Analisis genetik escherichia coli dari biawak (varanus spp.) asal indonesia: Analisis profil DNA dan karakterisasi integron
GENETIC ANALYSIS OF Escherichia coli FROM INDONESIAN
MONITOR LIZARDS (Varanus spp.) :
DNA PROFILING AND INTEGRON CHARACTERIZATION
BY
Diana Elizabeth Waturangi
GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
2002
ABSTRAK
Diana Elizabeth Waturangi. Analisis Genetik Escherichia coli dari
Biawak (Varanus spp.) asal Indonesia: Analisis profil DNA dan
Karakterisasi Integron.
Escherichia (E.). coli merupakan bakteri komensal pada saluran pencernaan
manusia dan hewan. Studi yang ada banyak dilakukan untuk E. coli yang
berasal dari manusia, bahan pangan serta hewan peliharaan, sedangkan
penelitian tentang keragaman genetik serta resistensi terhadap antibiotik
pada E. coli yang berasal dari reptil yang hidup di lingkungan alami, masih
sangat sedikit. Pada studi ini, isolat-isolat E. coli dari feses Varanus spp.
dianalisis keragaman genetiknya menggunakan metode ARDRA, MFLP,
ERlC serta melihat profil dari plasmid. Analisis resistensi terhadap antibiotik
dilakukan dengan mendeteksi keberadaan integron serta gen penyandi
resistensi terhadap antibiotik.
lsolasi E. coli dilakukan dari feses Varanus spp. yang berasal dari
beberapa daerah di Indonesia. DNA genom dari E. coli diamplifikasi dengan
PCR menggunakan primer 63f dan 1387r untuk analisa ARDRA, sedangkan
untuk analisis ERIC, digunakan primer ERlC 1 dan ERlC 2. Pada analisis
MFLP, DNA genom didigesti dengan
Blnl dan dipisahkan dengan
elektroforesis PFGE. Seluruh isolat diuji resistensinya terhadap beberapa
agen antimikrobe. Deteksi integron dilakukan dengan primer yang bersifat
spesifik untuk integron kelas 1. Amplikon yang dihasilkan dilakukan kloning
dan sekuensing. Plasmid dari isolat E. coli ditransformasi dan diseleksi pada
medium yang mengandung antibiotik tetrasiklin. Gen tet yang didapat lalu
dilakukan kloning dan sekuensing.
ARDRA tidak dapat menunjukkan keragaman genetik dari isolat E.
coli. Bagaimanapun juga analisis dengan MFLP dan ERlC menunjukkan
bahwa isolat E. coli beragam, dengan tingkat keragaman yang tinggi bahkan
pada isolat yang berasal dari spesimen yang sama. Analisis MFLP lebih
diskriminatif dibandingkan dengan ERIC. Seluruh isolat E. coli menunjukkan
resistensi terhadap agen antimikroba. Tiga isolat E. coli memiliki integron.
Dua diantaranya mengandung kaset gen dfrA5. Pada integron lainnya
memiliki dua kaset gen dfrA1 dan aadA1. Plasmid dari isolat ECVi6a
mengandung operon TetA yang berasosiasi dengan Tn1721. Penemuan
integron kelas 1 dengan kaset gen serta plasmid penyandi tet(A) yang
berasosiasi dengan Tn 1721, menunjukkan penyebaran yang luas dari
elemen tersebut, bahkan pada E. coli yang berasal dari hewan yang hidup di
lingkungan alami.
STATEMENT OF RESEARCH ORIGINALITY
This is to verify that the dissertation entitled :
Genetic Analysis of Escherichia coli from Indonesian Monitor
Lizards (Varanus spp.) : DNA Profiling and lntegron
Characterization
Is my own work which has never previously been published. All of the
incorporated data and information are validated and stated clearly.
Bogor, July 2002
Diana Elizabeth Waturangi
GENETIC ANALYSIS OF Escherichia coli FROM INDONESIAN
MONITOR LIZARDS (Vamnus spp.) : DNA PROFILING AND
INTEGRON CHARACTERIZATION
BY
DIANA ELIZABETH WATURANGI
A DISSERTATION
Submitted to the Bogor Agricultural University
In Partial Fulfilment of the Requirements for
the Degree Doctor of Microbiology
GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
2002
This is to certify that the dissertation
Title
Name
Student number
Study Program1
Sub Program
: Genetic Analysis of Escherichia coli from Indonesia
Monitor Lizards (Varanus spp.): DNA Profiling and
lntegron Characterization
: Diana Elizabeth Waturangi
: 99.5129
: BiologylMicrobiology
has been accepted toward fulfilment of the requirements for Doctorate degree
in Biology/Microbiology
1. Committee members
n
Dr. Antonius Suwanto. MSc
Chairman
P
Prof. Dr. Bibiana W. Lav. MSc
Graduate Program
c?
Dr. Dedv Durvadi S.
Examination Date: May 3, 2002
GENETIC ANALYSIS OF Escherichia coli FROM INDONESIAN
MONITOR LIZARDS (Vamnus spp.) : DNA PROFILING AND
INTEGRON CHARACTERIZATION
BY
DIANA ELIZABETH WATURANGI
A DISSERTATION
Submitted to the Bogor Agricultural University
In Partial Fulfilment of the Requirements for
the Degree Doctor of Microbiology
GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
2002
vii
ACKNOWLEDGMENTS
This study could not have been accomplished without the help and
support of many people. In the area of the academic study, I am especially
indebted to my dissertation committee members.
First, I would like to
express my gratitude to my mentor Dr. Antonius Suwanto, MSc for his
constant and valuable guidance in the process of the making of the
dissertation.
I would like also to express my sincere thanks to my other committee
members, Prof. Dr. Maggy T. Suhartono, MSc and Prof. Dr. Bibiana W. Lay,
MSc for their support, and encouragement.
I am also grateful for Prof. Dr. Stefan Schwarz in Institute for Animal
Science and Animal Behaviour of the Federal Research Center for
Agricultural (FAL), Celle, Germany and Prof. Dr. Walter Erdelen in UNESCO
Paris, France, for their insight and guidance during the course of my work.
A special tribute is due my colleagues and technician in Laboratory of
Molecular Biology, South East Asian Region Center for Tropical Biology,
SEAMEO-BIOTROP, Etty Pratiwi, Dinamella Wahjuningrum, Yogiara, Dwi
Suryanto, Rina Martini, Esti Puspitasari, lrawan Tan, Widanarni, Amarilla
Malik, Husen Samhari, and my best friends Nesti Sianipar, Yuhlanny, Yossy
and Lilieanny.
I think also of my gifted and faithful colleagues in Germany Kayode K.
Ojo and Corinna Kehrenberg, and for excellent technical assistance of Vera
Noding, Erika NuTJbeck, and Gisela Niemann.
Special thanks to Centre for Microbial Diversity, FMIPA, IPB for its
research support and for DAAD (German Academic Exchange Service) for
scholarship during my research in Germany.
Here, I need to express my deepest gratitude to my parents and all of
m y brothers and sisters for their prayers and encouragement.
Finally, I am very much aware that above everything else, it is God
himself who has work in the many different people, situasions, and in my self,
to make all these possible. And to Him is all the glory and praise.
CONTENTS
Page
LIST OF TABLES................................................................................xii
LIST OF FIGURES................. ..
....................................................... xiii
LIST OF APPENDICES ...........................................................................xiv
1. INTRODUCTION............................................................................................ 1
Background........................................................................................ 1
Objective....................................................................................................4
2 . LITERATURE................................................................................................. 5
2.1 Monitor Lizard........................................................................................... 5
2.2 Indigenous microorganism....................................................................... 6
2.3 Genetic Diversity of microorganism........................................................ 7
2.4
DNA profile analysis................................................................................ 8
2.4.1 ARDRA .................................................................................. 10
2.4.2 MFLP analysis............................................................................. 11
2.4.3 Analysis of repetitive DNA sequences.......................................... 12
2.5 Antibiotic resistance.................................................................................
13
....................................................................................
2.5.1 Tetracycline
14
2.5.2 Resistance to tetracycline............................................................. 16
2.5.2.1 Efflux protein.......................................................... 17
2.5.2.2 Ribosomal protection proteins.................................... 18
2.5.2.3 Enzymatic inactivation of tetracycline.......................... : 19
2.5.2.4 Mobility of tet gene...................................................19
2.5.3 Streptomycin.....................................................................20
2.5.4. Resistance to aminoglycoside............................................... 22
.......................... 22
2.5.4.1 Enzymatic inactivation................
.
............................................
2.5.4.2 Decreased permeability
23
2.5.4.3 Ribosome alteration.................................................23
2.5.4.4 Active efflux............................................................24
2.5.5 Sulfonamide......................................................................24
2.5.6 Trimethoprim ....................................................................25
2.5.7 Resistance to trimethoprim ...................................................26
2.5.7.1 Chromosomal resistance.........................................
26
2.5.7.2 Plasmid borne-resistance.......................................... 26
2.6 Tranfer of antibiotic resistance genes.............................................. 27
2.7 Integron.....................................................................................29
2.7.1 Class 1 integron................................................................. 30
2.7.2 The other classes of integrals...............................................32
Analysis of extracellular protease.................................................... 85
Antibiotic resistance analysis...........................................................86
Detection of integron..................................................................... 91
Cloning of integron........................................................................92
4.9.1 Single gene cassette.............................................................
93
4.9.2 Double gene cassettes.........................................................97
4.9.3 dfrA genecassette..........................................................................101
4.9.4 aadA I gene cassette........................................................... 102
4.10 Transformation of plasmid from E.coli..............................................103
4.1 1 Restriction mapping...................................................................... 104
4.1 1.1 Restriction analysis of pDEWTS 1......................................... 104
4.1 1.2 Restriction mapping of pDEWT 1..........................................107
4.12 Analysis of tet gene from pDEWT 1.................................................. 108
4.12.1 Cloning and sequencing of fragment from Smal digestion ........... 108
4.12.2 Cloning and sequencing of fragment from EcoRl digestion ......... 112
4.6
4.7
4.8
4.9
5 . CONCLUSION............................................................................ 118
6. REFERENCES.............................................................................
7. APPENDICES ............................................................................ 131
LIST OF TABLES
Page
2-1.
2.2 .
2-3
Position substitution R1-R4 of derivative......................................... 15
Mechanism of resistance for characterized tet and otr genes ............... 16
Gene cassette........................................................................... 33
3-1
3-2
Sequence of the primers and size of amplicon.................................. 52
53
Cycles of PCR............................................................................
4.1 .
