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Plasmid
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Figure 1: Schematic drawing of a bacterium with plasmids enclosed. (1)Chromosomal
DNA. (2) Plasmids
Plasmids are (typically) circular double-stranded DNA molecules that are separate from
the chromosomal DNA (Fig. 1). They usually occur in bacteria, sometimes in eukaryotic
organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae). Their size varies
from 1 to over 400 kilobase pairs (kbp). There are anywhere from one copy, for large
plasmids, to hundreds of copies of the same plasmid present in a single cell.
Contents
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1 Antibiotic resistance
2 Episomes
3 Vectors
4 Types of plasmid
5 Applications of plasmids
6 Plasmid DNA extraction
7 Conformations
8 See also
[edit]
Antibiotic resistance
1
Figure 2: Schematic drawing of a plasmid with antibiotic resistances
Plasmids often contain genes or gene-cassettes that confer a selective advantage to the
bacterium harboring them, e.g., the ability to make the bacterium antibiotic resistant.
Every plasmid contains at least one DNA sequence that serves as an origin of replication
or ori (a starting point for DNA replication), which enables the plasmid DNA to be
duplicated independently from the chromosomal DNA (Fig. 2)
[edit]
Episomes
Episomes are plasmids that can integrate themselves into the chromosomal DNA of the
host organism (Fig. 3). For this reason, they can stay intact for a long time, be duplicated
with every cell division of the host, and become a basic part of its genetic makeup. This
term is no longer commonly used for plasmids, since it is now clear that a region of
homology with the chromosome such as a transposon makes a plasmid into an episome.
[edit]
Vectors
2
Figure 3: Comparison of non-integrating plasmids (top) and episomes (bottom). 1
Chromosomal DNA. 2 Plasmids. 3 Cell division. 4 Chromosomal DNA with integrated
plasmids
Plasmids used in genetic engineering are called vectors. They are used to transfer genes
from one organism to another and typically contain a genetic marker conferring a
phenotype that can be selected for or against. Most also contain a polylinker or multiple
cloning site (MCS), which is a short region containing several commonly used restriction
sites allowing the easy insertion of DNA fragments at this location. See also
'Applications of plasmids', below.
[edit]
Types of plasmid
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative
plasmids contain so-called tra-genes, which perform the complex process of conjugation,
the sexual transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids
are incapable of initiating conjugation, hence they can only be transferred with the
assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are
mobilisable, and carry only a subset of the genes required for transfer. These plasmids
can 'parasitise' another plasmid, transferring at high frequency in the presence of a
conjugative plasmid.
It is possible for several different types of plasmids to coexist in a single cell, e.g., seven
different plasmids have been found in E. coli. On the other hand, related plasmids are
3
often 'incompatible', resulting in the loss of one of them from the cell line. Therefore,
plasmids can
Figure 4 : Schematic drawing of bacterial conjugation. 1 Chromosomal DNA. 2
Plasmids. 3 Pilus.
be assigned into incompatibility groups, depending on their ability to coexist in a single
cell. These incompatibility groupings are due to the regulation of vital plasmid functions.
An obvious way of classifying plasmids is by function. There are five main classes:
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Fertility-(F)plasmids, which contain tra-genes.
They are capable of conjugation.
Resistance-(R)plasmids, which contain genes that
can build a resistance against antibiotics or poisons.
Historically known as R-factors, before the nature
of plasmids was understood.
Col-plasmids, which contain genes that code for
(determine the production of) colicines, proteins
that can kill other bacteria.
Degrative plasmids, which enable the digestion of
unusual substances, e.g., toluene or salicylic acid.
Virulence plasmids, which turn the bacterium into a
pathogen.
Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are, upon cell division,
in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have
systems which attempt to actively distribute a copy to both daughter cells.
Some plasmids include an addiction system. These plasmids produce both a long-lived
poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive,
4
while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate
because of the lingering poison from the parent cell. This is an example of plasmids as
selfish DNA.
[edit]
Applications of plasmids
Plasmids serve as important tools in genetics and biochemistry labs, where they are
commonly used to multiply (make many copies of) or express particular genes. There are
many plasmids that are commercially available for such uses. Initially, the gene to be
replicated is inserted in a plasmid. These plasmids contain, in addition to the inserted
gene, one or more genes capable of providing antibiotic resistance to the bacterium that
harbors them. The plasmids are next inserted into bacteria by a process called
transformation, which are then grown on specific antibiotic(s). Bacteria which took up
one or more copies of the plasmid then express (make protein from) the gene that confers
antibiotic resistance. This is typically a protein which can break down any antibiotics that
would otherwise kill the cell. As a result, only the bacteria with antibiotic resistance can
survive, the very same bacteria containing the genes to be replicated. The antibiotic(s)
will, however, kill those bacteria that did not receive a plasmid, because they have no
antibiotic resistance genes. In this way the antibiotic(s) acts as a filter selecting out only
the modified bacteria. Now these bacteria can be grown in large amounts, harvested and
lysed to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case you
grow the bacteria containing a plasmid harboring the gene of interest. Just as the bacteria
produces proteins to confer its antibiotic resistance, it can also be induced to produce
large amounts of proteins from the inserted gene. This is a cheap and easy way of massproducing a gene or the protein it then codes for--for example, insulin or even antibiotics.
[edit]
Plasmid DNA extraction
As alluded to above, plasmids are often used to purify a specific sequence, since they can
easily be purified away from the rest of the genome. For their use as vectors, and for
molecular cloning, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, the archaetypes of
which are the miniprep and the maxiprep. The former can be used to quickly find out
whether the plasmid is correct in any of several bacterial clones. The yield is a small
amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and
for some cloning techniques. In the latter, much larger volumes of bacterial suspension
are grown from which a maxi-prep can be performed. Essentially this is a scaled-up
5
miniprep followed by additional purification. This results in relatively large amounts
(several ug) of very pure plasmid DNA.
