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

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 activ

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

3.3.2 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

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

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

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

selected in this study could serve as valuable sources for discovering new antibiotic substances in Vietnam

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

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

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

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]

1.1.2 History of the development of antibiotics

Antibiotics have been major weapon of human against infectious diseases Looking back at the history of human diseases, infectious diseases have accounted for a very large proportion of diseases as a whole Until the latter half of the 19th century, microorganisms were found to be responsible for a variety of infectious diseases Accordingly, chemotherapy aimed at the causative organisms was developed as the main therapeutic strategy [42]

In 1928, Fleming discovered penicillin He found that the growth of

Staphylococcus aureus was inhibited in a zone surrounding a contaminated

blue mold (a fungus from the Penicillium genus) in culture dishes, leading to

the finding that a microorganism would produce substances that could inhibit the growth of other microorganisms The antibiotic was named penicillin, and

it came into clinical use in the 1940s Penicillin, which is an outstanding agent

in terms of safety and efficacy, led in the era of antimicrobial chemotherapy

by saving lives of many wounded soldiers during World War II [33]

Figure 1: Discovery of important antibiotics and other natural products over the years [17]

During the subsequent two decades, from late 1940s to the late 1960s, many new antibiotics were identified, mostly from the actinomycetes, leading

to a golden age of antimicrobial chemotherapy (Fig 1)

In 1944, streptomycin was obtained from the soil bacterium

Streptomyces griseus Thereafter, chloramphenicol, tetracycline, macrolide,

and glycopeptide (e.g., vancomycin) were discovered from soil bacteria The synthesized antibiotic such as nalidixic acid, a quinolone antimicrobial drug, was obtained in 1962 Improvements in each class of antibiotics continued to achieve a broader antimicrobial spectrum and higher antimicrobial activity, for example β-lactam antibiotics β-lactam class includes penicillins, cephems, carbapenems, and monobactams Penicillins were originally

effective against Gram-positive organisms such as S aureus Later, S aureus

produces the penicillin-hydrolysing enzyme penicillinase, methicillin was developed On the other hand, attempts to expand the antimicrobial spectrum yielded ampicillin, which is also effective against Gram-negative

Enterobacteriaceae, and piperacillin, which is effective even against Pseudomonas aeruginosa (Fig 2)

Figure 2: Evolution of penicillin G [42]

The discovery, development, and clinical use of highly effective antibiotics during the 20th century have decreased substantially the mortality from bacterial infections However, since 1980 the introduction of new antibiotics for clinical use has declines, in part because of enormous expense

of developing and testing new drugs Parallel to this, there has been an alarming increase in bacterial resistance to the existing antibiotics Currently, bacterial resistance is combated by the discovery of new drugs However, microorganisms are becoming resistant more quickly than new drugs are made available Thus, future research in antimicrobial therapy may focus on finding how to overcome the 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 action may operate simultaneously making such method of classification difficult to sustain In some cases antibiotics have been classified on the basis of the producing organisms But the same organism may produce several antibiotics, e.g the

production of penicillin N and cephalosporin by a Streptomyces sp The same

antibiotics may also be produced by different organisms Antibiotics have been classified by routes of biosynthesis, yet several different biosynthetic routes often have large areas of similarity The spectra of organisms attacked have also been used, e.g those affecting bacteria, fungi, protozoa, etc However, antibiotics belonging to one group, e.g aminoglycosides, may have different spectra [30], [43]

The classification presented here is based on the chemical structure of the antibiotics and according to that antibiotics are classified into 13 groups (Table 1.1) [30]

Table 1.1 Groups of antibiotics based on their chemical structures

As mentioned above, there are different ways to classify antibiotics, non

of which could perfectly cover the diversity of antibiotics Not being an exception, the classification based on chemical structure has relative character, and generally a well-known example is given to facilitate recognition of the group it belongs to Here characteristics of few common groups of antibiotics are discussed in more details

a Aminoglycosides is a group of antibiotics in which amino sugars are

linked by glycoside bonds This class of antibiotic binds to the bacterial 30S ribosomal subunit, interfering with protein synthesis By binding to the ribosome, aminoglycosides inhibit the translocation of tRNA during translation and leaving the bacterium unable to synthesize proteins necessary for growth Representative aminoglycosides include kanamycin, streptomycin, gentamicin and a whole slew of other "-mycins" Streptomycin and gentamicin are well-known examples of the group Streptomycin is still

used as alternative drug in the treatment of tuberculosis, but rapid development of resistance and serious toxic effects have diminished its usefulness The aminoglycosides inhibit protein synthesis in many Gram-negative and some Gram-positive bacteria They are sometimes used in combination with penicillin Members of this group tend to be more toxic than other antibiotics [43]

b Beta-lactam antibiotics include the well-established and clinically

important penicillins and cephalosphorins as well as some relatively newer members such as cephamycins, nocardicins, thienamycins, and clavulanic acid Penicillins and cephalosphorins work by interfering with interpeptide linking of peptidoglycan, a strong, structural molecule found specifically bacterial cell walls Cell walls without intact peptidoglycan cross-links are structurally weak, prone to collapse and disintegrate when the bacteria attempts to divide [30]