4-2
4-3
4-4
4-5
4-6
Physiology test of bacteria............................................................ 68
Resistance phenotype. genotype of E.coli........................................ 89
Percentage of antibiotic resistance according to origin of isolates .......... 90
Approximate size of amplicons....................................................... 91
Gene cassette. position. length. and coding region............................. 93
Phenotypic and genotypic resistance of transformants............................ 103
LIST OF FIGURES
Page
Structure formula of tetracycline.................................................... 15
Structure formula of streptomycin enzymatic.................................... 22
Structural of sulfonamide. trimethoprim and dihydrofolic acid............... 25
General structure of integron 1...................................................... 30
Structure of gene cassette............................................................ 34
Model for cassette acquisition.........................................................37
Boundaries of gene cassettes....................................................... 38
Cassette fusion................................................................................40
Comparison of attl and 59-be........................................................ 42
Model for replacement of 59-be....................................................... 43
Fecal collection of Varanus spp ....................................................... 68
ARDRA employing Haelll and Sau3A.............................................. 71
ARDRA employing Cfol and Rsal ...................................................72
ARDRA employing Cfol................................................................73
75
Analysis of MFLP.........................................................................
Dendogram from MFLP analysis..................................................... 77
ERIC analysis..............................................................................81
Dendogram of ERIC analysis.......................................................... 82
Plasmid profile............................................................................. 83
Analysis of proteolytic....................................................................85
Percentage of antibiotic resistance................................................... 86
Schematic presentation of dfrA5 cassette.......................................... 95
Schematic presentation of dfrA5 cassette.......................................... 99
Restriction digest of pDEWTS 1...................................................... 105
Restriction map of pDEWTS 1.........................................................106
Restriction digest of pDEWT 1......................................................... 107
Comparison of Tn 7 727 and pDEWT 1............................................... 110
Estimation of site of recombination in pDEWT 1..................................114
1. INTRODUCTION
The natural habitats of microorganism are exceedingly diverse. The
wide spread occurrence of bacteria extends to regions ranging from the
upper atmosphere to sediments on the ocean bed. Any habitat suitable
for the growth of higher organisms can also support growth of
microorganism. But in addition, there are many habitats where, because
of some physical or chemical extreme, higher organism are absent yet
.microorganism exist and occasionally even flourish.
Bacteria occur most abundantly in habitats where they find nutrition,
moisture and a temperature appropriate for their growth and replication.
The conditions that favor the survival and growth of many microorganisms
are those under which higher organism normally live.
Microorganism inhabit the surface of higher organism and in some
cases live within plants, humans and animals. Microorganisms frequently
reach large numbers in such habitats and may benefit the animal in
nutritionally significant way (Brock & Madigan, 1997).
Animal body
provides favorable environments for the growth of microorganisms, and
microorganisms enter into various degrees of symbiotic relationship with
the host.
The microorganisms present at any site in a healthy animal are
collectively referred to as the normal microbiota.
Interaction between
normal microbiota and the host can be divided into different kinds of
relationship such as mutualisms, commensals and parasitisms.
The normal microbiota usually inhabits the skin, and the mucosal
surface of the oral cavity, the upper respiratory tract, the urinary tract and
the gastrointestinal tract. Microbiota residing in the intestinal tract usually
provide beneficial effects to the host. They are responsible for a wide
variety of metabolic reactions and assist in the enzymatic break down of
food and the production of certain vitamins that can be utilized by the host
like niacin, vitamin B1, 82, B6, 812 and vitamin K as well as folic acid and
biotin.
They play a considerable role as one of the major defense
mechanisms that protects the animal body against colonization by
invading pathogens, and stimulate the immune systems (Berg, 1996).
Intestinal microbiota also have a direct impact on the morphology of the
intestine; e.g. the degradation of mucus glycoproteins produced by the
epithelium is assigned to components of the intestinal microbiota (Falk et
a/. 1998).
The composition of the normal microbiota may vary with the animal
species, feed and environment.
Various skin surfaces and mucosal
membranes in animals including the contents of the digestive tract have a
microbiota with a characteristic composition. Microbiota of the intestines
contain various groups of bacteria among which is Enterobacteriaceae, for
instance Escherichia (E.) coli and various anaerobic bacteria.
E. coli is a microbiota of the human and animal digestive.
Nevertheless, pathogenic strains are an important cause of sickness and
mortality throughout the world.
Surprisingly, little is known about the
genetic diversity of E. coli in population of wild animal. Studies about
genetic diversity is important for identification
(Graf, 1999), typing
(Saverkoul et al., 1999), evolution (Belicum et al. 1998), epidemiology and
pathogenicity of the bacteria (Beltran et al. 1999; Nagy et a/. 1999).
A high degree of genetic diversity has been found in E. coli isolated
from sewage (Pupo and Richardon, 1995) and E. coli from mammalian
species in different continents (Souza et a/. 1999).
Yogiara et a/. (2001) reported that E. coli isolated from the buccal of
Varanus showed extensive genetic diversity. However, analysis of genetic
diversity of E. coli obtained from the gastrointestinal microbiota of Varanus
is required, because microbiota in the gastrointestinal tract might have
more specific relationship with their host than bacteria from mouth. In
addition, characteristics of the indigenous bacteria species might be able
to show the diversity and grouping of Varanus.
In this study, we used Amplified Ribosomal DNA Restriction
Analysis (ARDRA) (Graf, 1999), Macrorestriction Fragment Length
Polymorphism (MFLP) analysis employing
PFGE (Pulsed Field Gel
Electrophoresis) (Hudson et a/. 2000),
Enterobacterial Repetitive
Intergenic Consensus (ERIC) analysis (Lupsky and Weinstock, 1992) and
plasmid profiles to determine the genetic diversity of E. coli isolates
obtained from Varanus spp. In Indonesia.
Isolated bacteria were screened for
their
resistances to
antimicrobial agents, and the resistance genes were characterized. Since,
bacteria in their natural environment often exhibit resistance to many types
of antibiotics (Desselberger, 1998).
Data on antibiotic resistance in enterobacteria are usually found
only for clinical strains. Very patchy data on enterobacteria in the fecal
samples have been published, and thus there is only few data available on
how their resistance is acquired and maintained (Dunlop et a/. 1989).
Our preliminary study indicated that there are many isolates of
E. coli from feces of Varanus spp. exhibited resistance to many types of
antibiotics. Thus far antibiotic resistance was only characterized
phenotypically, which might not indicate their genetic properties. More
information is needed about the nucleotide sequences, diversity, stability
and movement of antibiotic resistance genes. Based on our results, we
thought it is necessary to characterize the genetic basis of these antibiotic
resistances and the mobile genetic elements which play a major role in the
transfer of the resistance genes.
The objectives of this research are:
1. To Analyze genetic diversity of E. coli isolated from feces of Varanus
spp. employing Analysis of ARDRA, Macrorestiction Fragment Length
Polymorphism (MFLP), Enterobacterial Repetitive Intergenic Consensus
(ERIC) and plasmid profile.
2. To study antibiotic resistance and genetic basis of the resistance
through detection of integrons 1 gene cassettes and analysis of the
antimicrobial resistance genes.
2. LITERATURE
Monitor lizard
2.1
Varanids are more widely known as monitor. In some places, they are
also known as leguaans or goannas, terms derived from the carib "iguana"
for the distantly related large iguana. The taxonomy of this organism is
described below.
Kingdom
: Animal
Phylum
: Chordata
Class
: Reptilia
Order
: Squamata
Suborder
: Sauria
Family
: Varanidae
Genus
: Varanus
Monitors are characterized by elongate heads, moveable eyelids,
external ear openings, four-well developed limbs with five digit and claws,
a long, bifid, and refractile tongue, and a visible parietal eye. Ventral
scales are rectangular and arranged in rows, all monitors are egg layers
(Bennett, 1998).
In Indonesia there is a type of lizards that can reach sizes that may
have thought only to exist in fairy tales. These unique reptiles have been
able to develop in an area where there is little for large animals to live on.
Monitor lizards of lndonesia that have been reported are Varanus
doreanus (The Blue-tailed monitor); V. indicus (The Mangrove monitor);
V. jobiensis (The Peach-Throated monitor); V. bengalensis nebilosus (The
clouded monitor); V. dumerii; V. gouldii (panoptes) (The New Guinea Sand
monitor). V. prasinus (The green tree monitor); V rudicolis (The black
roughneck monitor); V. salvadorii (The crocodile monitor); V. salvator (The
Asian water monitor); V. timorensis (The Timor monitor); V. melinus (The
yellow-headed monitor); V. yuwonoi (The black-backed mangrove monitor)
and V. komodoensis. The Varanus as it is with all vertebrates owe their
existence at least partially, to several biological processes catalyzed by
enzymes produced by bacteria commensals within their alimentary tract.
Hence, studies on these reptiles will be incomplete without a thorough
understanding of their normal microbiota.
2.2 Indigenous microorganisms
lndigenous microorganism normally inhabit the skin, oral cavity,
upper respiratory tract, gastrointestinal tract, and urinary tract of an
animal. The indigenous microorganism does not appear spontaneously in
newborn humans or animals; instead, certain microbes colonize particular
intestinal habitats at various times after birth.
These microbes are
characteristic for that particular habitat and animal host.
Interaction
between normal microbiota and the host can be divided into different kinds
of relationship. Mutualism in which both the microbes and the host benefit
from each other, commensalism which might be beneficial for
microorganism, but does not seem to be of any benefit to the host, and
parasitism which may be harmful to the host and produce disease under
certain circumstances (Berg, 1996).
The majority of the normal microbiota exhibit mutualistic or
communal relationship with the host. The relationship can change if local
anatomic barrier are breached (trauma) or changing in many chemical,
cellular and immunological mechanisms that have evolved to prevent
bacterial infections (Salyers and Whitt, 1994).
Escherichia coli is a member of the normal microbiota that inhabits
the gastrointestinal tract of humans and animals.
Nevertheless,
pathogenic strains are important since they cause serious and sometimes
fatal disease conditions in humans and animals throughout the world.
Some strains of E. coli have acquired multiple resistance to antibiotics
(Neu, 1992) and infections caused by these E. coli strains are difficult to
control. E. coli regarded as an important source of antibiotic resistance
determinants for other human or animal pathogenic bacteria (Dunlop et al.
1998).
2.3 Genetic Diversity of microorganism
Various influences on bacterial diversity have been identified, such
as spatial separation, and specific bacterium-host interaction (Souza et al.
1992). These influences can apparently differ between one species and
another.
Diversity in symbiotic and pathogenic bacteria seems to be lower
than in 'free living' bacteria (Latour et al. 1996). It may be due to the
unidirectional and predominating influence of the host, whereas bacteria
exposed to a variety of selective forces may have maintained a higher
adaptive capacity (Schloter et a/. 2000).
With the rise of molecular genetic tools in microbial ecology, it
became apparent that we know only a very small part of the diversity in
the microbial world, a bacterial species might compose of different genoand phenotypes. There is even more structural and functional diversity
below the species level.
Detailed molecular genetic information may help us to understand
evolution of the functionality of microbes in their particular environment,
creating new genetic variants below the species level (Schloter et a/.
I
2000).
2.4 DNA profile analysis
The analysis of DNA is an increasingly important tool in studies of
I
evolutionary developments, ecology, population genetics and systematics.