In recent times many commercial kits have been created to perform plasmid extraction at
various scales, purity and levels of automation.
[edit]
Conformations
When performing DNA_electrophoresis, plasmid DNA may appear in the following five
conformations:
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"Supercoiled" (or "Covalently Closed-Circular")
DNA is fully intact with both strands uncut.
"Relaxed Circular" DNA is fully intact with both
strands uncut, but has been enzymatically "relaxed"
(supercoils removed).
"Supercoiled Denatured" DNA, is not a "natural"
form present in vivo. It is a contaminent often
produced in small quantities following excessive
alkaline lysis; both strands are uncut but are not
correctly paired, resulting in a compacted plasmid
form.
"Nicked Open-Circular" DNA has one strand cut.
"Linearized" DNA has both strands cut site at only
one site.
The relative electrophoretic mobility (speed) of these DNA conformations in a gel are as
follows:
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Nicked Open Circular (slowest)
Linear
Relaxed Circular
Supercoiled Denatured
Supercoiled (fastest)
The rate of migration for small linear fragments is directly proportional to the voltage
applied at low voltages. At higher voltages, larger fragments migrate at continually
increasing yet different rates. Therefore the resolution of a gel decreases with increased
voltage.
At a specified, low voltage, the migration rate of small linear DNA fragments is a
function of their length. Large linear fragments (over 20kb or so) migrate at a certain
fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the
molecule following the leading end through the gel matrix. Restriction digests are
6
frequently used to analyse purified plasmids. Enzymes specifically break the DNA at
certain short sequences. The resulting linear fragments form 'bands' after gel
electrophoresis.
[edit]
See also
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Bacterial artificial chromosome
Retrieved from "http://en.wikipedia.org/wiki/Plasmid"
Category: Molecular biology
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8
PLASMIDS
When a virus gets its genetic material into a cell, one of the first things that most
do is make a dsDNA circle of it. And is not just a plain circle, but rather one with a
couple of extra twists in it. It is still a mystery as to why there must be no more
nor no less than TWO twists. This is called a "supercoil", and is illustrated at the
left:
It is from this that most of the mRNA's are made, and from this that the new
copies of genetic material are made also. This is called the rfDNA (replicative
form-DNA).
Interestingly, plasmids also have this form at some point in their existence. While
some plasmids like to insert themselves into the chromosome as "episomes"
(what's an epi-phyte or an epi-dermis?), these still have a phase in which they
are by themselves in the cytoplasm - and they are circular double=helices.
How these circular helices get from cell to cell depends on whether they are viral
or plasmid. If they are viral they code for extracellular packaging layers so that
they can float through the environment and attach to another appropriate cell.
If it is plasmid DNA, then there are genes coding for cellular equipment that can
be used in sort of a sexual way to duplicate the plasmid DNA and then send one
copy through a tube into a cell that doesn't have that particular type of plasmid.
Obviously, the plasmid DNA must also code for some surface components of the
host cell so that other donors of this plasmid don't try to "mate" with this cell.
While most viruses make their cells "sick" or even kill them, plasmids rarely do
harm to their host cells. Usually they multiply up to some small number (called
the "copy number") and stop reproducing. They usually confer some sort of
benefit to their hosts - new metabolic capacities, or changing the surfaces of the
hosts so that fewer types of viruses can infect them.
9
JUMPING GENES! (Transposons)
Evolutionary Leaps - Part Two
We have seen how viruses and plasmids can move genes around within the
branches of the Shrub of Life. But let us suppose that somewhere along this
convoluted pathway by which some block of genes are moving around, it comes
to a cell that contains a transposon. That transposon often can pick up a
neighboring host gene and jump with it into either a plasmid or a virus genome.
And that plasmid or virus containing the transposon then is moved to a new
closely related cell type, where the transposon and its associated gene, jump off
(damaging the viral or plasmid DNA, but nevertheless getting inserted into the
new host's chromosome - conferring it with a new gene).
Whether that gene or block of genes is used immediately or not, determines the
rate of this step of evolution. Most of us carry large amounts of unused DNA just waiting for some future eon to be found useful and cause a leap in evolution.
Genetics Images/plasmids.jpg
10
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Resources Journal
The Yeast Two-Hybrid Assay
An Exercise in Experimental Eloquence
Solmaz Sobhanifar
Graphics: Jiang Long
Once upon a time, it was believed that proteins were isolated entities, floating
in the cytosol and, for the most part, acting independently of surrounding
proteins. Proteins were thought to diffuse freely, and reactions occurred as a
result of proteins A and B randomly colliding with one another. Today we know
this picture to be far too simplistic to account for the complex processes that all
coalesce to become 'life'. Instead, the majority of cellular phenomena are
carried out by protein 'machines', or aggregates of ten or more proteins1.
These protein-protein interactions are critical to all cellular processes, and
understanding them is key to understanding any biological system. One
11
technique that can be used to study protein-protein interactions is the "yeast
two hybrid" system.
Yeasty goodness
A protein is composed of modules or domains, which are individually folded
units within the same polypeptide (protein) chain. The presence of these
individual domains allow the same protein to perform different functions. The
yeast two-hybrid technique uses two protein domains that have specific
functions: a DNA-binding domain (BD), that is capable of binding to DNA, and
an activation domain (AD), that is capable of activating transcription of the
DNA.
Figure 1. Normal Transcription. Normal transcription requires both the
DNA-binding domain (BD) and the activation domain (AD) of a
transcriptional activator (TA).
