư gan, ung thư phổi và ung thư cơ vân tim. 4 Nghiên cứu phân loại dựa trên các ñặc ñiểm hình thái và trình tự gen 16S rDNA cho thấy xạ khuẩn phân lập từ Cát Bà tương ñối ña dạng, trong ñó nhóm Streptomyces chiếm ña số (70%) và nhóm xạ khuẩn hiếm (non- Streptomyces) chiếm 30%, gồm các chi chính là Micromonospora, Nonomureae và Nocardia. 10 trong số 17 chủng lựa chọn có hoạt tính kháng khuẩn cao ñược xếp vào chi Streptomyces (dựa vào các ñặc ñiểm hình thái), 7 chủng còn lại ñược xếp vào chi Nonomuraea (dựa trên so sánh trình tự gen 16S rDNA). Các chủng lựa chọn từ nghiên cứu này có thể sử dụng làm ñối tượng ñể nghiên cứu phát hiện các chất kháng sinh mới ở Việt Nam. 5 FOREWORD Vietnam is located in the tropical to subtropical region, has 1,700 km coastline from the north to the south with many islands that are known for highly rich biodiversity. Some islands have been recognized as national parks for conservation and sustainable use of bioresources. Since microorganisms play a crucial role in the development of biotechnology, a great interest has been given to microbial sources in these conserved areas of the country. Whereas diversity of plants and animals in these conserved areas has been intensively studied, there is still not much known about the diversity and applicable capability of microorganisms there. Cat Ba Island presents the biggest island in Halong Bay, a World Natural Heritage in the North of Vietnam. The National park on the island has been reported to contain 620 species of higher plants, 32 species of mammals, 69 species of birds and 20 species of reptiles. Similar to other conserved areas in Vietnam, the National Park on Cat Ba Island has not yet been investigated on microbial diversity and their potential use. The study “Biodiversity and antibiotic activity of actinomycetes isolated from Catba island, Vietnam” presented here was conducted for providing first information about the actinomycete community on the island, one of the most abundant group of microbes with high applicable potential. Thus, a high number of actinomycete isolates were obtained from diverse soil and litter samples collected at Cat Ba island by using different isolation methods. Taxonomical diversity of the isolates was assessed via morphological classification as well as comparative study of the 16S rDNA sequences. On the other hand, potential use of the isolates was preliminarily investigated based on their activity in production of useful secondary metabolites such as antibiotic and antitumor compounds. Main part of the research was carried out at the Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi. 6 CHAPTER 1. INTRODUCTION 1. 1 Antibiotic 1.1.1 General introduction Microbes produce an extraordinary array of defense systems. These include broad-spectrum of classical antibiotics, metabolic by products, such as lactic acid produced by lactobacilli, lytic agents like lysozymes, numerous types of protein exotoxins, bacteriocins, etc, which are loosely defined as bioactive compounds with a bacteriocidal mode of action. This biological arsenal is striking not only in its diversity, but also in its natural abundance [26]. According to current definition, antibiotics are chemical compounds produced by microorganisms and in low concentrations they are capable of inhibiting the growth of, or killing, other microorganisms [30]. Antibiotics are also produced by higher plants and animals. Such substances are however excluded by this definition. Although produced by microorganisms, bacteriocins are also not included in this definition because they are not only larger in molecular size than the usual antibiotics, furthermore they affect mainly organisms related to the producing organism. In comparison to bacteriocins, conventional antibiotics however are more diverse in their chemical nature and attack organisms distantly related to themselves. Most importantly, while the information specifying the formation of ‘regular’ antibiotics is carried on several genes, bacteriocins need single genes [30]. Currently about 16,500 antibiotics have been discovered from microorganisms, and every year dozens of new antibiotic are discovered [17]. [...]