I
DNA has several significant advantages over alternatives such as proteins
for molecular systematics : The genotype rather than the phenotype is
assayed,
one or more sequences appropriate to a problem can be
1
selected on the basis of evolutionary rate, the methods are generally
applicable to any type of DNA, and DNA can be prepared from small
,
j
I
amounts of tissue or bacteria and DNA is also relatively stable when
handled appropriately (Hillis et a/. 1996).
Various methods have been developed for the identification, typing
and studies of the diversity of prokaryotic and eukaryotic organisms at the
DNA level. These methods differ in their taxonomic range, discriminatory
power, reproducibility and ease of interpretation and standardization. The
ideal method produces results that are invariable from laboratory to
laboratory and allows unambiguous comparative analyses and the
establishment of reliable databases (Morrel, 1997).
Genotyping methods differ in their power of discrimination,
depending on the taxonomic level and category.
In bacteriology,
discrimination to the species level is mostly referred to as identification,
while typing denotes differentiation to the strain level (Savelkoul et a/.
1999).
Some techniques have the capability to differentiate until
subspecies level, such as ARDRA (Hudson et a/. 2000). Other methods
obviously have the capacity to differentiate until strain level, such as DNA
sequencing, MFLP analysis using PFGE (Graf, 1999) and PCR analysis of
repetitive DNA such as Repetitive Extragenic Palindromic (REP) element
and Enterobacterial Repetitive lntergenic Consensus (ERIC) sequences
(Lupsky and Weinstock, 1992).
Differences in the number or pattern of DNA fragments can arise
through a number of distinct processes, including the fragment size, the
structure of DNA and the number or distribution of restriction sites. In
restriction site variation, the variation in fragment pattern revealed
following digestion with one or more restriction enzymes are referred to as
restriction fragment length polymorphisms (RFLPs) (Pousser et 81. 2000).
Base substitutions can create or eliminate cleavage sites for a particular
enzyme, thereby altering the number and size of fragments detected by
that enzyme alone (Hillis et a/. 1996).
All of these methods are based on mutations in restriction sites or
length variation of restriction fragment. But PFGE and repetitive PCR also
correspond to mutations which are dispersed over the genome (Savelkoul
et 81. 1999). Combination of several techniques might result in higher
discrimination power.
2.4.1 ARDRA
Cells contain three types of RNA: messenger RNA (mRNA),
transfer RNA (tRNA), and ribosomal RNA (rRNA). All these forms of RNA
are present in both prokaryotic and eukaryotic cells.
Ribosomal RNA
molecules constitute the bulk (75%) of cellular RNA. They are present in
ribosomes as three different molecules, classified by size: 28S, 18S, and
5s in the eukaryotic ribosome, or 23S, 16s and 5s in the prokaryotic
ribosome (Krieg, 1996). The comparison of rRNA sequences is a powerful
method for deducing phylogenetic and evolutionary relationships among
Bacteria, Archaea, and Eukarya (Ross et 81.2000).
The
ability to
establish
phylogenetic relationships among
microorganisms using ribosomal RNA sequences has been well
documented (Braun-Howland et 81. 1992). Ribosomal RNAs are excellent
molecules for discerning evolutionary relationship among living organisms.
These molecules are functionally and evolutionarily homologous in
different organisms; ancient molecules with extremely conserved
structures and nucleotide sequences; very abundant within cells; making
them easy to isolate from all types of organisms; large enough to permit
statistically significant comparisons of their sequences (Clayton et a/.
1995). A method has been published that relies on the use of a single pair
of primers to amplify the DNA encoding a variable region of the 16s rRNA
gene (Borrell et a/. 1997). The amplified DNA is subsequently subjected
to RFLP analysis.
2.4.2 MFLP analysis
Pulsed-field gel electrophoresis (PFGE) currently offers the most
expedient means to both analyze genome size and construct lowresolution physical maps of bacterial chromosomes (Bergthorsson and
Ochman, 1995).
Digestion of bacterial genomic DNA with rare-cutting restriction
endonucleases and separation of the fragments by PFGE is also now
used extensively for bacterial-strain typing (Maule, 1998). The DNA
fragment patterns obtained with this method, provide an estimate of the
degree of genomic relationship between strains that are important for most
epidemiological or clinical purposes (Suwanto, 1994).
Genomic
fingerprinting employing PFGE is a reliable technique that generates
reproducible results, which make
PFGE analysis a suitable general
technique that deserves consideration for investigating the epidemiology
of most microorganisms.
2.4.3 Analysis of repetitive DNA sequences
Repeated sequences are present in the genomes of all organisms.
The best known of these Alu are family of sequences identified in
mamalian species. Prokaryotic genomes are much smaller than the
genomes of mammalian species that may have been maintained through
selective pressure for rapid DNA replication and cell reproduction.
Noncoding repetitive DNA would likely be kept to a minimum under natural
selection for rapid growth, unless these sequences maintain themselves
as 'selfish' DNA (Vefsalovic et al. 1991).
The ubiquitous nature and seemingly random chromosomal
distribution of these repeats in prokaryotic genomes suggest that like
more-complex eukaryotes, the DNA sequences rearrangement in the
genomes of eubacteria may consist of short repeats interspersed with
longer single-copy sequence (Belikum et a/. 1998). Although the precise
function of repetitive sequences in prokaryotic genomes are obscure, their
presence can be exploited for several applications and molecular genetic
manipulations (Lupsky and Weinstock, 1992).
To assess the distribution and evolutionary conservation of two
distinct prokaryotic repetitive elements, consensus oligonucleotides were
used in PCR amplification. In this study, the distribution of repetitive DNA
sequence was examined by analysis of the ERIC sequences. These
sequences are loated in non-coding transcribed regions of the
chromosome, in either orientations with respect to transcription, and
include a conserved inverted repeat.
2.5 Antibiotic resistance
Acquisition of additional genes into the chromosomal DNA of
bacteria is one of the factors which causes genetic diversity in bacteria.
The acquired gene usually enables the bacteria to live in specific
ecological niches. Such acquired genes are considered as auxiliary genes
and their function include for example ability to degrade specific
substrates, resistance to heavy metals and also resistance to antibiotics.
Recent data indicated that antibiotic resistance has spread quickly
and dramatically among bacteria. The dissemination is an increasing
problem for the efficient control of infectious diseases. In their natural
environment there ils a scarcity of data on the occurrence of antibiotic
resistant-bacteria (Diesselberger, 1998).
High frequency of antibiotic resistance have been found in
enterobacteria in fecal flora as well as in clinical isolates. Each antibiotic
class has their own target and specificity. The mechanisms of antibiotic
resistance in bacteria are also different for each antibiotic. Sometimes for
one antibiotic there is only one mechanism of resistance known, for others
there are up to seven different mechanism known (Schwarz and Chaslus-
Danda, 2001).
2.5.1 Tetracycline
Tetracycline inhibit bacterial protein synthesis by preventing the
association of aminoacyl-tRNA with the bacterial ribosome. The most
simple tetracycline to display detectable antibacterial activity is 6-deoxy-6demethyltetracycline (Fig. 2-1) and substitution derivatives of this antibiotic
are described in Table 2-1.
To interact with their target sites, these
molecules need to cross one or more membrane systems depending on
whether the susceptible organism is gram-positive or gram-negative
(Chopra et 81. 1992).
Tetracycline cross the outer membrane of gram-negative enteric
bacteria through the OmpF and OmpC porin channels, as positively
charged
cation
probably
(magnesium-tetracycline)
coordination
complexes. The cationic metal ion-antibiotic complex is attracted by the
Donnan potential across the outer membrane, leading to accumulation in
the periplasm, where the metal ion-tetracycline complex probably
dissociates to liberate uncharged tetracycline, a weakly liphopilic molecule
able to diffuse through the lipid bilayer regions of the inner (cytoplasmic)
membrane (Schnappinger and Hillen, 1996).
I
Figure 2-1. Structure formula of tetracycline (Walter and Meyer, 1987).
I
Table 2-1. Position substitution R1-R4 of derivative
---- --
Derivative
R1
R2
R3
R4
Tetracycline
H
CH3
OH
H
Chlortetracycline CI
CH3
OH
H
Oxytetracycline
H
CH3
OH
OH
Doxycycline
H
CH3
H
OH
Minocyclin
N(CHs)2
H
H
H
--
2.5.2 Resistance to tetracycline
Three major mechanisms of resistance to tetracycline have been
described i.e. efflux, ribosomal protection, and enzymatic inactivation
(Table 2-2).
Table 2-2. Mechanism of resistance for characterized tet and otr genes
(Levy et 81. 1999)
Genes
Efflux
t e W , tet(B),tet(C),teqD), tet(E),tet(G),teqH),teql),
tet(J),tet(Z),tet(30)
tet(K), tet(L)
otr(B),tcn
tet(P)A
teto
tet(Y)
Ribosomal protection
tet(M),tet(O),teqs), teyw)
tet(Q),tet(T)
oWA), tet(P)B
Enzymatic inactivation
tet()o
Unknown
tet(U),
-- -- - --- --
- --- - ---- -
--
2.5.2.1 Efflux protein
All the tet efflux genes encode for membrane-associated proteins
which export tetracycline from the cell. Export of tetracycline reduces the
intracellular drug concentration and thus protects the ribosomes within the
cell.
The genes encoding efflux proteins belong to the major facilitator
superfamily (MFS), whose product include over 300 individual proteins
(Paulsen et a/. 1996). All the tet efflux genes code for membraneassociated proteins which export tetracycline, but no other substances
from the cell. Efflux genes are found in both gram negative and gram
positive species. The gram negative efflux genes are widely distributed
and normally associated with large plasmids, most of which are
conjugative (Jones et a/. 1992).
Each of the efflux genes codes for an approximately 46-kDa
membrane-bound efflux protein. These proteins have been divided into
six groups based on amino acid sequence identity.
Group 1 contain
Tet(A), Tet(B), Tet (C), Tet (D), Tet (E), Tet (G), Tet (H), Tet (Z), and
probably Tet (I), Tet (J). The tetracycline resistance protein in this group
have 41 to 78% amino acid identity (Tauch et a/. 2000). Most of these
efflux proteins appear to reside in the lipid bilayer, with the hydrophilic
amino acid loops protruding into the periplasmic and cytoplasmic space.
The efflux proteins exchange a proton for a tetracycline cation complex
against a concentration gradient (Yamaguchi et a/. 1990). The efflux
genes from gram-negative bacteria have two functional domains, a and
P,
which correspond to the N-and C-terminal halves of the protein (Rubin and
Levy, 1991).
Group 2 includes Tet (K) and Tet (L) , with 58 to 59% amino acid
identity; these proteins are found primarily in gram-positive species. This
group has 14 predicted transmembrane a-helices. These genes code for
proteins which confer resistance to tetracycline and chlortetracycline.
Group 3 includes Otr(B) and Tcr3, both found in Streptomyces spp. These
proteins have topology similar to group 2 proteins, with 14 predicted
transmembrane a -helices. Group 4 includes TetA(P) from Clostridium
spp., with 12 predicted transmembrane a-helices, while group 5 includes
T e t o from Mycobacterium smegmatis.