Both of these domains are required for transcription, whereby DNA is copied in
the form of mRNA, which is later translated into protein. In order for DNA to be
transcribed, it requires a protein called a transcriptional activator (TA). This
protein binds to the "promoter", a region situated upstream from the gene
(coding region of the DNA) that serves as a docking site for the transcriptional
protein (Figure 1). Once the TA has bound to the promoter, it is then able to
activate transcription via its activation domain. Hence, the activity of a TA
requires both a DNA binding domain and an activation domain. If either of
these domains is absent, then transcription of the gene will fail.
Furthermore, the binding domain and the activation domain do not necessarily
have to be on the same protein. In fact, a protein with a DNA binding domain
can activate transcription when simply bound to another protein containing an
activation domain; this principle forms the basis for the yeast two-hybrid
technique2.
In the two-hybrid assay, two fusion proteins are created: the protein of interest
(X), which is constructed to have a DNA binding domain attached to its Nterminus, and its potential binding partner (Y), which is fused to an activation
domain. If protein X interacts with protein Y, the binding of these two will form
an intact and functional transcriptional activator2. This newly formed
transcriptional activator will then go on to transcribe a reporter gene, which is
simply a gene whose protein product can be easily detected and measured. In
this way, the amount of the reporter produced can be used as a measure of
interaction between our protein of interest and its potential partner (Figure 2).
12
Figure 2. Yeast two-hybrid transcription. The yeast two-hybrid
technique measures protein-protein interactions by measuring
transcription of a reporter gene. If protein X and protein Y interact, then
their DNA-binding domain and activation domain will combine to form a
functional transcriptional activator (TA). The TA will then proceed to
transcribe the reporter gene that is paired with its promoter.
The Recipe for Successful Interactions
First, it is necessary to construct the 'bait' and 'hunter' fusion proteins. The
'bait' fusion protein is the protein of interest (or 'bait') linked to the GAL4
binding domain, or GAL4 BD. This is done by inserting the segment of DNA
encoding the bait into a plasmid, which is a small circular molecule of doublestranded DNA that occurs naturally in both bacteria and yeast. This plasmid
will also have inserted in it a segment of Gal4 BD DNA next to the site of bait
DNA insertion. Therefore, when the DNA from the plasmid is transcribed and
converted to protein, the bait will now have a binding domain attached to its
end (Figure 3). The same procedure is used to construct the 'hunter' protein,
where the potential binding partner is fused to the GAL4 AD.
13
Figure 3. Plasmid construction. The 'bait' and 'hunter' fusion proteins
are constructed in the same manner. The 'bait' DNA is isolated and
inserted into a plasmid adjacent to the GAL4 BD DNA. When this DNA
is transcribed, the 'bait' protein will now contain the GAL4 DNA-binding
domain as well. The 'hunter' fusion protein contains the GAL4 AD.
In addition to having the fusion proteins encoded for, these plasmids will also
contain selection genes, or genes encoding proteins that contribute to a cell's
survival in a particular environment. An example of a selection gene is one
encoding antibiotic resistance; when antibiotics are introduced, only cells with
the antibiotic resistance gene will survive. Yeast two-hybrid assays typically
use selection genes encoding proteins capable of synthesizing amino acids
such as histidine, leucine and tryptophan (Figure 4).
14
Figure 4. Bait and Hunter Plasmids. The yeast two-hybrid assay uses two
plasmid constructs: the bait plasmid, which is the protein of interest fused to a
GAL4 binding domain, and the hunter plasmid, which is the potential binding
partner fused to a GAL4 activation domain.
Once the plasmids have been constructed, they must next be introduced into a
host yeast cell by a process called "transfection". In this process, the outermembrane of a yeast cell is disturbed by a physical method, such as
sonification or chemical disruption. This disruption produces holes that are
large enough for the plasmid to enter, and in this way, the plasmids can cross
the membrane and enter the cell (Figure 5).
15
Figure 5. Transfection. The 'bait' and 'hunter' plasmids are introduced into
yeast cells by transfection. In this process, the plasma membrane is disrupted
to yield holes, through which the plasmids can enter. Once transfection has
occurred, cells containing both plasmids are selected for by growing cells on
minimal media. Only cells containing both plasmids have both genes
encoding for missing nutrients, and consequently, are the only cells that will
survive.
Once the cells have been transfected, it is necessary to isolate colonies that
have both 'bait' and 'hunter' plasmids. This is because not every cell will have
both plasmids cross their plasma membrane; some will have only one plasmid,
while others will have none. Isolation of transfected cells involves identifying
cells containing plasmids by virtue of their expressing the selection genes
mentioned previously. After the cells have been transfected and allowed to
recover for several days, they are then plated on minimal media, or media that
is lacking one essential nutrient, such as tryptophan. The cells used for
transfection are called auxotrophic mutants; these cells are deficient in
producing nutrients required for their growth. By supplying the gene for the
deficient nutrient in the 'bait' or 'hunter' plasmid, cells containing the plasmid
are able to survive on the minimal media, whereas untransfected cells cannot
(Figure 5). Selection in this way occurs in two rounds: first on one minimal
media plate, to select for the 'bait' plasmid, and then on another minimal media
plate, to select for the 'hunter'4.
Once inside the cell, if binding occurs between the hunter and the bait,
transcriptional activity will be restored and will produce normal Gal4 activity.
16
The reporter gene most commonly used in the Gal4 system is LacZ, an E. coli
gene whose transcription causes cells to turn blue 4. In this yeast system, the
LacZ gene is inserted in the yeast DNA immediately after the Gal4 promoter,
so that if binding occurs, LacZ is produced. Therefore, detecting interactions
between bait and hunter simply requires identifying blue versus non-blue.
What Can I Do With My Very Own Yeast Two-Hybrid??
