How Do You Know if Genes Are Plasmid Encoded

Br J Pharmacol. 2008 Mar; 153(Suppl i): S347–S357.

Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria

P M Bennett

iEmeritus Professor of Bacterial Genetics, University of Bristol, Bristol, UK

Received 2007 Jul 31; Accustomed 2007 Nov 5.

Abstruse

Bacteria have existed on Earth for three billion years or and then and take go adept at protecting themselves against toxic chemicals. Antibiotics take been in clinical use for a little more than than 6 decades. That antibiotic resistance is now a major clinical problem all over the world attests to the success and speed of bacterial accommodation.

Mechanisms of antibiotic resistance in bacteria are varied and include target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic aggregating. Acquisition of genes needed to elaborate the various mechanisms is greatly aided by a variety of promiscuous cistron transfer systems, such every bit bacterial conjugative plasmids, transposable elements and integron systems, that motion genes from i DNA organization to another and from one bacterial jail cell to another, not necessarily 1 related to the factor donor. Bacterial plasmids serve as the scaffold on which are assembled arrays of antibiotic resistance genes, by transposition (transposable elements and ISCR mediated transposition) and site-specific recombination mechanisms (integron gene cassettes).

The evidence suggests that antibody resistance genes in human bacterial pathogens originate from a multitude of bacterial sources, indicating that the genomes of all bacteria can be considered as a single global genetic pool into which most, if not all, bacteria tin can dip for genes necessary for survival. In terms of antibiotic resistance, plasmids serve a central function, as the vehicles for resistance gene capture and their subsequent dissemination. These various aspects of bacterial resistance to antibiotics volition be explored in this presentation.

Keywords: antibiotic resistance, plasmid, transposon, integron, resistance factor cassette, ISCR element, recombination, conjugation

Introduction

Mail-World State of war ii generations born in the developed countries of the globe take been highly privileged. Forget the astounding increase in affluence over the terminal 6 decades, put on one side the staggering technological advances and the dubious benefits of email and mobile phones; the immediate mail service-World War two generation was the kickoff to bask, from nascency, the benefits of a modern health system that has largely abolished the hurting and heartache of many infectious diseases, peculiarly those of childhood, that ravaged societies in the by. This was made possible past the discovery and development of a multitude of antibiotics. Directly and indirectly, their use has transformed medical practice to the point where, at least in the developed world, many, if not all, bacterial infections, so long the scourge of humanity, have, until recently, been considered to be little more than a nuisance, rather than the life-threatening conditions they were and can be. Nonetheless, over the last x–xv years, this comfortable perception has changed, as illustrated past regular reports in the printing, of the rise of 'superbugs' and speculation as to the foreseeable end of the antibiotic era. What has gone wrong in and then short a time, lilliputian more than two man generations? Are the doom-mongers to be believed, or are the pronouncements of media correspondents largely exaggeration, driven by the want for a 'good story'? Although antibiotic use clearly has a continuing major and effective role in current medicine, the commentators accept besides probably got it correct in the medium to long term. The ability of leaner to rapidly evolve into strains that are resistant to antibiotics, although foreseen by Alexander Fleming, has continually been underestimated. We tend to forget that bacteria have inhabited the planet for approximately three and a half billion years, somewhat longer than mankind, and in that time accept had to suit on innumerable occasions to toxic substances all of a sudden introduced into their environments. Indeed, almost antibiotics used today have their origins in antibacterial compounds produced by other microbes as weapons with which to protect their territories; that bacteria accept non only survived merely adjusted and proliferated impressively to colonize some of the most inhospitable parts of the planet attests to their powers of accommodation. It should come as no real surprise that they have adult powerful Deoxyribonucleic acid-modifying strategies that profoundly facilitate the process of adaptation and, hence, development.