Group 6 includes unnamed
determinants from Corynebacterium striatum (Chopra and Roberts, 2001).
2.5.2.2 Ribosomal protection proteins
These are cytoplasmic proteins that protect the ribosome from the
action of tetracycline. The binding of these proteins to the ribosome is
believed to cause an alteration in ribosomal conformation which prevents
tetracycline from binding to the ribosome, without altering or inhibiting
protein synthesis. The hydrolysis of GTP may provide the energy for the
ribosomal conformational change. The ribosomal protection proteins also
need to dissociate from the ribosome to allow EF-G to bind, since they
have overlapping binding sites on the ribosome (Triber et a/. 1998)
2.5.2.3 Enzymatic inaativation of tetracycline
The tet(X) gene encodes the only example of tetracycline
resistance due to enzymatic alteration of tetracycline. The tet(X) gene
product is a 44-kDa cytoplasmic protein that chemically modifies
tetracycline in the presence of both oxygen and NADPH (Speer et a/.
1991).
2.5.2.4 Mobility of tet genes
Tetracycline resistance genes are found in many bacteria isolated
from human, animals and environment.
The widely distribution of tet
genes is based on their association with mobile genetic elements like
plasmids orland transpasons (Jones et a/. 1992). The gram-positive efflux
genes tet(K) and tet(L) are associated with small plasmids (Schwarz et a/.
1992). The gram-negative tet efflux genes are often found on transposons
inserted into a diverse group of plasmids from a variety of incompatibility
groups (Jones et a/. 1992). The ribosomal protection genes tet(S) and
tet(Q) can be found oln conjugative plasmids, or in the chromosome,
where they are non self-mobile (Charpentier et a/. 1994). The tet(M) and
tet(Q) genes are generally associated with conjugative chromosomal
elements, which code for their own transfer (Salyers et a/. 1995). These
conjugative transposons are also able to transfer mobilizable plasmids to
other isolates and species and even unlinked genomic DNA (Shows et a/.
1992).
2.5.3
Streptomycin
Streptomycin is the first aminoglycoside antibiotic.
The basic
chemical structure required for both potency and the spectrum of antibiotic
activity of aminoglycoside is that of one or several aminated sugars joined
in glycosidic linkages to a dibasic cyclitol (Fig. 2-2). Their bacterial activity
is attributed to the irreversible binding to the ribosomes although their
interaction with other cellular structures and metabolic process has also
been considered. They have a broad spectrum (Mingeot-Leclercq et a/.
1999). Spectinomycin which is an aminocyclitol devoid of aminosugars is
by extension included in the family of aminoglycosides. It also differs from
them by its bacteriostatic activity and by its way of action. Spectinomycin
acts on protein synthesis during the mRNA-ribosome interaction and it
does not lead to mistranslation like some aminoglycosides do.
Transport of aminoglycosides across the cytoplasmic membrane is
dependent upon electron transport and is termed energy dependent phase
I (EDP I) (Bryan and Kwan, 1983). In the cytosol, aminoglycosides bind to
the 30s sub-unit of ribosomes, again through an energy-dependent
process (EDP-II) while this binding does not prevent formation of the
inhibition complex of peptide synthesis, (Binding of mRNA, fMetRNA, and
association of the 50s sub unit). It causes misreading and or premature
termination (Melaneon et a/. 1992). The aberrant proteins may be inserted
into the cell membranes leading to altered permeability and further
stimulation of aminoglycoside transport (Busse et a/. 1992).
R=
Shvtptomycin
CHO
Dih~dmstreptomycin CH20H
Figure 2-2. Structure formula of streptomycin and its derivative.
2.5.4 Resistance to aminoglycoside
Four mechanisms have been recognized, namely enzymatic
inactivation, ribosome alteration, decreased permeability, and efflux
system. The first mechanism is of most clinical importance since the
genes encoding aminoglycoside modifying enzymes can be disseminated
by plasmids or transposons.
2.5.4.1 Enzymatic inactivation
Enzymatic Inactivation of aminoglycosides is conferred by Nacetyltransferases, O-adenyltransferasesor or O-phosphotransferases.
These enzymes are classified into three major classes according to the
type modification: AAC (acetyltransferase) which use acetyl-coenzyme A
as donor and affect amino functions. ANT (nucleotidyltransferases also
referred to as AAD adenyltransferases), APH (phosphotransferases)
which both use ATP as a donor and affect hydroxyl functions (Shaw et a/.
1993).
New aminoglycoside-inactivating enzymes have been identified, the
genes of some of them are part of integrons (Sandvang, 1999).
Aminoglycoside-modifying enzymes catalyze the covalent modification of
specific amino or hydrolize functions, leading to a chemically modified
drug which binds poorly to ribosomes and for which the EDP-II of
accelerated drug uptake also fails to occur, thereby resulting most often in
high-level resistance (Mingeot-Leqlercq et a/. 1999).
The genes of aminoglycoside-modifying enzymes are often plasmid
encoded but are also associated with transposable elements. Plasmid
exchange and dissemination of tranposons facilitate the rapid acquisition
of a drug resistance phenotype not only within a given species but among
a large variety of bacterial species.
There are some reports about
evidence of aad encoding resistance to streptomycin and spectinomycin
and aac genes as parts of gene cassettes in integrons (Centron and Roy,
1998; Sandvang, 19919; Adrian et a/. 2000). The association of these
antibiotic resistance genes with integrons allows transfer of these
resistance genes from one bacterium to others.
2.5.4.2 Decreased permeability
Absence of or alteration in the aminoglycosides transport system,
inadequate membrane potential, modification in the LPS phenotype can
result in a cross resistance to all aminoglycoside (Salyers and Whitt,
1995). Membrane impermeabilization with reduced drug uptake has been
found in Pseudomonas (Chambers and Sande, 1995).
2.5.4.3 Ribosome alteration
High level resistance to streptomycin and spectinomycin can be
resulted from single step mutations in chromosomal genes encoding
ribosomal proteins rpsL (or strA), rpsD (or ramA or sud2), rpsE (eps or spc
or spcA). Mutation in strC (or strB) generate a low-level streptomycin
resistance (Eliopoulos et a/. 1989).
2.5.4.4 Active efflux
Resistance by active efflux has been reported in E. coli for
neomycin, kanamycin, and hygromycin A antibiotics (Nikaido, 1996), Such
as AcrD in E. coli (Rosenberg et a/. 2000), and M e w in Pseudomonas
(Aires etal. 1999).
2.5.5 Sulfonamides
Sulfonamides are one of the antimicrobials that were used in the
treatment of a variety of bacterial infections. The target of this group of
antimicrobial is dihydropteroate synthase (DHPS), which catalyzes the
formation of dihydropteroic acid in bacteria and some eucaryotic cell
(Brown, 1962). This molecule is not present in human cells (Huovinen et
a/. 1995)
The structure of sulfonamides is analogous to that of p-amino
benzoic acid (PABA), the substrate of the DHPS. Sulfonamides act as an
inhibitor and work competitively with the original substrate (Fig. 2-3).
The antimicrobial resistance gene of sulfonamide can be located on
chromosome. It appears because of the mutation in the folP gene that
result in low level of sulfonamide resistance (Huovinen et al. 1995).
Resistance to high concentration of this antibiotic has been
observed in gram-negative and the genes were located on plasmids. Two
such plasmid borne-resistance genes have been found. The gene sull is
found almost exclusively on integron structures carried by large
conjugative plasmids (Sundstrom et a/. 1988) and the other gene is su12
that is usually found on small mobilizable plasmids (Rgdstorm et a/. 1988).
2.5.6 Trimethoprim
Trimethoprim is an antibiotic commonly used in the treatment of
infections caused by gflam-negative bacteria. It is a synthetic antibacterial
agent that belongs to the diaminopyrimidine group of compounds. It can
be regarded as an a~ntifolate,a structural analogue to folic acid and
competitively inhibits the reduction of dihydrofolate to tetrahydrofolate by
dihydrofolate reductase (DHFR), an enzymatic reaction for synthesis of
thymine (Fig. 2-3).
Figure 2-3. Structural of sulfonamide(a), dihydrofolic acid(b), and
trimethoprim(c). demonstrating structural similarities relative to
competitive ilnhibition.
2.5.7 Resistance to trimethoprim
2.5.7.1 Chromosomal resistance
Resistance to trimethoprim due to mutational changes in the
intrinsic dfr gene has been reported in several pathogens.
Several
changes were observed in different parts of the structural gene in different
isolates including the C-terminal part not known to participate in the
binding of substrates or inhibitors. These changes lead to an altered
secondary structure of the enzyme causing a loss or decrease in site of
binding of trimethoprim (Skbld, 2001).
2.5.7.2 Plasmid borne-resistance
The gene of the trimethoprim-resistant can be located on plasmids
or in the chromosomarl DNA.
The most common mechanism is the
production an additional plasmid-mediated DHFR (Amyes and Smith,
1976).
At least sixteen trimethoprim resistance enzymes in enterobacteria
have been characterized and grouped, based on the kinetic properties and
the nucleotide sequences of their genes.
The largest and the most
prevalent of these groups are type I-like enzyme, which include dfrl, dfrlb,
dfr5, dfrfi, and dfr7 (Huovinen et
81.
1995). This group of enzyme is
identified by an open reading frame (ORF) of 157 amino acid residues,
and the members of this group share between 64 and 88 % amino acid
sequence identity in this ORF. Most of them are associated with integrons
as a gene cassette (Recchia and Hall, 1995).
The most prevalent trimethoprim resistance gene is dfrl, which
occurs in a cassette in both class 1 and class 2 integrons. This is also the
reason why the trimelthoprim resistance genes have spread quickly,
because via integron, transfer can happen horizontally.
2.6 Transfer of antibiotic resistance genes
Bacteria have access, in principle, to a large selection of resistance
genes scattered throughout the bacterial kingdom and mechanisms have
evolved to reassort these genes, moving them genetically from one DNA
molecule to another and physically from one bacterial cell to another.
Several mechanlisms for the acquisition and dissemination of
resistant determinants involve DNA exchange and in this way resistance
genes can spread efficiently among bacterial populations from animals
and humans (Davies, 1994).
The former type involves conjugation,
transformation and tranlsduction mechanism have been demonstrated to
be important in the movement of resistance gene among bacteria. DNA
integration into their host genome could be achieved through: (i) classical
recombination, which is RecA-dependent and requires extensive
homology between the recombining DNA molecules; (ii) transposition,
which normally involves a discrete transposable element and requires no
homology between the transposable element and the molecule in which it
integrates; (iii) site-specific recombination, which involves recombination
between short homologous sequences mediated by recombination
enzymes specific for recombination site.
This mechanism has been
shown to be involved in the dissemination of resistance gene via integrons
and gene cassettes.
An efficient route of acquisition and vertical and horizontal
dissemination of resistance determinants is through mobile genetic
elements including plasmids, transposons, and integrons, they usually
cany one or more antibiotic resistance genes (Francia et a/. 1999).