Generally the yeast two-hybrid assay can identify novel protein-protein
interactions. By using a number of different proteins as potential binding
partners, it is possible to detect interactions that were previously
uncharacterized3. Secondly, the yeast two-hybrid assay can be used to
characterize interactions already known to occur. Characterization could
include determining which protein domains are responsible for the interaction,
by using truncated proteins, or under what conditions interactions take place,
by altering the intracellular environment.
The last and most recent application of the yeast two-hybrid involves
manipulating protein-protein interactions in an attempt to understand its
biological relevance. For example, many disorders arise due to mutations
causing the protein to be non-functional, or have altered function. Such is the
case of some cancers; a mutation in a pro-growth pathway does not allow for
the binding of negative regulatory proteins, resulting in the pro-growth pathway
never turning 'off'. The yeast two-hybrid is one means of determining how
mutation affects a protein's interaction with other proteins. When a mutation is
identified that affects binding, the significance of this mutation can be studied
further by creating an organism that has this mutation and characterizing its
phenotype.
Conclusion…
The yeast two-hybrid assay is an elegant means of investigating proteinprotein interactions. A fairly new addition to the family of microbiological
studies, these interactions have become increasingly important to our
understanding of biological systems in the past few years. While isolation of a
protein in an attempt to understand its function still has its place in biological
research, we now understand that biological reactions do not occur in isolation.
A protein is constantly interacting with other proteins in what we now know to
be a delicate balance — to ignore this wealth of information would be to deny
ourselves the opportunity to fully appreciate the stuff that life is made of.
References
1. Alberts, B. (1998). The Cell as a Collection of Protein Machines:
Preparing the Next Generation of Molecular Biologists. Cell 92: 291-4.
2. Fields S, Song O.K. (1989). A novel genetic system to detect proteinprotein interactions. Nature 340:245-6.
3. Fields S, Sternglanz R. (1994). The two-hybrid system: an assay for
protein-protein interactions. Trends in Genetics 10:286-92.
4. Two-hybrid analysis of genetic regulatory networks — online protocol:
http://cmmg.biosci.wayne.edu/finlab/YTHnetworks.html
17
Additional Reading
1. Bartel P, Fields S. (eds). (1997). The yeast two-hybrid system. New
York: Oxford University Press.
Contact us:
[email protected]
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Protein Identification
using SDS-PAGE and Mass
Spectrometry
Molecular Techniques
real molecular biology experiments
targetted for Biology 11/12 students
SAGE
painless gene expression
profiling.
The Southern Blot
a very brief overview
-----------------
18
Some potentially useful plasmid maps
from Rein Aasland
Department of Molecular Biology
University of Bergen
Bergen, Norway
e-mail:[email protected]
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pCMV-GFP-LpA a CMV-based expression vector for the green fluorescent
protein, GFP.
pS65T-C1, Clontech's CMV-driven vector for expression of Red-Shifted GFP.
LpA vectors (polyA signals from SV40)
Map of subclones from the murine Hoxa-locus
A guestimate of pBTM116, a LexA-fusion vector for Two-hybrid screening in
yeast (Bartel and Fields)
Stratagene's pBS(-) general purpose cloning vector.
A guestimate of Danny Huylebroeck's pSV51L.
Huylebroeck et al. Gene 66(2):163-181 1998
The Vaillancourt Laboratory
Department of Plant Pathology
19
University of Kentucky
Plasmid Vector Catalog

pGEM-3Zf+: Promega
This vector can be used as a standard cloning vector, as templates for
in vitro transcription, and for production of circular ssDNA for
sequencing due to the presence of the origin of replication of the
filamentous phage f1. The presence of the gene encoding the lacZ apeptide allows recombinants to be selected by blue-white screening.

pT7blue: Novagen
pT7blue contains the pUC19 backbone, a T7 promoter, f1 origin of
replication, and modified multiple cloning region. The multiple
cloning region contains an EcoRV site used for blunt cloning flanked
by an NdeI site, which allows PCR fragments to be conveniently
subloned into the NdeI sites of many pET vectors.
20

pUC19
pUC19 is a small, high copy number E.coli plasmid cloning vector
that is part of a series of related plasmids constructed by Messing and
co-workers (Yanisch-Perron et al., Gene 33, 103-119). The pUC
plasmids contain portions of pBR322 and M13mp19.
21

pZL1: Life Technologies
pZL1 is an E. coli cloning vector derived from pSPORT 1 containing
a loxP sequence installed at one of the BspHI sites and the phage P1
incA incompatibility locus at the other BspHI site. All other features
of pSPORT are preserved in pZL1. This vector is excised from 
ZIPLOXTM DNA by cre-mediated recombination.
No picture available.

pBR322
pBR322 carries genes that confer tetracycline and ampicillin
resistance. It was constructed from several naturally occuring
plasmids (Balbas et al., Gene 50: 3).
No picture available.

pBluescript II KS+: Stratagene
The pBluescript phagemid was derived from pUC19. The KS
designation indicates that the polylinker is oriented so that lacZ
transcription proceeds from KpnI to SacI. The phagemid contains an
f1 origin of replication, a ColE1 origin, the lacZ gene interupted by
the polylinker region to facilitate blue-white screening, and an
ampicillin resistance gene.