Penicillins are bactericidal, inhibiting formation of the cell wall There are four types of penicillins: the narrow-spectrum penicillin-G types, ampicillin and its relatives, the penicillinase-resistant penicillins, and the extended spectrum penicillins that are active against pseudomonads Penicillin-G types are effective against gram-positive strains of streptococci, staphylococci, and some gram-negative bacteria such as meningococcus Penicillin-G is used to treat such diseases as syphilis, gonorrhea, meningitis, anthrax, and yaws The related penicillin V has a similar range of action but is less effective Ampicillin and amoxicillin have a range of effectiveness similar to that of penicillin-G, with a slightly broader spectrum, including some Gram-negative bacteria [43]

c Chloramphenicol inhibits growth of many Gram-positive and

Gram-negative bacteria; it was the first broad-spectrum antibiotic to be used clinically Chloramphenicol was originally obtained from cultures of the soil

bacterium Streptomyces venezuelae Chloramphenicol inhibits bacterial

protein biosynthesis by interfering with the intrinsic catalytic activity of the

peptidyl-transferase of the ribosome during the elongation phase of translation Because of its small molecular size, it promotes diffusion into areas of the body that are normally inaccessible to many other drugs However, chloramphenicol has serious adverse effects, the most important is the suppression of bone marrow activity, therefore affecting the formation of

blood cells [6], [30]

d Macrolides are a group of antibiotics named for the presence of a

macrocyclic lactone ring in their molecules Macrolides exert their bateriostatic effect by binding irreversibly to the 50S subunit of bacterial ribosomes By binding to the ribosome, macrolides inhibit translocation of tRNA during translation (the production of proteins under the direction of DNA) Although the cells of humans also have ribosomes, the eukaryotic cellular protein factories differ in size and structure from the ribosomes of prokaryotes Erythromycin, azithromycin and clarithromycin are a few examples of macrolide antibiotics Erythromycin is effective against Gram-positive cocci and is often used as a substitute for penicillin against streptococcal and pneumococcal infections Other uses for macrolides include diphtheria and bacteremia Side effects may include nausea, vomiting, and diarrhea; infrequently, there may be temporary auditory impairment [30]

e Tetracyclines are a group of closely related broad-spectrum

antibiotics produced by Streptomyces spp The tetracyclines interfere with the

attachment of the tRNA carrying the amino acids to the ribosome at the 30S subunit of the 70S ribosome, preventing the addition of amino acids to the growing polypeptide chain Tetracyclines are broad-spectrum antibiotics effective against strains of streptococci, gram-negative bacilli, rickettsia (causing typhoid fever), and spirochetes (causing syphilis) They are also used

to treat urinary-tract infections and bronchitis Because of their wide range of effectiveness, tetracyclines can sometimes upset the balance of resident

bacteria that are normally held in check by the body's immune system, leading

to secondary infections in the gastrointestinal tract and vagina, for example Tetracycline use is now limited because of the increase of resistant bacterial strains [30]

1.1.4 Anti-tumor antibiotics

Each cell in higher organisms has a definite function which is carried out

in cooperation with other cells Sometimes, a cell lost the cooperation with other cells surrounding it, starting to divide indiscriminately and independently to form a structure called a tumor or neoplasm Neoplasms are treated by one or more of three methods i.e by surgery to remove the tumor,

by radiation to selectively destroy the cancer cells or by chemotherapy Many

of chemotherapeutic agents used in cancer treatment are secondary metabolites produced by microorganisms (so called anti-tumor antibiotics), especially strains ofthe genus Streptomyces [4], [30] Some of the best known

groups used in clinical practice include anthracyclines, actinomycins and bleomycins

Anthracyclines have been widely

used in the treatment of hematological

and solid neoplasms such as leukemias,

lymphomas, breast cancer, prostate

cancer and bladder cancer [12] The

anthracycline antibiotics are produced by

Streptomyces coeruleorubidus or

Streptomyces peucetius and include

daunorumycin (DNR), doxorubicin,

epirubicin and idarumycin (IDA) (Fig 3)