Plasmids are extrachromosomal DNA elements, capable of
autonomous replication due to their replication systems. The properties of
these elements are not essential for the bacteria under physiological
conditions, but may be of benefit for the bacterium
MONITOR LIZARDS (Varanus spp.) :
DNA PROFILING AND INTEGRON CHARACTERIZATION
BY
Diana Elizabeth Waturangi
GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
2002
ABSTRAK
Diana Elizabeth Waturangi. Analisis Genetik Escherichia coli dari
Biawak (Varanus spp.) asal Indonesia: Analisis profil DNA dan
Karakterisasi Integron.
Escherichia (E.). coli merupakan bakteri komensal pada saluran pencernaan
manusia dan hewan. Studi yang ada banyak dilakukan untuk E. coli yang
berasal dari manusia, bahan pangan serta hewan peliharaan, sedangkan
penelitian tentang keragaman genetik serta resistensi terhadap antibiotik
pada E. coli yang berasal dari reptil yang hidup di lingkungan alami, masih
sangat sedikit. Pada studi ini, isolat-isolat E. coli dari feses Varanus spp.
dianalisis keragaman genetiknya menggunakan metode ARDRA, MFLP,
ERlC serta melihat profil dari plasmid. Analisis resistensi terhadap antibiotik
dilakukan dengan mendeteksi keberadaan integron serta gen penyandi
resistensi terhadap antibiotik.
lsolasi E. coli dilakukan dari feses Varanus spp. yang berasal dari
beberapa daerah di Indonesia. DNA genom dari E. coli diamplifikasi dengan
PCR menggunakan primer 63f dan 1387r untuk analisa ARDRA, sedangkan
untuk analisis ERIC, digunakan primer ERlC 1 dan ERlC 2. Pada analisis
MFLP, DNA genom didigesti dengan
Blnl dan dipisahkan dengan
elektroforesis PFGE. Seluruh isolat diuji resistensinya terhadap beberapa
agen antimikrobe. Deteksi integron dilakukan dengan primer yang bersifat
spesifik untuk integron kelas 1. Amplikon yang dihasilkan dilakukan kloning
dan sekuensing. Plasmid dari isolat E. coli ditransformasi dan diseleksi pada
medium yang mengandung antibiotik tetrasiklin. Gen tet yang didapat lalu
dilakukan kloning dan sekuensing.
ARDRA tidak dapat menunjukkan keragaman genetik dari isolat E.
coli. Bagaimanapun juga analisis dengan MFLP dan ERlC menunjukkan
bahwa isolat E. coli beragam, dengan tingkat keragaman yang tinggi bahkan
pada isolat yang berasal dari spesimen yang sama. Analisis MFLP lebih
diskriminatif dibandingkan dengan ERIC. Seluruh isolat E. coli menunjukkan
resistensi terhadap agen antimikroba. Tiga isolat E. coli memiliki integron.
Dua diantaranya mengandung kaset gen dfrA5. Pada integron lainnya
memiliki dua kaset gen dfrA1 dan aadA1. Plasmid dari isolat ECVi6a
mengandung operon TetA yang berasosiasi dengan Tn1721. Penemuan
integron kelas 1 dengan kaset gen serta plasmid penyandi tet(A) yang
berasosiasi dengan Tn 1721, menunjukkan penyebaran yang luas dari
elemen tersebut, bahkan pada E. coli yang berasal dari hewan yang hidup di
lingkungan alami.
STATEMENT OF RESEARCH ORIGINALITY
This is to verify that the dissertation entitled :
Genetic Analysis of Escherichia coli from Indonesian Monitor
Lizards (Varanus spp.) : DNA Profiling and lntegron
Characterization
Is my own work which has never previously been published. All of the
incorporated data and information are validated and stated clearly.
Bogor, July 2002
Diana Elizabeth Waturangi
GENETIC ANALYSIS OF Escherichia coli FROM INDONESIAN
MONITOR LIZARDS (Vamnus spp.) : DNA PROFILING AND
INTEGRON CHARACTERIZATION
BY
DIANA ELIZABETH WATURANGI
A DISSERTATION
Submitted to the Bogor Agricultural University
In Partial Fulfilment of the Requirements for
the Degree Doctor of Microbiology
GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
2002
This is to certify that the dissertation
Title
Name
Student number
Study Program1
Sub Program
: Genetic Analysis of Escherichia coli from Indonesia
Monitor Lizards (Varanus spp.): DNA Profiling and
lntegron Characterization
: Diana Elizabeth Waturangi
: 99.5129
: BiologylMicrobiology
has been accepted toward fulfilment of the requirements for Doctorate degree
in Biology/Microbiology
1. Committee members
n
Dr. Antonius Suwanto. MSc
Chairman
P
Prof. Dr. Bibiana W. Lav. MSc
Graduate Program
c?
Dr. Dedv Durvadi S.
Examination Date: May 3, 2002
GENETIC ANALYSIS OF Escherichia coli FROM INDONESIAN
MONITOR LIZARDS (Vamnus spp.) : DNA PROFILING AND
INTEGRON CHARACTERIZATION
BY
DIANA ELIZABETH WATURANGI
A DISSERTATION
Submitted to the Bogor Agricultural University
In Partial Fulfilment of the Requirements for
the Degree Doctor of Microbiology
GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
2002
vii
ACKNOWLEDGMENTS
This study could not have been accomplished without the help and
support of many people. In the area of the academic study, I am especially
indebted to my dissertation committee members.
First, I would like to
express my gratitude to my mentor Dr. Antonius Suwanto, MSc for his
constant and valuable guidance in the process of the making of the
dissertation.
I would like also to express my sincere thanks to my other committee
members, Prof. Dr. Maggy T. Suhartono, MSc and Prof. Dr. Bibiana W. Lay,
MSc for their support, and encouragement.
I am also grateful for Prof. Dr. Stefan Schwarz in Institute for Animal
Science and Animal Behaviour of the Federal Research Center for
Agricultural (FAL), Celle, Germany and Prof. Dr. Walter Erdelen in UNESCO
Paris, France, for their insight and guidance during the course of my work.
A special tribute is due my colleagues and technician in Laboratory of
Molecular Biology, South East Asian Region Center for Tropical Biology,
SEAMEO-BIOTROP, Etty Pratiwi, Dinamella Wahjuningrum, Yogiara, Dwi
Suryanto, Rina Martini, Esti Puspitasari, lrawan Tan, Widanarni, Amarilla
Malik, Husen Samhari, and my best friends Nesti Sianipar, Yuhlanny, Yossy
and Lilieanny.
I think also of my gifted and faithful colleagues in Germany Kayode K.
Ojo and Corinna Kehrenberg, and for excellent technical assistance of Vera
Noding, Erika NuTJbeck, and Gisela Niemann.
Special thanks to Centre for Microbial Diversity, FMIPA, IPB for its
research support and for DAAD (German Academic Exchange Service) for
scholarship during my research in Germany.
Here, I need to express my deepest gratitude to my parents and all of
m y brothers and sisters for their prayers and encouragement.
Finally, I am very much aware that above everything else, it is God
himself who has work in the many different people, situasions, and in my self,
to make all these possible. And to Him is all the glory and praise.
CONTENTS
Page
LIST OF TABLES................................................................................xii
LIST OF FIGURES................. ..
....................................................... xiii
LIST OF APPENDICES ...........................................................................xiv
1. INTRODUCTION............................................................................................ 1
Background........................................................................................ 1
Objective....................................................................................................4
2 . LITERATURE................................................................................................. 5
2.1 Monitor Lizard........................................................................................... 5
2.2 Indigenous microorganism....................................................................... 6
2.3 Genetic Diversity of microorganism........................................................ 7
2.4
DNA profile analysis................................................................................ 8
2.4.1 ARDRA .................................................................................. 10
2.4.2 MFLP analysis............................................................................. 11
2.4.3 Analysis of repetitive DNA sequences.......................................... 12
2.5 Antibiotic resistance.................................................................................
13
....................................................................................
2.5.1 Tetracycline
14
2.5.2 Resistance to tetracycline............................................................. 16
2.5.2.1 Efflux protein.......................................................... 17
2.5.2.2 Ribosomal protection proteins.................................... 18
2.5.2.3 Enzymatic inactivation of tetracycline.......................... : 19
2.5.2.4 Mobility of tet gene...................................................19
2.5.3 Streptomycin.....................................................................20
2.5.4. Resistance to aminoglycoside............................................... 22
.......................... 22
2.5.4.1 Enzymatic inactivation................
.
............................................
2.5.4.2 Decreased permeability
23
2.5.4.3 Ribosome alteration.................................................23
2.5.4.4 Active efflux............................................................24
2.5.5 Sulfonamide......................................................................24
2.5.6 Trimethoprim ....................................................................25
2.5.7 Resistance to trimethoprim ...................................................26
2.5.7.1 Chromosomal resistance.........................................
26
2.5.7.2 Plasmid borne-resistance.......................................... 26
2.6 Tranfer of antibiotic resistance genes.............................................. 27
2.7 Integron.....................................................................................29
2.7.1 Class 1 integron................................................................. 30
2.7.2 The other classes of integrals...............................................32
Analysis of extracellular protease.................................................... 85
Antibiotic resistance analysis...........................................................86
Detection of integron..................................................................... 91
Cloning of integron........................................................................92
4.9.1 Single gene cassette.............................................................
93
4.9.2 Double gene cassettes.........................................................97
4.9.3 dfrA genecassette..........................................................................101
4.9.4 aadA I gene cassette........................................................... 102
4.10 Transformation of plasmid from E.coli..............................................103
4.1 1 Restriction mapping...................................................................... 104
4.1 1.1 Restriction analysis of pDEWTS 1......................................... 104
4.1 1.2 Restriction mapping of pDEWT 1..........................................107
4.12 Analysis of tet gene from pDEWT 1.................................................. 108
4.12.1 Cloning and sequencing of fragment from Smal digestion ........... 108
4.12.2 Cloning and sequencing of fragment from EcoRl digestion ......... 112
4.6
4.7
4.8
4.9
5 . CONCLUSION............................................................................ 118
6. REFERENCES.............................................................................
7. APPENDICES ............................................................................ 131
LIST OF TABLES
Page
2-1.
2.2 .
2-3
Position substitution R1-R4 of derivative......................................... 15
Mechanism of resistance for characterized tet and otr genes ............... 16
Gene cassette........................................................................... 33
3-1
3-2
Sequence of the primers and size of amplicon.................................. 52
53
Cycles of PCR............................................................................
4.1 .
4-2
4-3
4-4
4-5
4-6
Physiology test of bacteria............................................................ 68
Resistance phenotype. genotype of E.coli........................................ 89
Percentage of antibiotic resistance according to origin of isolates .......... 90
Approximate size of amplicons....................................................... 91
Gene cassette. position. length. and coding region............................. 93
Phenotypic and genotypic resistance of transformants............................ 103
LIST OF FIGURES
Page
Structure formula of tetracycline.................................................... 15
Structure formula of streptomycin enzymatic.................................... 22
Structural of sulfonamide. trimethoprim and dihydrofolic acid............... 25
General structure of integron 1...................................................... 30
Structure of gene cassette............................................................ 34
Model for cassette acquisition.........................................................37
Boundaries of gene cassettes....................................................... 38
Cassette fusion................................................................................40
Comparison of attl and 59-be........................................................ 42
Model for replacement of 59-be....................................................... 43
Fecal collection of Varanus spp ....................................................... 68
ARDRA employing Haelll and Sau3A.............................................. 71
ARDRA employing Cfol and Rsal ...................................................72
ARDRA employing Cfol................................................................73
75
Analysis of MFLP.........................................................................