Development of resistance to antibiotics by bacteria that threaten homo well-existence constitutes, arguably, the well-nigh serious challenge to the continuing efficacy of much of mod medical practice, such every bit complex surgical procedures and organ transplants. Antibiotic therapy, as nearly if not all in the medical profession appreciate, is one of the foundation stones of modern medicine. Without effective procedures to limit bacterial infection, many modernistic medical procedures would be considerably more risky, if not a consummate waste of fourth dimension and resources, and rates of morbidity and mortality from bacterial infection would exist considerably higher than at nowadays. Yet, the ground won in the hard-fought boxing for supremacy over these microscopic opponents is in danger of being ceded. Bacteria are non quiescent regarding their fates when faced with annihilation with antibiotics. Over several millennia, bacteria have become proficient at dealing with innumerable substances that threaten their survival. For them, antibiotics are just another group of poisonous compounds, the lethal effects of which have to exist neutralized in some way. The effectiveness of the diverse strategies employed is attested by the impressive speed with which resistant versions of human pathogens accept emerged to every antibiotic that has been introduced into clinical practise throughout the last 6 to vii decades. What drives the process? The reply is simple—utilise of antibiotics and the more the use the greater the likelihood that resistant strains of bacterial pathogens will emerge (Levy, 2002). Obviously, none of the changes that confer antibiotic resistance is caused by blueprint; rather, changes to the genetic blueprints of all bacteria are made at random. The vast majority make no improvement and are lost from the population over time; nevertheless, those that confer advantage are conserved and, given appropriate selection, undergo clonal amplification, a striking example of the Darwinian hypothesis 'survival of the fittest'. Indeed, the whole flow of antibody use can be considered as one large ongoing experiment designed to test the hypothesis. To date, Darwin'southward insight has been strikingly confirmed.

Changes can be made to a bacterium's genetic inheritance in two ways; (1) by mutations that change the pre-existing Deoxyribonucleic acid of the prison cell—these alterations, base of operations changes and Dna deletions and inversions (Avison and Bennett, 2005), change genes already possessed but do non add new genes, that is new Dna, to the prison cell genome—and (2) by acquisition of new genetic material, that is capture of genes new to the prison cell, which expands the genome. This commodity examines the latter machinery, which is largely, although not exclusively, responsible for the evolution of antibiotic-resistant variant strains of bacteria, which cause infections of man and animals.

The miracle of factor acquisition implies cistron transfer from some outside source; this source is other bacteria. Bacteria have three methods by which DNA may be transferred from one cell to some other; transformation, transduction and conjugation. This article will be concerned only with the last—conjugation—and more specifically with the elements that promote it, namely, bacterial plasmids. For more detailed descriptions of transformation, transduction and conjugation meet Bennett et al. (2004).

Plasmids are the platforms on which factor arrays are assembled and reassorted. The accession of potentially useful genes on these platforms, promoted by a variety of recombination systems, can allow a bacterial strain to aggrandize its expanse of functioning into niches that were previously denied to it because they were too hazardous, if not lethal. The development of multiple antibiotic resistance is a particularly striking example, which has allowed many bacterial species to gain a tenacious foothold in many of our hospitals, particularly big hospitals where antibiotics are used in large quantities. Accordingly, understanding how antibody resistance develops and is spread by mobile genetic elements is a desirable pre-requisite to the design and development of intervention strategies intended to minimize the threat of bacterial infections.

Mobile bacterial genetic elements

Mobile genetic elements autumn into two general types; elements that can motility from one bacterial jail cell to some other, which in terms of antibiotic resistance includes resistance plasmids and conjugative resistance transposons, and elements that can move from one genetic location to another in the aforementioned cell. The latter elements include resistance transposons, gene cassettes and ISCR-promoted cistron mobilization. Plasmids and conjugative transposons transfer from 1 prison cell to another by mechanisms that involve replication. Transposons, gene cassettes and ISCR-mediated factor transfer between sites on the same or on unlike Deoxyribonucleic acid molecules require some form of recombination, which may or may non also include some form of replication (Bennett, 2005). Plasmids accumulate antibody resistance genes as a consequence of the activities of at to the lowest degree these three recombination systems.

Bacterial plasmids

The elements that move many bacterial genes from one bacterial cell to another, the so-called horizontal factor transfer, are bacterial plasmids, specifically conjugative plasmids, that is those able to promote their own transfer and the transfer of other plasmids from i bacterial prison cell to some other. Plasmids are best thought of equally small-scale, auxiliary, dispensable chromosomes (Figure 1). In general, they exist separately from and are replicated independently of the chief bacterial chromosome, although the majority of replication functions are provided by the host cell. They practise not suit any of the set of core genes needed by the cell for basic growth and multiplication, but rather carry genes that may be useful periodically to enable the jail cell to exploit detail environmental situations, for example in the current context, survive and thrive in the presence of a potentially lethal antibiotic. Hence, plasmids carry a considerable variety of genes, including those that confer antibiotic resistance and resistance to a number of toxic heavy metals, such as mercury, cadmium and argent, those that provide enzymes that aggrandize the nutritional ability of the cell, virulence determinants that permit invasion of and survival in animate being systems and functions that enhance the chapters to repair DNA damage (Stanisich, 1988). Most plasmids investigated so far are circular, double-stranded Deoxyribonucleic acid molecules (Effigy i) that range in size from those with but 2 or 3 genes (ii–3 kb) to elements that are equivalent to 10% or more of the host cell chromosome, that is suit 400 genes or more (cf Escherichia coli chromosome that encodes approximately 4700 genes; Charlebois, 1999). A resistance plasmid is whatever plasmid that carries one or more antibiotic resistance genes (it may too be, for example, a metabolic plasmid, because information technology encodes a metabolic function, or a virulence plasmid, because it possesses 1 or more virulence genes. Wagon of 1 blazon of gene does not preclude carriage of other types that do not contribute towards maintenance and spread of the plasmid).