Daunorumycin and adriamycin link up

base pairs and thus inhibit RNA and

DNA synthesis Mithramycin and

Figure 3: Chemical structures of

daunorumycin and idarumycin

chromomycin A3 which are actinomycins, inhibit DNA-dependent RNA synthesis Bleomycins react with DNA and cause it to break [30], [4]

1.1.5 The need of developing new antibiotics

One of the triumphs of modern medicine is the development of antibiotics and other antimicrobial substances However, it is clear that microbes have developed and will continue to develop resistance to currently available antibiotics by either new mutations or exchange of genetic information [2], [8] New antibiotics that are active against resistant bacteria are required, especially those are anti-tumor and anti-parasitic compounds The search for anti-tumor antibiotics however is more difficult than that for antibacterial or antifungal in terms of methodology and interpretation [30] Moreover, new antibiotics are required for the use in agriculture as drugs for plant or animal diseases, since they could have effect on human through the 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 grows

critically both on the surface of the solid substrata (forming aerial mycelia) as

well as into it, leading to formation of substrate mycelia [3] Most of actinomycetes produce spores varying widely in shape and size, which can

serve as taxonomical characteristics

Actinomycetes are Gram positive bacteria having high G+C content (>55%) in their DNA The majority of actinomycetes has free living, saprophytic life form and is widely distributed in soil, water and plant litter

Actinomycetes play an ecologically important role in recycling substances in nature They decompose and utilize difficult-to-degrade organic matters such

as humic acid in the soil Many strains have the ability to solubilize lignin and degrade lignin-related compounds by producing cellulose- and hemicellulose-degrading enzymes and extracellular peroxidases [24], [32] Some actinomycete strains also occur in other environments rich in organic matter such as composts, in both the mesophilic and thermophilic phases [38], and sewage sludge, where, notably the mycolic acid-containing actinomycetes are associated with the extensive and undesirable formation of stable foams and scum [14], [36]

In general, mesophilic temperatures of 25-30°C and neutral pH are optimal conditions for the growth of actinomycetes Nevertheless, many species have been isolated from extreme environments, for example the

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, the Streptomyces and non-Streptomyces [7], [28]

The best known actinomycete genus is Streptomyces, which contains

approximately 500 species, all with a characteristically high GC content (69–

73 %) in the DNA Streptomyces are extremely prevalent in soil, where they

saprophically degrade a wide range of complex organic substrates by means

of extracellular enzymes Indeed, the characteristic musty smell of many soils

is due to the production of a volatile organic compound called geosmin A

high proportion of therapeutically useful antibiotics derive from Streptomyces

species, well-known examples are streptomycin, erythromycin and tetracycline [39]

1.2.2 Actinomycetes and secondary metabolites

Special interest in actinomycetes lies in their ability to produce secondary metabolites of highly applicable values Of all the reported natural products from microbes 45% are produced by actinomycetes, 38% by fungi and 17% by unicellular bacteria [2] Two-thirds of known antibiotics are

produced by actinomycetes, mainly by Streptomyces species [28] Various

antibiotic substances from actinomycetes have been characterized, including aminoglycosides, anthracyclins, glycopeptides, β-lactams, macrolides, nucleosides, peptides, polyenes, polyester, polyketides, 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 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 cell lines

- Study property of antibiotics produced by the selected actinomycetes via thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC)

- Study taxonomy of actinomycetes based on morphological characteristics and 16S rDNA gene sequences

CHAPTER 2 MATERIALS AND METHODS

2.1 Work flow

Collection and isolation of actinomycetes

Collection of samples from Catba island

Isolation by dry – heating and rehydration – centrifugation

Screening of antibiotic producing actinomycetes

Primary screening by agar disc method

Secondary screening by culture broth method

Chromatography analyses of antibiotics

Ethyl-acetate extraction

Analysis via thin-layer chromatography (TLC)

Analysis via high performance liquid chromatography (HPLC)

Primary study on cytotoxicity activity

Color test method

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 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 method [15]

In the dry-heating method, soil and littler samples were first dried at room temperature for 5 – 7 days, then possessed a heat treatment at 90 – 110 0

C for 10 – 30 minutes for killing non-spore forming bacteria (most actinomycete spores did not die at this condition) Afterward, the samples were spreaded onto HV medium (Humic acid - vitamin agar medium; humid acid 1 g, CaCO3 0.02 g, FeSO4.7H2O 0.01 g, KCl 1.71 g, MgSO4.7H2O 0.05

g, Na2HPO4 0.5 g, cycloheximine 50 mg, nalidixic acid 20 mg, kabicidine 14

mg, agar 18 g, H2O 1 L, pH 7.2) and incubated at 28 – 30 0C for 7 to 14 days Actinomycete colonies of different morphology were picked up and transferred to new HV agar medium for the second purification The isolates were then transferred to YS medium (yeast extract – starch agar medium; glucose 10 g, yeast extract 2 g, agar 17 g, distilled water 1 L, pH 7.0) for producing higher cell mass