Dendogram from MFLP analysis..................................................... 77
ERIC analysis..............................................................................81
Dendogram of ERIC analysis.......................................................... 82
Plasmid profile............................................................................. 83
Analysis of proteolytic....................................................................85
Percentage of antibiotic resistance................................................... 86
Schematic presentation of dfrA5 cassette.......................................... 95
Schematic presentation of dfrA5 cassette.......................................... 99
Restriction digest of pDEWTS 1...................................................... 105
Restriction map of pDEWTS 1.........................................................106
Restriction digest of pDEWT 1......................................................... 107
Comparison of Tn 7 727 and pDEWT 1............................................... 110
Estimation of site of recombination in pDEWT 1..................................114
1. INTRODUCTION
The natural habitats of microorganism are exceedingly diverse. The
wide spread occurrence of bacteria extends to regions ranging from the
upper atmosphere to sediments on the ocean bed. Any habitat suitable
for the growth of higher organisms can also support growth of
microorganism. But in addition, there are many habitats where, because
of some physical or chemical extreme, higher organism are absent yet
.microorganism exist and occasionally even flourish.
Bacteria occur most abundantly in habitats where they find nutrition,
moisture and a temperature appropriate for their growth and replication.
The conditions that favor the survival and growth of many microorganisms
are those under which higher organism normally live.
Microorganism inhabit the surface of higher organism and in some
cases live within plants, humans and animals. Microorganisms frequently
reach large numbers in such habitats and may benefit the animal in
nutritionally significant way (Brock & Madigan, 1997).
Animal body
provides favorable environments for the growth of microorganisms, and
microorganisms enter into various degrees of symbiotic relationship with
the host.
The microorganisms present at any site in a healthy animal are
collectively referred to as the normal microbiota.
Interaction between
normal microbiota and the host can be divided into different kinds of
relationship such as mutualisms, commensals and parasitisms.
The normal microbiota usually inhabits the skin, and the mucosal
surface of the oral cavity, the upper respiratory tract, the urinary tract and
the gastrointestinal tract. Microbiota residing in the intestinal tract usually
provide beneficial effects to the host. They are responsible for a wide
variety of metabolic reactions and assist in the enzymatic break down of
food and the production of certain vitamins that can be utilized by the host
like niacin, vitamin B1, 82, B6, 812 and vitamin K as well as folic acid and
biotin.
They play a considerable role as one of the major defense
mechanisms that protects the animal body against colonization by
invading pathogens, and stimulate the immune systems (Berg, 1996).
Intestinal microbiota also have a direct impact on the morphology of the
intestine; e.g. the degradation of mucus glycoproteins produced by the
epithelium is assigned to components of the intestinal microbiota (Falk et
a/. 1998).
The composition of the normal microbiota may vary with the animal
species, feed and environment.
Various skin surfaces and mucosal
membranes in animals including the contents of the digestive tract have a
microbiota with a characteristic composition. Microbiota of the intestines
contain various groups of bacteria among which is Enterobacteriaceae, for
instance Escherichia (E.) coli and various anaerobic bacteria.
E. coli is a microbiota of the human and animal digestive.
Nevertheless, pathogenic strains are an important cause of sickness and
mortality throughout the world.
Surprisingly, little is known about the
genetic diversity of E. coli in population of wild animal. Studies about
genetic diversity is important for identification
(Graf, 1999), typing
(Saverkoul et al., 1999), evolution (Belicum et al. 1998), epidemiology and
pathogenicity of the bacteria (Beltran et al. 1999; Nagy et a/. 1999).
A high degree of genetic diversity has been found in E. coli isolated
from sewage (Pupo and Richardon, 1995) and E. coli from mammalian
species in different continents (Souza et a/. 1999).
Yogiara et a/. (2001) reported that E. coli isolated from the buccal of
Varanus showed extensive genetic diversity. However, analysis of genetic
diversity of E. coli obtained from the gastrointestinal microbiota of Varanus
is required, because microbiota in the gastrointestinal tract might have
more specific relationship with their host than bacteria from mouth. In
addition, characteristics of the indigenous bacteria species might be able
to show the diversity and grouping of Varanus.
In this study, we used Amplified Ribosomal DNA Restriction
Analysis (ARDRA) (Graf, 1999), Macrorestriction Fragment Length
Polymorphism (MFLP) analysis employing
PFGE (Pulsed Field Gel
Electrophoresis) (Hudson et a/. 2000),
Enterobacterial Repetitive
Intergenic Consensus (ERIC) analysis (Lupsky and Weinstock, 1992) and
plasmid profiles to determine the genetic diversity of E. coli isolates
obtained from Varanus spp. In Indonesia.
Isolated bacteria were screened for
their
resistances to
antimicrobial agents, and the resistance genes were characterized. Since,
bacteria in their natural environment often exhibit resistance to many types
of antibiotics (Desselberger, 1998).
Data on antibiotic resistance in enterobacteria are usually found
only for clinical strains. Very patchy data on enterobacteria in the fecal
samples have been published, and thus there is only few data available on
how their resistance is acquired and maintained (Dunlop et a/. 1989).
Our preliminary study indicated that there are many isolates of
E. coli from feces of Varanus spp. exhibited resistance to many types of
antibiotics. Thus far antibiotic resistance was only characterized
phenotypically, which might not indicate their genetic properties. More
information is needed about the nucleotide sequences, diversity, stability
and movement of antibiotic resistance genes. Based on our results, we
thought it is necessary to characterize the genetic basis of these antibiotic
resistances and the mobile genetic elements which play a major role in the
transfer of the resistance genes.
The objectives of this research are:
1. To Analyze genetic diversity of E. coli isolated from feces of Varanus
spp. employing Analysis of ARDRA, Macrorestiction Fragment Length
Polymorphism (MFLP), Enterobacterial Repetitive Intergenic Consensus
(ERIC) and plasmid profile.
2. To study antibiotic resistance and genetic basis of the resistance
through detection of integrons 1 gene cassettes and analysis of the
antimicrobial resistance genes.
2. LITERATURE
Monitor lizard
2.1
Varanids are more widely known as monitor. In some places, they are
also known as leguaans or goannas, terms derived from the carib "iguana"
for the distantly related large iguana. The taxonomy of this organism is
described below.
Kingdom
: Animal
Phylum
: Chordata
Class
: Reptilia
Order
: Squamata
Suborder
: Sauria
Family
: Varanidae
Genus
: Varanus
Monitors are characterized by elongate heads, moveable eyelids,
external ear openings, four-well developed limbs with five digit and claws,
a long, bifid, and refractile tongue, and a visible parietal eye. Ventral
scales are rectangular and arranged in rows, all monitors are egg layers
(Bennett, 1998).
In Indonesia there is a type of lizards that can reach sizes that may
have thought only to exist in fairy tales. These unique reptiles have been
able to develop in an area where there is little for large animals to live on.
Monitor lizards of lndonesia that have been reported are Varanus
doreanus (The Blue-tailed monitor); V. indicus (The Mangrove monitor);
V. jobiensis (The Peach-Throated monitor); V. bengalensis nebilosus (The
clouded monitor); V. dumerii; V. gouldii (panoptes) (The New Guinea Sand
monitor). V. prasinus (The green tree monitor); V rudicolis (The black
roughneck monitor); V. salvadorii (The crocodile monitor); V. salvator (The
Asian water monitor); V. timorensis (The Timor monitor); V. melinus (The
yellow-headed monitor); V. yuwonoi (The black-backed mangrove monitor)
and V. komodoensis. The Varanus as it is with all vertebrates owe their
existence at least partially, to several biological processes catalyzed by
enzymes produced by bacteria commensals within their alimentary tract.
Hence, studies on these reptiles will be incomplete without a thorough
understanding of their normal microbiota.
2.2 Indigenous microorganisms
lndigenous microorganism normally inhabit the skin, oral cavity,
upper respiratory tract, gastrointestinal tract, and urinary tract of an
animal. The indigenous microorganism does not appear spontaneously in
newborn humans or animals; instead, certain microbes colonize particular
intestinal habitats at various times after birth.
These microbes are
characteristic for that particular habitat and animal host.
Interaction
between normal microbiota and the host can be divided into different kinds
of relationship. Mutualism in which both the microbes and the host benefit
from each other, commensalism which might be beneficial for
microorganism, but does not seem to be of any benefit to the host, and
parasitism which may be harmful to the host and produce disease under
certain circumstances (Berg, 1996).
The majority of the normal microbiota exhibit mutualistic or
communal relationship with the host. The relationship can change if local
anatomic barrier are breached (trauma) or changing in many chemical,
cellular and immunological mechanisms that have evolved to prevent
bacterial infections (Salyers and Whitt, 1994).
Escherichia coli is a member of the normal microbiota that inhabits
the gastrointestinal tract of humans and animals.
Nevertheless,
pathogenic strains are important since they cause serious and sometimes
fatal disease conditions in humans and animals throughout the world.
Some strains of E. coli have acquired multiple resistance to antibiotics
(Neu, 1992) and infections caused by these E. coli strains are difficult to
control. E. coli regarded as an important source of antibiotic resistance
determinants for other human or animal pathogenic bacteria (Dunlop et al.
1998).
2.3 Genetic Diversity of microorganism
Various influences on bacterial diversity have been identified, such
as spatial separation, and specific bacterium-host interaction (Souza et al.
1992). These influences can apparently differ between one species and
another.
Diversity in symbiotic and pathogenic bacteria seems to be lower
than in 'free living' bacteria (Latour et al. 1996). It may be due to the
unidirectional and predominating influence of the host, whereas bacteria
exposed to a variety of selective forces may have maintained a higher
adaptive capacity (Schloter et a/. 2000).
With the rise of molecular genetic tools in microbial ecology, it
became apparent that we know only a very small part of the diversity in
the microbial world, a bacterial species might compose of different genoand phenotypes. There is even more structural and functional diversity
below the species level.
Detailed molecular genetic information may help us to understand
evolution of the functionality of microbes in their particular environment,
creating new genetic variants below the species level (Schloter et a/.
I
2000).
2.4 DNA profile analysis
The analysis of DNA is an increasingly important tool in studies of
I
evolutionary developments, ecology, population genetics and systematics.
I
DNA has several significant advantages over alternatives such as proteins
for molecular systematics : The genotype rather than the phenotype is
assayed,
one or more sequences appropriate to a problem can be
1
selected on the basis of evolutionary rate, the methods are generally
applicable to any type of DNA, and DNA can be prepared from small
,
j
I
amounts of tissue or bacteria and DNA is also relatively stable when
handled appropriately (Hillis et a/. 1996).