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An electron microscope movie of a small-scale bacterial plasmid.

Plasmid-encoded antibiotic resistance encompasses most, if not all classes of antibiotics currently in clinical employ and includes resistance to many that are at the forefront of antibody therapy. Notable among these are commonly used cephalosporins, fluoroquinolones and aminoglycosides. Many resistance plasmids are conjugative, that is they encode the functions necessary to promote cell-to-cell DNA transfer, peculiarly their own transfer. Others are mobilizable when helped by a conjugative plasmid co-resident in the cell. In general, mobilizable plasmids lack the genes that encode the functions that enable cells to couple prior to DNA transfer (which are provided past the conjugative plasmid) but do encode the functions needed specifically for transfer of their own DNA. Accordingly, mobilizable resistance plasmids tend to be relatively pocket-size, ofttimes less than 10 kb in size, encoding simply a scattering of genes including the resistance cistron(s), whereas conjugative plasmids tend to exist somewhat larger, 30 kb or more than (resistance plasmids of 100 kb or more are non unusual), reflecting the sizable amount of DNA (20–thirty kb) needed to encode the conjugation functions that permit cell-to-cell coupling, particularly between Gram-negative bacteria. Such coupling is mediated past an external filamentous appendage called a sex activity pilus, which substantially acts similar a grappling hook to join donor and recipient cells and which is then retracted into the donor to effect envelope-to-envelope contact, when a DNA transfer pore forms to bridge the cytoplasmic compartments of the conjoined cells (Wilkins, 1995). Conjugative plasmids in Gram-positive bacteria tend to be smaller than those in Gram-negative bacteria, reflecting a somewhat different mechanism of cell-to-cell coupling (see Bennett, 2005), which requires less genetic data. Conjugation is a replicative process that leaves both donor and recipient cells with a re-create of the plasmid (Wilkins, 1995).

Conjugative plasmids tin can showroom broad or narrow host range. For the latter, transfer is restricted generally to and betwixt a small number of like bacterial species. Broad host range denotes an element able to transfer between widely different bacterial species and, indeed, some broad host range plasmids from Gram-negative leaner announced to accept no host limitation within the division and, using genetic constructs assembled in the test tube, have been shown to be able to transfer to, but not survive in both Gram-positive leaner and unicellular eukaryotic microbes such equally yeast. Ane broad host range plasmid is the resistance plasmid RP1 (also known as RP4 and RK4), first identified in a clinical strain of Pseudomonas aeruginosa. This plasmid appears to be able to transfer productively to most, if not all Gram-negative bacteria. Many other, unrelated resistance plasmids are also known to take broad host ranges (Thomas, 1989). What determines host range has not been widely investigated, but 1 possibility is that it reflects the nature of the surface receptor on the potential recipient cell needed by the particular conjugation machinery of the plasmid. If the potential recipient prison cell lacks this structure, and then plasmid transfer to it will not occur. If distribution of the receptor is limited, and so the plasmid will exhibit a narrow host range. Another possibility is that although transfer of the plasmid is successful, the recipient cell is unable to back up its replication. Both broad host range and narrow host range plasmids are mutual. Indeed, plasmids are common in nearly bacterial species investigated to date, identifying a big pool of mobile genetic information. Farther, multiple plasmid railroad vehicle is not at all uncommon.

Transposons

Resistance transposons are essentially jumping gene systems that incorporate a resistance cistron inside the element. They come up in many forms, distinguished by structure, genetic relatedness and mechanism of transposition and tin can carry a diversity of resistance genes (Bennett, 2005). What is illustrated (Tables ane and two) are simply a few of those known. All of these elements have the power to motility both intra- and inter-molecularly, that is they can leap from one site to another within a DNA molecule or from ane DNA molecule to some other, for instance from one plasmid to another, or from a plasmid to a bacterial chromosome and vice versa. These mechanisms generally do non require Deoxyribonucleic acid homology betwixt the element and the sites of insertion and although there are examples where a detail transposon has a strong preference for a detail nucleotide sequence at an insertion site, many others show no obvious preference and insert into new sites more or less at random (Craig, 1997).