The rehydration–centrifugation method is an isolation method specific for motile actinomycetes The sequential steps are as following:

- Take 0.5 g of air–dried soil or litter samples in 100 ml beakers, gently added 50 ml of 10 mM phosphate buffer (pH = 7) containing 10% soil extract solution (mix 500 g soil and 500 ml distilled water in a flask, autoclave at 121

°C for 30 minutes, afterward filtrate twice, autoclave again and kept the solution at 4 °C)

- Cover the beakers with aluminium foil and incubate statically at 30 °C for 90 minutes to allow liberation of motile zoospores

- Transfer 8 ml of the supernatant into 15 ml falcon tubes, centrifuge at room temperature for 20 minutes at 1,500 g to eliminate streptomycetes and other non-motile actinomycetes from the supernatant The tubes were then allowed to settle for 30 minutes

- Take 1 ml of the supernatant for serial dilution in sterile tap water, aliquots 0.2 ml of serial dilution of 10-2, 10-3 and 10-4 were plated onto HV agar and incubated at 28 – 30 °C for 2 – 3 weeks to produce colonies

- Actinomycete colonies were then picked up, transferred to new HV agar medium for second purification and then to YS agar medium for biomass production

All actinomycete strains isolated by two methods described above were maintained as stock cultures frozen at -80 °C in 20% glycerol solution at

Vietnam Type Culture Collection (VTCC)

2.2.2 Targeted microorganisms

Four microorganism strains, including Micrococcus luteus (a Gram positive bacterium), Escherichia coli (a Gram negative bacterium), Candida

albicans (a yeast) and Fusarium oxysporium (a filamentous fungus) were used

as targets in screening for antimicrobial activity These strains were cultivated

in proper nutrient media, i.e Mueller-Hinton medium (MHA; meat extract

0.3%, hydrolysis casein 1.75%, starch 0.15%, pH 7.4) for E coli and M

luteus, yeast/malt extract medium (YM; glucose 1%, peptone 0.5%, yeast

extract 0.3%, malt extract 0.3%) for C albicans and F oxysporum The

cultures were incubated under shaking condition at 37 °C for E coli and M

luteus or 30 °C for C albicans and F oxysporum These strains were provided by the VTCC

2.2.3 Screening antibiotic producing actinomycetes

Before using in experiments, all actinomycete strains were refreshed on

YS medium plates at 30 °C for 3–4 days After reactivation, the strains were cultivated in liquid soybean meal medium (starch 25 g, soybean meal 15 g, yeast extract 1 g, CaCO3 4 g, distilled water 1 L, pH 6.2) at 30 °C for 3 days

on shaker at 100 rpm Culture broths were then centrifuged at 8,000 rpm for

15 minutes, the supernatants were used to evaluate antimicrobial activity

Agar disc method

Agar discs (5 mm in diameter) picked from YS agar plates of well grown actinomycete cultures were placed onto surface of agar plates previously seeded with a target microorganism and incubated at proper conditions for 2 days The inhibitory effect was assessed based on the formation of clear inhibition zones around the agar discs and the activity was measured as the diameter of these zones [19]

Culture broth method

Small wells were aseptically created on solidified agar plates previousely seeded with a target microorganism Approximately 25 µl of solutions collected from the culture broths of the actinomycetes after centrifugation were applied into the wells and incubated for 2 days at proper condition to the target microorganism Antibiotic activity was assessed through the inhibitory zones formed around the wells The experiments were performed in duplicates in all cases, sterilized culture broth was used as negative control [1]

2.2.4 Ethyl-acetate extraction

Culture broths of the actinomycetes grown on soybean meal liquid medium were centrifuged at 8000 rpm for 15 min at room temperature and the supernatants were collected for the extraction To extract the antibiotic substances, equal volume of ethyl-acetate was added and the mixtures were shacked violently for 1 h The solvent was collected by using separation funnels, afterward sodium sulfate was added into solvent at 5 g.L-1 The elimination of solvent was performed in rotary evaporator and the obtained precipitates were dissolved in 1 ml of chloroform [45] Prior to chromatography, the solutions of crude extracts in chloroform were tested again for the antibiotic activity by using the culture broth method, however in this case reaction with chloroform served as negative control

2.2.5 Chromatography analyses of antibiotics

In chromatography analysis of antibiotics and cytotoxicity assay, raw extract of strain A16 which produces multi-components of anthracyclines was used as a standard of anthracyclines This strain was supplied by National Institute of Technology and Evaluation (NITE) in Japan