Various methods have been developed for the identification, typing
and studies of the diversity of prokaryotic and eukaryotic organisms at the
DNA level. These methods differ in their taxonomic range, discriminatory
power, reproducibility and ease of interpretation and standardization. The
ideal method produces results that are invariable from laboratory to
laboratory and allows unambiguous comparative analyses and the
establishment of reliable databases (Morrel, 1997).
Genotyping methods differ in their power of discrimination,
depending on the taxonomic level and category.
In bacteriology,
discrimination to the species level is mostly referred to as identification,
while typing denotes differentiation to the strain level (Savelkoul et a/.
1999).
Some techniques have the capability to differentiate until
subspecies level, such as ARDRA (Hudson et a/. 2000). Other methods
obviously have the capacity to differentiate until strain level, such as DNA
sequencing, MFLP analysis using PFGE (Graf, 1999) and PCR analysis of
repetitive DNA such as Repetitive Extragenic Palindromic (REP) element
and Enterobacterial Repetitive lntergenic Consensus (ERIC) sequences
(Lupsky and Weinstock, 1992).
Differences in the number or pattern of DNA fragments can arise
through a number of distinct processes, including the fragment size, the
structure of DNA and the number or distribution of restriction sites. In
restriction site variation, the variation in fragment pattern revealed
following digestion with one or more restriction enzymes are referred to as
restriction fragment length polymorphisms (RFLPs) (Pousser et 81. 2000).
Base substitutions can create or eliminate cleavage sites for a particular
enzyme, thereby altering the number and size of fragments detected by
that enzyme alone (Hillis et a/. 1996).
All of these methods are based on mutations in restriction sites or
length variation of restriction fragment. But PFGE and repetitive PCR also
correspond to mutations which are dispersed over the genome (Savelkoul
et 81. 1999). Combination of several techniques might result in higher
discrimination power.
2.4.1 ARDRA
Cells contain three types of RNA: messenger RNA (mRNA),
transfer RNA (tRNA), and ribosomal RNA (rRNA). All these forms of RNA
are present in both prokaryotic and eukaryotic cells.
Ribosomal RNA
molecules constitute the bulk (75%) of cellular RNA. They are present in
ribosomes as three different molecules, classified by size: 28S, 18S, and
5s in the eukaryotic ribosome, or 23S, 16s and 5s in the prokaryotic
ribosome (Krieg, 1996). The comparison of rRNA sequences is a powerful
method for deducing phylogenetic and evolutionary relationships among
Bacteria, Archaea, and Eukarya (Ross et 81.2000).
The
ability to
establish
phylogenetic relationships among
microorganisms using ribosomal RNA sequences has been well
documented (Braun-Howland et 81. 1992). Ribosomal RNAs are excellent
molecules for discerning evolutionary relationship among living organisms.
These molecules are functionally and evolutionarily homologous in
different organisms; ancient molecules with extremely conserved
structures and nucleotide sequences; very abundant within cells; making
them easy to isolate from all types of organisms; large enough to permit
statistically significant comparisons of their sequences (Clayton et a/.
1995). A method has been published that relies on the use of a single pair
of primers to amplify the DNA encoding a variable region of the 16s rRNA
gene (Borrell et a/. 1997). The amplified DNA is subsequently subjected
to RFLP analysis.
2.4.2 MFLP analysis
Pulsed-field gel electrophoresis (PFGE) currently offers the most
expedient means to both analyze genome size and construct lowresolution physical maps of bacterial chromosomes (Bergthorsson and
Ochman, 1995).
Digestion of bacterial genomic DNA with rare-cutting restriction
endonucleases and separation of the fragments by PFGE is also now
used extensively for bacterial-strain typing (Maule, 1998). The DNA
fragment patterns obtained with this method, provide an estimate of the
degree of genomic relationship between strains that are important for most
epidemiological or clinical purposes (Suwanto, 1994).
Genomic
fingerprinting employing PFGE is a reliable technique that generates
reproducible results, which make
PFGE analysis a suitable general
technique that deserves consideration for investigating the epidemiology
of most microorganisms.
2.4.3 Analysis of repetitive DNA sequences
Repeated sequences are present in the genomes of all organisms.
The best known of these Alu are family of sequences identified in
mamalian species. Prokaryotic genomes are much smaller than the
genomes of mammalian species that may have been maintained through
selective pressure for rapid DNA replication and cell reproduction.
Noncoding repetitive DNA would likely be kept to a minimum under natural
selection for rapid growth, unless these sequences maintain themselves
as 'selfish' DNA (Vefsalovic et al. 1991).
The ubiquitous nature and seemingly random chromosomal
distribution of these repeats in prokaryotic genomes suggest that like
more-complex eukaryotes, the DNA sequences rearrangement in the
genomes of eubacteria may consist of short repeats interspersed with
longer single-copy sequence (Belikum et a/. 1998). Although the precise
function of repetitive sequences in prokaryotic genomes are obscure, their
presence can be exploited for several applications and molecular genetic
manipulations (Lupsky and Weinstock, 1992).
To assess the distribution and evolutionary conservation of two
distinct prokaryotic repetitive elements, consensus oligonucleotides were
used in PCR amplification. In this study, the distribution of repetitive DNA
sequence was examined by analysis of the ERIC sequences. These
sequences are loated in non-coding transcribed regions of the
chromosome, in either orientations with respect to transcription, and
include a conserved inverted repeat.
2.5 Antibiotic resistance
Acquisition of additional genes into the chromosomal DNA of
bacteria is one of the factors which causes genetic diversity in bacteria.
The acquired gene usually enables the bacteria to live in specific
ecological niches. Such acquired genes are considered as auxiliary genes
and their function include for example ability to degrade specific
substrates, resistance to heavy metals and also resistance to antibiotics.
Recent data indicated that antibiotic resistance has spread quickly
and dramatically among bacteria. The dissemination is an increasing
problem for the efficient control of infectious diseases. In their natural
environment there ils a scarcity of data on the occurrence of antibiotic
resistant-bacteria (Diesselberger, 1998).
High frequency of antibiotic resistance have been found in
enterobacteria in fecal flora as well as in clinical isolates. Each antibiotic
class has their own target and specificity. The mechanisms of antibiotic
resistance in bacteria are also different for each antibiotic. Sometimes for
one antibiotic there is only one mechanism of resistance known, for others
there are up to seven different mechanism known (Schwarz and Chaslus-
Danda, 2001).
2.5.1 Tetracycline
Tetracycline inhibit bacterial protein synthesis by preventing the
association of aminoacyl-tRNA with the bacterial ribosome. The most
simple tetracycline to display detectable antibacterial activity is 6-deoxy-6demethyltetracycline (Fig. 2-1) and substitution derivatives of this antibiotic
are described in Table 2-1.
To interact with their target sites, these
molecules need to cross one or more membrane systems depending on
whether the susceptible organism is gram-positive or gram-negative
(Chopra et 81. 1992).
Tetracycline cross the outer membrane of gram-negative enteric
bacteria through the OmpF and OmpC porin channels, as positively
charged
cation
probably
(magnesium-tetracycline)
coordination
complexes. The cationic metal ion-antibiotic complex is attracted by the
Donnan potential across the outer membrane, leading to accumulation in
the periplasm, where the metal ion-tetracycline complex probably
dissociates to liberate uncharged tetracycline, a weakly liphopilic molecule
able to diffuse through the lipid bilayer regions of the inner (cytoplasmic)
membrane (Schnappinger and Hillen, 1996).
I
Figure 2-1. Structure formula of tetracycline (Walter and Meyer, 1987).
I
Table 2-1. Position substitution R1-R4 of derivative
---- --
Derivative
R1
R2
R3
R4
Tetracycline
H
CH3
OH
H
Chlortetracycline CI
CH3
OH
H
Oxytetracycline
H
CH3
OH
OH
Doxycycline
H
CH3
H
OH
Minocyclin
N(CHs)2
H
H
H
--
2.5.2 Resistance to tetracycline
Three major mechanisms of resistance to tetracycline have been
described i.e. efflux, ribosomal protection, and enzymatic inactivation
(Table 2-2).
Table 2-2. Mechanism of resistance for characterized tet and otr genes
(Levy et 81. 1999)
Genes
Efflux
t e W , tet(B),tet(C),teqD), tet(E),tet(G),teqH),teql),
tet(J),tet(Z),tet(30)
tet(K), tet(L)
otr(B),tcn
tet(P)A
teto
tet(Y)
Ribosomal protection
tet(M),tet(O),teqs), teyw)
tet(Q),tet(T)
oWA), tet(P)B
Enzymatic inactivation
tet()o
Unknown
tet(U),
-- -- - --- --
- --- - ---- -
--
2.5.2.1 Efflux protein
All the tet efflux genes encode for membrane-associated proteins
which export tetracycline from the cell. Export of tetracycline reduces the
intracellular drug concentration and thus protects the ribosomes within the
cell.
The genes encoding efflux proteins belong to the major facilitator
superfamily (MFS), whose product include over 300 individual proteins
(Paulsen et a/. 1996). All the tet efflux genes code for membraneassociated proteins which export tetracycline, but no other substances
from the cell. Efflux genes are found in both gram negative and gram
positive species. The gram negative efflux genes are widely distributed
and normally associated with large plasmids, most of which are
conjugative (Jones et a/. 1992).
Each of the efflux genes codes for an approximately 46-kDa
membrane-bound efflux protein. These proteins have been divided into
six groups based on amino acid sequence identity.
Group 1 contain
Tet(A), Tet(B), Tet (C), Tet (D), Tet (E), Tet (G), Tet (H), Tet (Z), and
probably Tet (I), Tet (J). The tetracycline resistance protein in this group
have 41 to 78% amino acid identity (Tauch et a/. 2000). Most of these
efflux proteins appear to reside in the lipid bilayer, with the hydrophilic
amino acid loops protruding into the periplasmic and cytoplasmic space.
The efflux proteins exchange a proton for a tetracycline cation complex
against a concentration gradient (Yamaguchi et a/. 1990). The efflux
genes from gram-negative bacteria have two functional domains, a and
P,
which correspond to the N-and C-terminal halves of the protein (Rubin and
Levy, 1991).
Group 2 includes Tet (K) and Tet (L) , with 58 to 59% amino acid
identity; these proteins are found primarily in gram-positive species. This
group has 14 predicted transmembrane a-helices. These genes code for
proteins which confer resistance to tetracycline and chlortetracycline.
Group 3 includes Otr(B) and Tcr3, both found in Streptomyces spp. These
proteins have topology similar to group 2 proteins, with 14 predicted
transmembrane a -helices. Group 4 includes TetA(P) from Clostridium
spp., with 12 predicted transmembrane a-helices, while group 5 includes
T e t o from Mycobacterium smegmatis.
Group 6 includes unnamed
determinants from Corynebacterium striatum (Chopra and Roberts, 2001).
