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

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
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
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

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]. 7

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]

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].
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 11

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

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 13

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 of the genus Streptomyces [4], [30]. Some of the best known
groups used in clinical practice include anthracyclines, actinomycins and
bleomycins.
Anthracyclines have been widely

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. 15

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

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). 17

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

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


Cytotoxicity assay

Taxonomical identification of actinomycetes
 Morphological characterization

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, CaCO
3
0.02 g, FeSO
4
.7H
2
O 0.01 g, KCl 1.71 g, MgSO
4
.7H
2
O 0.05
g, Na
2
HPO
4
0.5 g, cycloheximine 50 mg, nalidixic acid 20 mg, kabicidine 14
20

mg, agar 18 g, H
2
O 1 L, pH 7.2) and incubated at 28 – 30
0

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). 21

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, CaCO
3
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