22
Back to Vaillancourt Home Page
Back to Protocols and Collections Page
Molecular
Microbiology
Selected poster
presentations
41st Interscience Conference on
Antimicrobial Agents and Chemotherapy
16-19 December, 2001, Chicago, USA
Poster # P006/51
Clonal Spread of Cefotaxime-Resistant
23
(CTX-R) Salmonella typhimurium in
Belarus: Epidemiology and Molecular
Analysis of Resistance Mechanisms
M. EDELSTEIN1, M. PIMKIN1, I. EDELSTEIN1, T. DMITRACHENKO2, V. SEMENOV2,
L. STRATCHOUNSKI1
1
2
Institute of Antimicrobial Chemotherapy, Smolensk, Russia
Medical University, Vitebsk, Belarus
The PDF format poster (45

Revised abstract

Introduction

Methods

Results and discussion

Conclusions
REVISED ABSTRACT
In the present study we explored the genetic relatedness and resistance mechanisms t
lactams of 15 cefotaxime-resistant (CTX-R) S.typhimurium isolated in 7 Belarussian
hospitals during 1994 - 2000. Five previously characterized CTX-M-4 -lactamaseproducing isolates from St.-Petersburg (Russia) with similar resistance phenotype as
as 3 unrelated susceptible strains were also included for comparison. Susceptibility te
using Etests revealed a common antibiogram in CTX-R strains: susceptibility to cefo
resistance to ceftriaxone, aztreonam, amoxicillin/clavulanate, piperacillin/tazobactam
decreased susceptibility to ceftazidime (TZ). An 8-16 fold reduction of MICs of TZ i
presence of clavulanate indicated a production of ESBL. Using molecular typing by E
PCR and RAPD with primers highly discriminative for S.typhimurium identical
fingerprints were obtained for all CTX-R isolates including those from St.-Petersburg
whereas susceptible strains were readily distinguished. The determinants of resistanc
transferred by conjugation to E.coli AB1456 (RifR). Two types of transconjugants (T
were selected on agar containing rifampin with either cefotaxime or ampicillin. The T
of type 1 expressed a -lactamase of pI 7.5 conferring resistance only to penicillins a
their combinations with inhibitors and generated a specific 755bp product upon PCR
primers for blaOXA-1 genes. The TRCs of type 2 acquired small (~8kb) plasmids with
similar but distinguishable PstI- and PvuII-digestion patterns and produced an ESBL
belonged to a CTX-M-type according to pI 8.4 and amplification of 543bp blaCTX-M
24
internal fragment. We conclude that the CTX-R isolates of S.typhimurium probably
represented a single clone and that its resistance to -lactams was attributed to
coproduction of a CTX-M-type ESBL and an OXA-1-like penicillinase.
INTRODUCTION
Multiple drug resistance in salmonellae have emerged as important problem in many
countries of the world. Development of resistance to oxyimino--lactams is especiall
alarming because these drugs have been successfully used for empirical treatment of
severe salmonellosis forms over the long time. Isolates of S.typhimurium resistant to
cefotaxime were first identified in Argentina in 1990. The resistance was attributed to
production of plasmid-mediated ESBL designated CTX-M-2 (A.Bauernfeind et al., 1
Subsequently there have been several reports of CTX-M-type -lactamase-producing
S.typhimurium strains isolated in East and South European countries, including Latvi
(P.Bradford et al., 1998), Greece (L.Tzouvelekis et al., 1998), Russia (M.Gazouli et a
1998) and Hungary (P.Tassios et al., 1999). The latter report have pointed out the sp
of single S.typhimurium clone resistant to expanded-spectrum cephalosporins in three
European countries. In the present study we describe a long-time countrywide outbre
salmonellosis in Belarus caused by CTX-R S.typhimurium clone and demonstrate its
genetic relatedness to the CTX-M-4 -lactamase-producing strains previously isolate
St.-Petersburg (Russia).
METHODS
Bacterial strains: This study was performed with 15 non-duplicate S.typhimurium is
obtained from hospitalized children either during local nosocomial outbreaks or from
sporadic cases of salmonellosis in 7 Belarussian hospitals located in Vitebsk, Rechits
Minsk, Gomel and Volcovisk in 1994-2000. Five previously characterized CTX-M-4
lactamase-producing isolates with similar resistance phenotype isolated in St.-Petersb
Russia (M.Gazouli et al., 1998) as well as 3 unrelated susceptible strains were also
included for comparison.
Susceptibility testing: MICs of ampicillin, amoxicillin/clavulanic acid (2:1), piperac
piperacillin/tazobactam (tazobactam fixed at 4mg/L), cefotaxime, ceftriaxone, ceftaz
ceftazidime/clavulanic acid (4:1), aztreonam, and cefoxitin were determined using Et
(AB Biodisk, Sweden) on Mueller-Hinton agar (Becton Dickinson, USA). Susceptib
non--lactam agents: tetracycline, chloramphenicol, gentamicin, tobramycin,
trimethoprim/sulfamethoxazole, and ciprofloxacin was determined by disk-diffusion
method. The results of susceptibility testing were interpreted according to the current
NCCLS standards. E.coli strains ATCC® 25922 and ATCC® 35218 were used for qu
25
control.
Bacterial strains typing by PCR-based methods: The genetic relatedness of
S.typhimurium clinical isolates was studied by two independent methods: 1) ERIC-PC
with primers ERIC1R (5'-atgtaagctcctggggattcac-3') and ERIC2 (5'aagtaagtgactggggtgagcg-3‘);
2) RAPD typing with primer OPB-17 (5’-agggaacgag-3’) described by A.Lin et al. (1
Template DNA was extracted from 3-4 colonies of each strain grown overnight on
MacConkey agar using the InstaGene matrix (BioRad, USA). The ERIC-PCR mixe
set up in Ready-To-Go PCR Bead format (Amersham Pharmacia Biotech, USA) prov
the following composition of reaction mixture: 10mM Tris-HCl (pH 9.0), 50mM KC
1.5mM MgCl2, 200M of each dNTP and 1.5U of Taq-polymerase after addition of
primers (50 pmoles each), 10l of template DNA and water to a final volume of 25
amplification was carried out in a PTC-200 thermocycler (MJ Research, USA) under
following conditions: 2 min 30 sec initial denaturation at 94oC followed by 35 cycles
sec denaturation at 94oC, 1 min annealing at 47oC, and 1 min elongation at 72oC with
final elongation step extended to 4 min. The RAPD mixes were also prepared with R
To-Go PCR Beads and contained 50pmoles of primer OPB-17 and 2l of template D
Thermal cycling was carried out as described for ERIC-PCR, except that annealing
temperature was set to 35oC. PCR products were analysed by electrophoresis in 1.3%
agarose gel and ethidium bromide staining.