2.5.2.2 Ribosomal protection proteins
These are cytoplasmic proteins that protect the ribosome from the
action of tetracycline. The binding of these proteins to the ribosome is
believed to cause an alteration in ribosomal conformation which prevents
tetracycline from binding to the ribosome, without altering or inhibiting
protein synthesis. The hydrolysis of GTP may provide the energy for the
ribosomal conformational change. The ribosomal protection proteins also
need to dissociate from the ribosome to allow EF-G to bind, since they
have overlapping binding sites on the ribosome (Triber et a/. 1998)
2.5.2.3 Enzymatic inaativation of tetracycline
The tet(X) gene encodes the only example of tetracycline
resistance due to enzymatic alteration of tetracycline. The tet(X) gene
product is a 44-kDa cytoplasmic protein that chemically modifies
tetracycline in the presence of both oxygen and NADPH (Speer et a/.
1991).
2.5.2.4 Mobility of tet genes
Tetracycline resistance genes are found in many bacteria isolated
from human, animals and environment.
The widely distribution of tet
genes is based on their association with mobile genetic elements like
plasmids orland transpasons (Jones et a/. 1992). The gram-positive efflux
genes tet(K) and tet(L) are associated with small plasmids (Schwarz et a/.
1992). The gram-negative tet efflux genes are often found on transposons
inserted into a diverse group of plasmids from a variety of incompatibility
groups (Jones et a/. 1992). The ribosomal protection genes tet(S) and
tet(Q) can be found oln conjugative plasmids, or in the chromosome,
where they are non self-mobile (Charpentier et a/. 1994). The tet(M) and
tet(Q) genes are generally associated with conjugative chromosomal
elements, which code for their own transfer (Salyers et a/. 1995). These
conjugative transposons are also able to transfer mobilizable plasmids to
other isolates and species and even unlinked genomic DNA (Shows et a/.
1992).
2.5.3
Streptomycin
Streptomycin is the first aminoglycoside antibiotic.
The basic
chemical structure required for both potency and the spectrum of antibiotic
activity of aminoglycoside is that of one or several aminated sugars joined
in glycosidic linkages to a dibasic cyclitol (Fig. 2-2). Their bacterial activity
is attributed to the irreversible binding to the ribosomes although their
interaction with other cellular structures and metabolic process has also
been considered. They have a broad spectrum (Mingeot-Leclercq et a/.
1999). Spectinomycin which is an aminocyclitol devoid of aminosugars is
by extension included in the family of aminoglycosides. It also differs from
them by its bacteriostatic activity and by its way of action. Spectinomycin
acts on protein synthesis during the mRNA-ribosome interaction and it
does not lead to mistranslation like some aminoglycosides do.
Transport of aminoglycosides across the cytoplasmic membrane is
dependent upon electron transport and is termed energy dependent phase
I (EDP I) (Bryan and Kwan, 1983). In the cytosol, aminoglycosides bind to
the 30s sub-unit of ribosomes, again through an energy-dependent
process (EDP-II) while this binding does not prevent formation of the
inhibition complex of peptide synthesis, (Binding of mRNA, fMetRNA, and
association of the 50s sub unit). It causes misreading and or premature
termination (Melaneon et a/. 1992). The aberrant proteins may be inserted
into the cell membranes leading to altered permeability and further
stimulation of aminoglycoside transport (Busse et a/. 1992).
R=
Shvtptomycin
CHO
Dih~dmstreptomycin CH20H
Figure 2-2. Structure formula of streptomycin and its derivative.
2.5.4 Resistance to aminoglycoside
Four mechanisms have been recognized, namely enzymatic
inactivation, ribosome alteration, decreased permeability, and efflux
system. The first mechanism is of most clinical importance since the
genes encoding aminoglycoside modifying enzymes can be disseminated
by plasmids or transposons.
2.5.4.1 Enzymatic inactivation
Enzymatic Inactivation of aminoglycosides is conferred by Nacetyltransferases, O-adenyltransferasesor or O-phosphotransferases.
These enzymes are classified into three major classes according to the
type modification: AAC (acetyltransferase) which use acetyl-coenzyme A
as donor and affect amino functions. ANT (nucleotidyltransferases also
referred to as AAD adenyltransferases), APH (phosphotransferases)
which both use ATP as a donor and affect hydroxyl functions (Shaw et a/.
1993).
New aminoglycoside-inactivating enzymes have been identified, the
genes of some of them are part of integrons (Sandvang, 1999).
Aminoglycoside-modifying enzymes catalyze the covalent modification of
specific amino or hydrolize functions, leading to a chemically modified
drug which binds poorly to ribosomes and for which the EDP-II of
accelerated drug uptake also fails to occur, thereby resulting most often in
high-level resistance (Mingeot-Leqlercq et a/. 1999).
The genes of aminoglycoside-modifying enzymes are often plasmid
encoded but are also associated with transposable elements. Plasmid
exchange and dissemination of tranposons facilitate the rapid acquisition
of a drug resistance phenotype not only within a given species but among
a large variety of bacterial species.
There are some reports about
evidence of aad encoding resistance to streptomycin and spectinomycin
and aac genes as parts of gene cassettes in integrons (Centron and Roy,
1998; Sandvang, 19919; Adrian et a/. 2000). The association of these
antibiotic resistance genes with integrons allows transfer of these
resistance genes from one bacterium to others.
2.5.4.2 Decreased permeability
Absence of or alteration in the aminoglycosides transport system,
inadequate membrane potential, modification in the LPS phenotype can
result in a cross resistance to all aminoglycoside (Salyers and Whitt,
1995). Membrane impermeabilization with reduced drug uptake has been
found in Pseudomonas (Chambers and Sande, 1995).
2.5.4.3 Ribosome alteration
High level resistance to streptomycin and spectinomycin can be
resulted from single step mutations in chromosomal genes encoding
ribosomal proteins rpsL (or strA), rpsD (or ramA or sud2), rpsE (eps or spc
or spcA). Mutation in strC (or strB) generate a low-level streptomycin
resistance (Eliopoulos et a/. 1989).
2.5.4.4 Active efflux
Resistance by active efflux has been reported in E. coli for
neomycin, kanamycin, and hygromycin A antibiotics (Nikaido, 1996), Such
as AcrD in E. coli (Rosenberg et a/. 2000), and M e w in Pseudomonas
(Aires etal. 1999).
2.5.5 Sulfonamides
Sulfonamides are one of the antimicrobials that were used in the
treatment of a variety of bacterial infections. The target of this group of
antimicrobial is dihydropteroate synthase (DHPS), which catalyzes the
formation of dihydropteroic acid in bacteria and some eucaryotic cell
(Brown, 1962). This molecule is not present in human cells (Huovinen et
a/. 1995)
The structure of sulfonamides is analogous to that of p-amino
benzoic acid (PABA), the substrate of the DHPS. Sulfonamides act as an
inhibitor and work competitively with the original substrate (Fig. 2-3).
The antimicrobial resistance gene of sulfonamide can be located on
chromosome. It appears because of the mutation in the folP gene that
result in low level of sulfonamide resistance (Huovinen et al. 1995).
Resistance to high concentration of this antibiotic has been
observed in gram-negative and the genes were located on plasmids. Two
such plasmid borne-resistance genes have been found. The gene sull is
found almost exclusively on integron structures carried by large
conjugative plasmids (Sundstrom et a/. 1988) and the other gene is su12
that is usually found on small mobilizable plasmids (Rgdstorm et a/. 1988).
2.5.6 Trimethoprim
Trimethoprim is an antibiotic commonly used in the treatment of
infections caused by gflam-negative bacteria. It is a synthetic antibacterial
agent that belongs to the diaminopyrimidine group of compounds. It can
be regarded as an a~ntifolate,a structural analogue to folic acid and
competitively inhibits the reduction of dihydrofolate to tetrahydrofolate by
dihydrofolate reductase (DHFR), an enzymatic reaction for synthesis of
thymine (Fig. 2-3).
Figure 2-3. Structural of sulfonamide(a), dihydrofolic acid(b), and
trimethoprim(c). demonstrating structural similarities relative to
competitive ilnhibition.
2.5.7 Resistance to trimethoprim
2.5.7.1 Chromosomal resistance
Resistance to trimethoprim due to mutational changes in the
intrinsic dfr gene has been reported in several pathogens.
Several
changes were observed in different parts of the structural gene in different
isolates including the C-terminal part not known to participate in the
binding of substrates or inhibitors. These changes lead to an altered
secondary structure of the enzyme causing a loss or decrease in site of
binding of trimethoprim (Skbld, 2001).
2.5.7.2 Plasmid borne-resistance
The gene of the trimethoprim-resistant can be located on plasmids
or in the chromosomarl DNA.
The most common mechanism is the
production an additional plasmid-mediated DHFR (Amyes and Smith,
1976).
At least sixteen trimethoprim resistance enzymes in enterobacteria
have been characterized and grouped, based on the kinetic properties and
the nucleotide sequences of their genes.
The largest and the most
prevalent of these groups are type I-like enzyme, which include dfrl, dfrlb,
dfr5, dfrfi, and dfr7 (Huovinen et
81.
1995). This group of enzyme is
identified by an open reading frame (ORF) of 157 amino acid residues,
and the members of this group share between 64 and 88 % amino acid
sequence identity in this ORF. Most of them are associated with integrons
as a gene cassette (Recchia and Hall, 1995).
The most prevalent trimethoprim resistance gene is dfrl, which
occurs in a cassette in both class 1 and class 2 integrons. This is also the
reason why the trimelthoprim resistance genes have spread quickly,
because via integron, transfer can happen horizontally.
2.6 Transfer of antibiotic resistance genes
Bacteria have access, in principle, to a large selection of resistance
genes scattered throughout the bacterial kingdom and mechanisms have
evolved to reassort these genes, moving them genetically from one DNA
molecule to another and physically from one bacterial cell to another.
Several mechanlisms for the acquisition and dissemination of
resistant determinants involve DNA exchange and in this way resistance
genes can spread efficiently among bacterial populations from animals
and humans (Davies, 1994).
The former type involves conjugation,
transformation and tranlsduction mechanism have been demonstrated to
be important in the movement of resistance gene among bacteria. DNA
integration into their host genome could be achieved through: (i) classical
recombination, which is RecA-dependent and requires extensive
homology between the recombining DNA molecules; (ii) transposition,
which normally involves a discrete transposable element and requires no
homology between the transposable element and the molecule in which it
integrates; (iii) site-specific recombination, which involves recombination
between short homologous sequences mediated by recombination
enzymes specific for recombination site.
This mechanism has been
shown to be involved in the dissemination of resistance gene via integrons
and gene cassettes.
An efficient route of acquisition and vertical and horizontal
dissemination of resistance determinants is through mobile genetic
elements including plasmids, transposons, and integrons, they usually
cany one or more antibiotic resistance genes (Francia et a/. 1999).
Plasmids are extrachromosomal DNA elements, capable of
autonomous replication due to their replication systems. The properties of
these elements are not essential for the bacteria under physiological
conditions, but may be of benefit for the bacterium