Table 1

Some examples of composite resistance transposons from Gram-negative and Gram-positive bacteria

Transposon Size (kb) Last elements Marker(s)
Gram-negative elements
 Tn5 5.7 IS50 (IR) KmBlSm
 Tn9 2.5 ISone (DR) Cm
 Tn10 ix.3 IS10 (IR) Tc
 Tn903 3.1 IS903 (IR) Km
 Tn1525 4.4 ISxv (DR) Km
 Tn2350 x.4 ISone (DR) Km
Gram-positive elements
 Tn4001 iv.7 IS256 (IR) GmTbKm
 Tn4003 3.six IS257 (DR) Tm

Table 2

Some examples of complex resistance transposons from Gram-negative and Gram-positive bacteria

Transposon Size (kb) Terminal IRs (bp) Mark(south)
Gram-negative elements
 Tni 5 38/38 Ap
 Tnthree v 38/38 Ap
 Tn21 20 35/38 SmSuHg
 Tn501 8.2 35/38 Hg
 Tn1721 11.4 35/38 Tc
 Tn3926 7.8 36/38 Hg
Gram-positive elements
 Tn551 5.iii 35 Ery
 Tn917 5.3 38 Ery
 Tn4451 6.2 12 Cm

Transposons belong to the set of mobile elements called transposable elements that encompasses small cryptic elements called insertion sequences (IS elements), transposons and transposing bacteriophages, such as bacteriophage μ (Bennett, 2004). This concluding type of chemical element is a bacterial virus that uses transposition to replicate. A transposon differs from an IS element in that it encodes at least one role that changes the phenotype of the cell in a predictable fashion, for instance a resistance transposon confers resistance to a particular antibiotic(due south).

Transposons are either modular systems, referred to as blended transposons, constructed from a pair of IS elements and a central DNA sequence that is not inherently able to transpose, the expression of which alters the cell phenotype (Figures 2 and 3) or circuitous systems where transposition and not-transposition functions have not plain been assembled in a modular way (Figure 4). For an IS element-dependent resistance transposon, two copies of the aforementioned IS chemical element are needed as flanking terminal structures, either as direct or inverted repeats, to the primal department that contains the cistron(s) that confer antibiotic resistance. Although the inverted system of IS elements is more stable genetically, the direct repeat arrangement offers opportunities to migrate to another site where the IS element is as well found. This occurs by a two-stage homologous recombination process whereby office of the composite structure is showtime excised from its existing site by a single crossover between the copies of the IS element releasing a round, double-stranded DNA species comprising the central section of the composite resistance transposon and one re-create of the IS element, the other remaining at the original genetic location. The released DNA tin can then be rescued, essentially by reversing the first recombination, by homologous recombination involving a unmarried crossover, using the copy of the IS sequence on the free intermediate and some other copy at the new location, which recreates the blended transposon at the new site. As IS elements can generally exist found at many unlike sites, particularly on different plasmids, the potential for moving resistance genes around by this method is considerable. Accordingly, there are pros and cons regarding both arrangements. The transposition modules, the IS elements, more often than not retain the ability to transpose as individual elements as well as part of the compound structure. This is not the case with complex elements, which are indivisible with respect to transposition. The well-known transposons Tn5, encoding resistance to aminoglycosides such every bit kanamycin and neomycin, and Tnx encoding resistance to tetracycline, are compound elements found in a number of Gram-negative bacteria, particularly members of the enterobacteriaceae. Such chemical compound structures are created past adventure and go established in a population of cells by the selective forces operating on the bacterial flora, for example exposure to kanamycin/neomycin or tetracycline, respectively, when the particular element confers a distinct survival advantage. With time, the construction tends to undergo changes that stabilize it. Many such elements may have a relatively recent genesis. In contrast, Tn3, encoding resistance to a number of β-lactam antibiotics, including ampicillin, and Tn21 (Effigy 5), encoding resistance to streptomycin, spectinomycin and sulphonamides equally well as mercuric ions, are examples of complex transposons and are also commonly institute on plasmids in members of the enterobacteriaceae. Construction of this type of element is less hands explained and no general prove-based model has yet been proposed, although aspects of construction of some complex transposons, such as Tn21, can exist deduced. Tn3, Tn21 and similar elements are likely to be of somewhat greater antiquity than most composite transposons and are probably the results of multiple recombination events, including both insertions and deletions, which get-go insert not-transposition functions into a cryptic element so refine the sequence past deletion to eliminate 'non-essential' functions. This refinement would make the element more compact and hence more than readily transposable. It has been demonstrated that increasing the size of a item transposable element reduces its frequency of transposition.