Isoelectric focusing: Crude sonic extracts containing -lactamases were examined o
PhastSystem apparatus on preformed polyacrilamide gels covering the pH ranges 5-8
3-9 (Amersham Pharmacia Biotech, USA) and stained with nitrocefin. -Lactamases
known pIs (TEM-1 (pI 5.4), TEM-2 (pI 5.6), TEM-3 (pI 6.3), SHV-1 (pI 7.6), and SH
(pI 8.2)) were used as standards.
PCR detection of -lactamase genes: A pair of primers (5’-ataaaattcttgaagacgaaa-3
5’-gacagttaccaatgcttaatca-3’) described by C.Mabilat and S.Goussard (1993) was use
amplify specific 1080-bp fragment of blaTEM gene. The PCR mixes contained: 12.5m
Tris-HCl (pH 8.3), 62.5mM KCl, 2mM MgCl2, 200M of each dNTP, 0.25M of ea
primer, 1.25U AmpliTaq DNA polymerase (Perkin-Elmer, USA) and 20l of templa
DNA prepared with InstaGene matrix in total volume of 50l. The PCR was carried
a PTC-200 thermocycler (MJ Research, USA) as follows: 1 min 50 sec initial denatu
at 94oC followed by 35 cycles of 10 sec denaturation at 94oC, 10 sec annealing at 54o
and 45 sec elongation at 72oC with final elongation step extended to 3 min.
PCR detection of blaCTX-M genes was performed using the primes (CTX-M/F: 5’tttgcgatgtgcagtaccag-3’ and CTX-M/R: 5’-gatatcgttggtggtgccat-3’) matching the con
sequences at positions 205 to 224 and 747 to 728, with respect to the CTX-M transla
starting point. The PCR mixes contained in 50l volumes: 50mM KCl, 10mM Tris-H
(pH 9), 0.1% TritonX-100, 1. 5mM MgCl2, 200M of each dNTP, 0.4M of each pr
1 TaqBead Hot Start Polymerase (Promega, USA) and 5l of template DNA prepare
Lyse-N-Go PCR reagent (PIERCE, USA) as recommended by manufacturer. To veri
26
the amplified sequences correspond to either the blaCTX-M-1- or blaCTX-M-2-related gen
PCR products purified by ethanol/sodium acetate precipitation were subjected to rest
enzyme digests with PstI and PvuII. The PCR products and restriction fragments wer
separated in 3.5% AmpliSize agarose (BioRad, USA) gel and stained with ethidium
bromide.
A PCR with primers (OXA-1/F: 5'-atgaaaaacacaatacatatcaac-3’ and OXA-1/R: 5'tttcctgtaagtgcggacac-3’) was used to detect 755-bp internal fragment of blaOXA-1-rela
genes. The composition of PCR mixes and amplification conditions were the same as
described for blaCTX-M genes except the concentration of primers – 0.5M (each) and
annealing temperature - 48oC.
Extraction and analysis of plasmid DNA: Plasmid DNA was extracted from three
S.typhimurium isolates as well as from E.coli transconjugants and transformants usin
Prep-A-Gene DNA Miniprep Kit (BioRad, USA) as recommended by manufacturer a
subjected to restriction enzyme digests with AvaII, PstI or PvuII (Amersham Pharma
Biotech, USA). Native plasmids and restriction products were analysed by electropho
in 1.3% agarose gel and ethidium bromide staining.
Transfer of resistance: All resistant S.typhimurium isolates were mated in broth wit
E.coli AB1456 (RifR). The transconjugants were selected on agar containing rifampin
(100g/ml) with either cefotaxime (10g/ml) or ampicillin (100g/ml). In addition, t
plasmid DNA was transformed into competent cells of E.coli TOP10 (Promega, USA
transformants were selected on agar containing cefotaxime (10g/ml).
RESULTS AND DISCUSSION
Susceptibility. The resistance phenotypes and other characteristics of S.typhimurium
isolates from St.-Petersburg and Belarus are shown in Table 1. All isolates were high
resistant to penicillins, cefotaxime, ceftriaxone and aztreonam, but susceptible to cefo
MICs of ceftazidime were generally below the resistance level, however a synergy
between all oxyimino--lactams and -lactamase-inhibitors (especially tazobactam)
suggested an ESBL-production. All salmonella from St.-Petersburg and 9 Belarussia
isolates demonstrated high-level resistance to penicillin-inhibitor combinations, the
remaining 6 isolates were fully susceptible to piperacillin/tazobactam and had the MI
amoxicillin/clavulanate close to the resistance breakpoints (8-32 mg/L). In addition,
isolates were resistant to tetracycline and chloramphenicol, 15 – to gentamicin and
tobramycin, and 9 – to trimethoprim/sulfamethoxazole.
ERIC-PCR and RAPD typing. The isolation of multiple S.typhimurium with simila
characteristic phenotype of -lactam resistance may indicate dissemination of either
single clone or a specific ESBL-species among different strains. Molecular typing by
ERIC-PCR and RAPD with primers highly discriminative for S.typhimurium (A.Lin
27
1996; ) showed that all CTX-R isolates from St.-Petersburg and Belarus were genetic
related whereas control susceptible strains were clearly distinguishable (Fig. 1).
