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Your continued donations keep Wikipedia running! Plasmid From Wikipedia, the free encyclopedia Jump to: navigation, search 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 [hide] 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: 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: "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: 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 Bacterial artificial chromosome Retrieved from "http://en.wikipedia.org/wiki/Plasmid" Category: Molecular biology Views Article Discussion Edit this page History Personal tools Sign in / create account Navigation Main Page Community Portal Current events Recent changes Random article Help Contact Wikipedia Donations Search Go Search Toolbox What links here 7 Related changes Upload file Special pages Printable version Permanent link Cite this article In other languages Česky Dansk Deutsch Español Français עברית Македонски Nederlands 日本語 Polski Português Suomi Русский Svenska Tiếng Việt 中文 This page was last modified 23:31, 9 February 2006. All text is available under the terms of the GNU Free Documentation License (see Copyrights for details). Wikipedia® is a registered trademark of the Wikimedia Foundation, Inc. Privacy policy About Wikipedia Disclaimers 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 ----------------------------------------------------------------------------------- Related Articles Related Resources « home : molecular biology Main 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] Related Articles Related Resources 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] 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, 200M of each dNTP and 1.5U of Taq-polymerase after addition of primers (50 pmoles each), 10l 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 2l 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, 200M of each dNTP, 0.25M of ea primer, 1.25U AmpliTaq DNA polymerase (Perkin-Elmer, USA) and 20l of templa DNA prepared with InstaGene matrix in total volume of 50l. 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 50l volumes: 50mM KCl, 10mM Tris-H (pH 9), 0.1% TritonX-100, 1. 5mM MgCl2, 200M of each dNTP, 0.4M of each pr 1 TaqBead Hot Start Polymerase (Promega, USA) and 5l 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 – 0.5M (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 (100g/ml) with either cefotaxime (10g/ml) or ampicillin (100g/ml). In addition, t plasmid DNA was transformed into competent cells of E.coli TOP10 (Promega, USA transformants were selected on agar containing cefotaxime (10g/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] 33 34 35