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A diagrammatic representation of the blended resistance transposon Tn5 that confers resistance to kanamycin, bleomycin and streptomycin. O, I, outside and within boundaries of the last inverted ISfifty elements accommodating the short final inverted repeats (IRs) that ascertain IS50L (left-hand copy of IS50) and IS50R (correct-hand copy of IS50) and that are essential for transposition of both ISl and Tn5; km, kanamycin resistance gene; bl, bleomycin resistance gene; sm, streptomycin resistance gene; tnp, gene encoding the IS50 transposase; inh, factor encoding an inhibitor of the IS50 transposase; bp, base pairs (for further data see Reznikoff, 2002).

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A diagrammatic representation of the composite resistance transposon Tn10 that confers resistance to tetracyclines. Tnten displays last inverted repeats of IS10; IS10L, left-hand re-create of IS10, a non-functional copy of ISten due to multiple mutations in the transposase gene; IS10R, right-manus copy of ISten that encodes a functional transposase and an antisense RNA molecule used for downregulated expression of the transposase factor; IR, brusk inverted repeat sequences institute at the extremities of IS10, which are essential for transposition of both IS50 and Tn10; tetA, gene encoding the tetracycline resistance efflux pump; tetC,D, genes co-regulated with tetA; tetR, gene encoding a transcriptional repressor necessary for inducible expression, by tetracycline, of tetracycline efflux pump TetA; bp, base of operations pairs (for farther data see Haniford, 2002).

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A diagrammatic representation of the complex resistance transposon Tn3 that confers resistance to ampicillin and another β-lactam antibiotics. IR, the short inverted repeated sequences found at the extremities of the transposon, which are essential for Tniii transposition; tnpA, gene encoding element's transposase; tnpR, cistron encoding a site-specific recombinase that resolves the transposition cointegrate structure generated by transposition; bla TEM-1, gene encoding the TEM-1 β-lactamase; bp, base of operations pairs; kb, kilobase.

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A diagrammatic representation of the complex resistance transposon Tn21 that confers resistance to streptomycin, spectinomycin, sulphonamides and mercuric ions. merTPCAD, genes encoding resistance to mercuric ions and some organo-mercurial compounds; merR, gene encoding the transcriptional repressor of the inducible mer operon; sul1, factor encoding resistance to sulphonamides; aadA1, cistron encoding resistance to streptomycin and spectinomycin; int, integrase gene; attI, integron gene cassette insertion site; tnpA, cistron encoding the Tn21 transposase; tnpR, gene encoding a site-specific recombinase responsible for resolution of the transposition cointegrate structure generated past transposition; p int , int promoter; pc, promoter for integron gene cassettes and suli.

Integrons and gene cassettes

Bacterial integrons are gene capture systems that utilize site-specific recombination, instead of transposition, mechanisms (Effigy 6). They incorporate a specialized recombination organisation consisting of a gene, int, which encodes a site-specific recombination enzyme called an integrase, and a site at which brusk Dna sequences called gene cassettes (Recchia and Hall, 1995), considering most accommodate only a single gene, are inserted by the integrase. In the procedure of moving from one integron to another, or from ane site in an integron to another in the same integron, a gene cassette exists as a small, autonomous non-replicating double-stranded circular Dna molecule. This state is an intermediate in the mechanism that mediates cassette transfer from 1 integron to another or the re-assortment of gene cassettes within a detail integron (Bennett, 1999).

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A diagrammatic representation of the elements of a class one resistance integron. 5′-CS, 5′ constant sequence; 3′-CS, 3′ constant sequence; int, integrase gene; attI, integron gene cassette integration site; attC, gene cassette insertion sequence (likewise called a 59 base element); qacEΔ, truncated version of the quaternary ammonium compound resistance gene qacEastward; sul1, cistron encoding resistance to sulphonamides; orfv,6, possible genes (open reading frames) of unknown function(for further information run across Hall, 1997).