Figure 1. ERIC-PCR (a) and RAPD profiles (b) of representative cefotaxime-resistan
susceptible S.typhimurium isolates. Lane M, -BstEII+pUC18-HaeIII; lanes 1 to 5, C
isolates; lanes 6 to 8, unrelated susceptible strains.
-lactamase characterization. Isoelectric focusing revealed the production of -lact
with a pI of approximately 8.4 in all S.typhimurium isolates. The resistance phenotyp
pI of the enzymes were indicative of a CTX-M-type ESBL. In support of this assump
the specific internal fragments of blaCTX-M genes were amplified by PCR from all iso
Digestion of PCR-products with PstI and PvuII restriction endonucleases, which allo
distinguish the groups of blaCTX-M-1 and blaCTX-M-2 related genes, demonstrated that a
isolates carried the genes of the latter group (Fig. 2). In addition, 14 isolates resistant
piperacillin/tazobactam produced a second -lactamase with pI 7.5, which correlated
OXA-4, and 2 isolates expressed a third enzyme with pI 5.4 (presumably TEM-1), w
apparently did not affect the resistance phenotype. The presence of TEM- and OXA-lactamases was confirmed by PCR with blaTEM and blaOXA-1 specific primers,
respectively.
28
Figure 2. PstI-PvuII double digests of blaCTX-M-gene amplification products. Lane M
pUC18-HaeIII; lanes 1 to 15, CTX-R Belarussian isolates; lane 16, C.freundii (CTXlane 17, S.typhimurium (CTX-M-4); lane 18, undigested 543-bp PCR-product.
Transfer of resistance. In broth mating experiments two different types of
transconjugants (TRCs) were obtained from isolates resistant to cefotaxime and
piperacillin/tazobactam. The TRCs of type 1 were selected on plates containing rifam
and ampicillin at a high frequency (10-3–10-4). The results for only two representative
TRCs of type 1 (AB1465/SP891 and AB1456/6570-1) are shown in table 1. All clon
this type produced an OXA-1-related -lactamase (most likely – OXA-4) providing
resistance only to penicillins and decreasing susceptibility to penicillin-inhibitor
combinations, but vary in the number of co-transferred non--lactam resistance mark
These data may suggest that different resistance determinants were located on multip
separate plasmids, and that the differences in antibiogram of genetically related
S.typhimurium isolates could be attributed to the variation in their plasmid spectrum.
TRCs of type 2 were obtained on selective plates containing rifampin and cefotaxime
very low frequency (~10-6). In spite of the multiple attempts and prolonged incubatio
mating mixtures only two isolates transferred the cefotaxime resistance to recipient s
The respective TRCs (AB1456/6570-2 and AB1456/1358-2) produced a single CTX
lactamase conferring ESBL phenotype of the donor strains, but were susceptible to
piperacillin/tazobactam and all non--lactam antibiotics. The laboratory clones
(TOP10/SP891 and TOP10/1358) obtained by plasmid-DNA transformation and sele
on cefotaxime-containing plates displayed the same resistance phenotype and produc
CTX-M--lactamase. Notably, one of these transformants additionally produced a TE
-lactamase (Table 1).
Analysis of plasmids carrying the blaCTX-M genes. Each of the CTX-M--lactamas
producing transconjugants and transformants acquired a single plasmid. The length o
CTX-M--lactamase encoding plasmids from different donor strains varied from 7.3
kb. When these plasmids were digested with restriction endonuclease PstI or PvuII, t
patterns obtained were rather similar differing by 3 bands at most (Fig. 3). All but on
plasmid originating from Russian isolate (SP891) did not contain AvaII restriction si
The digestion patterns and the presence of blaTEM-1 gene on this differing plasmid ind
29
a possible insertion of a TnA-type transposon. In addition to the identical ERIC-PCR
RAPD patterns, the similarity of CTX-M--lactamase-encoding plasmids further sup
the clonal origin of the CTX-R isolates. These plasmids were probably non-selftransferable, but their transmission in vitro was facilitated by coexisting conjugative
plasmids.
Figure 3. Digestion patterns of CTX-M--lactamase-encoding plasmids.
Lanes M, -BstEII+pUC18-HaeIII;
lanes 1 to 4, digestion with AvaII;
lanes 5 to 8, digestion with PstI;
lanes 9 to 12, digestion with PvuII;
lanes 1, 5, 9, E.coli TOP10/SP891;
lanes 2, 6, 10, E.coli TOP10/1358;
lanes 3, 7, 11, E.coli AB1465/1358-2;
lanes 4, 8, 12, E.coli AB1465/6570-2.
Possible relationship between CTX-R S.typhimurium isolates from Belarus and o
European countries. In this study we described a clonal spread of CTX-R S.typhimu
in Belarus. To our knowledge, this study revealed and characterized the biggest num
CTX-M--lactamase producing S.typhimurium that have been isolated in one country
a long period of time. Moreover, we demonstrated a possible clonal relationship betw
isolates from Belarus and St.-Petersburg (Russia). Notably, the Russian isolates inclu
this study were previously compared with Hungarian and Greek strains and all of the
were found to be highly related on the basis of PFGE typing (P.Tassios et al., 1999).
Unfortunately, direct comparison of Belarussian clone and Latvian CTX-M-5--lacta
producing isolates described by P.Bradford et al. (1998) was not possible in this stud
However, a number of common features, including resistance spectrum and propertie
30
CTX-M--lactamase encoding plasmids, suggests a possible relationship between CT
S.typhimurium isolates from nearby located Latvia and Belarus.
Table 1. Susceptibilities and other characteristics of S.typhimurium isolates and E.coli
transconjugants and transformants.