... Taxonomical identification of actinomycetes Morphological characterization DNA extraction PCR amplification of the 16S rDNA sequencing, and phylogenetic analysis 18 2.2 Methods 2.2.1 Isolation of actinomycetes Catba is an island with the territory of approximately 140 km2 in Halong Bay, Northern Vietnam It is the largest island in the Bay and the preserved National Park occupies about half of its area The... Study property of antibiotics produced by the selected actinomycetes via thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) 16 - Study taxonomy of actinomycetes based characteristics and 16S rDNA gene sequences 17 on morphological CHAPTER 2 MATERIALS AND METHODS 2.1 Work flow Collection and isolation of actinomycetes Collection of samples from Catba island Isolation by... with rich biodiversity in Vietnam The selected strains were then subjected to studies on the antibiotic substances they produced as well as the phylogenetic affiliation of selected strains The particular objectives were as follows: - Isolate actinomycetes from soil and litter samples on Catba island (Haiphong, Vietnam) - Screen actinomycetes having high antimicrobial activity and activity against human... 1000 replicates [9] 27 CHAPTER 3 RESULTS AND DISCUSSION 3.1 Biodiversity of the actinomycete isolated from Catba island In this study, total 424 actinomycete strains were isolated from the collected samples In regard to isolation sources, 353 strains (83.25%) were isolated from soil and 71 strains (16.75%) from plant – litter samples The isolates were first subjected to a morphological classification,... antibiotic resistance and new antibiotics with different mechanisms of action are also needed [10] 1.1.3 Classification of antibiotics There are several methods of antibiotic classification that have been adopted by various authors One of the methods, which has been used, is based on mode of action, e.g whether antibiotics act on the cell wall, or inhibit proteins, etc However, several mechanisms of. .. actinomycins and tetracyclines [13] These substances have been succesfully used as herbicides, anticancer agents, drugs, immunoregulators and antiparasitic agents [44] 1.3 Objectives of the study This study aimed to investigating biodiversity and to screening actinomycete strains with high antimicrobial activity among a collection of actinomycetes isolated from Catba island, a national park with rich biodiversity. .. The park contains both land and marine areas with high biodiversity Catba national park was recognized by UNESCO in December 2004 as a Biosphere reserve in the world Figure 4: Catba national park Soil and leaf litter samples were collected from Catba island and used as sources for actinomycete isolation Two isolation methods were used in this study, the dry-heating method [31] and the rehydration-centrifugation... fragmentation pattern of substrate mycelia, morphology of aerial mycelia, structure of spore chains and the spore forms were observed under the phase contrast microscope (Zeiss) connected with a camera and image controlling software The Atlas–Morphology of actinomycetes [11] and Identification 25 Manual of Actinomycetes [29] were used as references for the taxonomical determination of the isolated strains... food chain 1.2 Actinomycetes 1.2.1 General characteristics Actinomycetes make a big group of diverse bacteria, most of which grow aerobically and form branching mycelia similar to those of fungi The name actinomycete derives from the Greek “actys” (ray) and “mykes” (fungus) and actinomycetes were initially regarded as minute fungi because of their mycelium-like growth The branching network of hyphae usually... psychrophilic Arthrobacter ardleyensis isolated from sediment from an Antarctic lake was able to grow at temperatures as low as 0°C [5] and Nocardiopsis alkaliphila isolated from desert soil in Egypt could grow at pH 9.5-10 [18] At the presence actinomycetes are defined as the order Actinomycetales which consists of 13 suborders, 42 families and about 200 genera Actinomycetes are often divided into two categories, . discovering new antibiotic substances in Vietnam. 3 TÓM TẮT Tên luận văn: Đa dạng sinh học và hoạt tính kháng sinh của các chủng xạ khuẩn phân lập ở ñảo Cát Bà, Việt nam Người hướng. Viện Vi sinh vật và Công nghệ Sinh học, Đại học Quốc gia Hà nội Ngành: Công nghệ Sinh học Chuyên ngành: Công nghệ Sinh học Mã số: 60 42 80 Tổng số 424 chủng xạ khuẩn phân lập từ mẫu ñất và lá. investigated on microbial diversity and their potential use. The study Biodiversity and antibiotic activity of actinomycetes isolated from Catba island, Vietnam presented here was conducted