Etest MICs, mg/L
Strai
ns
A
M
X
L
P
P
P
T
c
C
T
T
X
T
Z
non-lactams
T
Z
L
F A
X T
ES
BL
T
S
C A
C
e
x
hl g
ip
t
t
PCR for bla
genes
T
E
M
OX CT
A- X1
M
pI of
lacta
mase
s
S.typhimurium clinical isolates
SP8
29*
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
4
0. 2
8
6
4
+
R
R S S
S
-
+
+
7.5;
~8.4
SP8
32*
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
6
0. 2
8
6
4
+
R
R S S
S
-
+
+
7.5;
~8.4
SP8
93*
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
4
0. 2
8
6
4
+
R
R S S
S
-
+
+
7.5;
~8.4
SP8
38*
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
8
1
2
6
4
+
R
R R S
S
+
+
+
5.4;
7.5;
~8.4
SP8
91*
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
2
5
6
1
2
1. 2
5
6
4
+
R
R R S
S
+
+
+
5.4;
7.5;
~8.4
657
0
2
5
6
9
6
2
5
6
1
2
8
2
5
6
2
5
6
8
1
2
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
307
8
2
5
6
1
9
2
2
5
6
1
2
8
2
5
6
2
5
6
4
1
2
6
4
+
R
R
R
S
-
+
+
7.5;
~8.4
132
05
2
5
6
6
4
2
5
6
2
5
6
2
5
6
2
5
6
8
0. 2
4
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
139
53
2
5
6
9
6
2
5
6
2
5
6
2
5
6
2
5
6
1
2
0. 2
8
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
135
26
5
2
9
6
5
2
5
2
5
2
5
2
1
2
0. 2
5
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
I
31
6
6
6
6
6
142
42
2
5
6
9
6
2
5
6
2
5
6
2
5
6
2
5
6
1
6
1
2
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
167
53
2
5
6
9
6
2
5
6
2
5
6
2
5
6
2
5
6
1
6
1
2
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
26
2
5
6
9
6
2
5
6
1
2
8
2
5
6
2
5
6
1
2
1
2
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
16
2
5
6
9
6
2
5
6
2
5
6
2
5
6
2
5
6
1
2
0. 2
8
6
4
+
R
R R R
S
-
+
+
7.5;
~8.4
135
8
2
5
6
3
2
2
5
6
2
2
5
6
2
5
6
1
2
0. 2
8
6
4
+
R
R R S
S
-
-
+
~8.4
160
3
2
5
6
1
6
2
5
6
2
2
5
6
2
5
6
1
6
1
2
6
4
+
R
R R S
S
-
-
+
~8.4
700
2
5
6
1
2
2
5
6
3
2
5
6
2
5
6
1
2
0. 2
5
6
4
+
R
R R S
S
-
-
+
~8.4
27
2
5
6
3
2
2
5
6
2
2
5
6
2
5
6
1
2
0. 2
8
6
4
+
S
S R S
S
-
-
+
~8.4
147
2
5
6
1
2
2
5
6
2
2
5
6
2
5
6
4
0. 2
8
6
4
+
S
S S S
S
-
-
+
~8.4
983
7
2
5
6
8
2
5
6
3
2
5
6
2
5
6
3
0. 2
8
6
4
+
S
S S S
S
-
-
+
~8.4
E.coli
TOP
10/
SP8
91
2
5
6
1
2
8
2
5
6
6
2
5
6
2
5
6
1
6
1. 8
5
6
4
+
S
S S S
S
+
-
+
5.4;
~8.4
TOP
10/
135
8
2
5
6
4
8
2
5
6
2
2
5
6
2
5
6
1
2
1. 8
5
6
4
+
S
S S S
S
-
-
+
~8.4
AB1
456/
2
5
6
4
2
5
2
2
5
2
5
1
6
1
6
4
+
S
S S S
S
-
-
+
~8.4
4
32
657
0-2
6
6
6
6
AB1
456/
SP8
91
2
5
6
1
9
2
2
5
6
6
4
0. 0.
5 1
0
.
5
0. 4
5
0
.
1
-
R
R S S
S
-
+
-
7.5
AB1
456/
657
0-1
2
5
6
2
5
6
2
5
6
6
4
0. 0.
8 1
0
.
5
0. 4
5
0
.
1
-
R
R R R
S
-
+
-
7.5
* Strains from St.-Petersburg (SP).
Susceptibility is colour-coded:
resistant (R)
intermediate (I)
susceptible (s)
AM – ampicillin, XL – amoxicillin/clavulanic acid, PP – piperacillin, PTc – piperacillin/tazobactam, CT –
cefotaxime, TX – ceftriaxone, TZ – ceftazidime, TZL – ceftazidime/clavulanic acid, AT – aztreonam, FX –
cefoxitin, Tet – tetracycline, Chl – chloramphenicol, Ag – aminoglycosides (gentamicin, tobramycin) Sxt
– trimethoprim/sulfamethoxazole, and Cip – ciprofloxacin.
CONCLUSIONS
Based on all assays performed, we conclude that the CTX-R isolates of S.typhimurium probably represented a single
clone broadly disseminated in Belarus and perhaps in some other countries of East and South Europe. The resistance
of this clone to extended-spectrum cephalosporins and penicillin-inhibitor combinations was attributed to
simultaneous production of a CTX-M-type ESBL and an OXA-1-related penicillinase.
ACKNOWLEDGEMENT: We wish to thank Dr. Nadezhda S. Kozlova and Dr. Dmitry Gladin (St.-Petersburg
Medical Academy) for providing S.typhimurium strains expressing a CTX-M-4 -lactamase.
© IAC SSMA 2000-2005 · [email protected]
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