Most resistance integrons conform to a structure known as a grade 1 integron. Such elements have a distinctive structure comprising two final invariable regions, termed constant sequences (CS), and a highly variable central section. At ane end is v′-CS, which accommodates int, the cassette insertion site attI and the promoter from which cassette genes are expressed. At the other stop is 3′-CS, which accommodates part of a gene, qacEΔ1, that intact confers resistance to quaternary ammonium compounds, followed by sul, a gene that confers resistance to sulphonamides and 2 other genes designated orf5 and orf6. These CS regions flank a variable one, both in terms of length and sequence, that comprises the gene cassettes of the particular integron. This region necessarily varies as the identities and number of the gene cassettes change from one integron to another.

Resistance gene cassettes lack promoters from which to express the genes they carry. Accordingly, a promoter is provided inside five′-CS for the cassettes that are part of the integron. Necessarily cassette insertion is strictly oriented so that the beginning of the cistron carried by the cassette is the nearest int. Gene cassettes tin can exist inserted one subsequently the other into the integron insertion site, attI (Figure 7), to produce impressive resistance cistron arrays (Effigy eight). Each insertion regenerates attI. Two or more are expressed in a polycistronic manner from the cassette promoter within 5′-CS and such expression displays polarity. The gild of the gene cassettes from v′-CS indicates the order of addition, the i nearest being the latest addition, considering each is inserted at the aforementioned point. Accordingly, each new cassette addition displaces the existing assortment from attI. The various resistance cassettes, of which 50–lx are known, confer resistance to several classes of antibiotics (Table three) (Recchia and Hall, 1995). More recently identified antibiotic resistance factor cassettes include those that encode the metallo-β-lactamases IMP and VIM (Laraki et al., 1999; Lauretti et al., 1999; Poirel et al., 2000; Riccio et al., 2000; Nordmann and Poirel, 2002) (Figure 8), which confer resistance to the potent carbapenem β-lactams imipenem and meropenem. Where individual cassettes originate from is unknown, but impressive chromosomal arrays of gene cassettes encoding a multiplicity of functions, known every bit super integrons and containing tens of factor cassettes, including resistance factor cassettes, have been found in a number of bacterial species (Rowe-Magnus et al., 2001). The private cassettes in these arrays tin migrate to the much smaller integrons found on plasmids in bacteria of clinical origin, most of which usually contain less than five gene cassettes.

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Cartoon of gene cassette capture by a bacterial integron.

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Some examples of resistance cistron arrays in grade i bacterial integrons. intane, form 1 integrase cistron; qac, gene encoding resistance to 4th ammonium compounds; attI, integron gene cassette insertion site; attC, cistron cassette insertion sequence; qacEΔ1, truncated version of qacE; sul1, gene conferring resistance to sulphonamides; orf (open reading frame), possible cistron of unknown function; bla VIM, cistron encoding a VIM metallo-β-lactamase; bla IMP, gene encoding an IMP metallo-β-lactamase; aacA, aadA, aph, genes encoding resistance to aminoglycosides; cat, cistron encoding resistance to chloramphenicol.

Table 3

Examples of resistance genes carried on gene cassettes in grade i bacterial resistance integrons

Resistance to β-lactams
 Course A β-lactamases: bla P family (P1,2,3)
 Form B β-lactamases:blaIMP family (1–8); blaVIM family unit (i,two,3,4)
 Class D β-lactamases: blaOXA family unit (1,two,three,v,7,9,10)
Resistance to aminoglycosides
 Aminoglycoside adenylyltransferases: aadA1a, aadA1b, aadA2, aadB
 Aminoglycoside acetyltransferases: aacA1, aacA4, aacA7,aacC1, aacC
Resistance to chloramphenicol
 Chloramphenicol acetyltransferases: catB2, catB3, catB5
 Chloramphenicol exporter: cmlA
Resistance to trimethoprim
 Course A dihydrofolate reductases: dfrA1, dfrA5, dfrA7, dfrA12, dfrA14
 Class B dihydrofolate reductases: dfrB1, dfrB2, dfrB3
Resistance to antiseptics/disinfectant
 Quaternary ammonium chemical compound exporter: qacE, qacG

ISCR-mediated cistron transfer

Although the movements of resistance transposons and gene cassettes can account for much of the recombination involved in resistance plasmid structure, it has recently become apparent that in that location is at to the lowest degree one other recombination system that contributes to the associates of banks of resistance genes on bacterial plasmids. This aspect of plasmid evolution is based on a set of mobile genetic elements chosen ISCR elements (Toleman, Bennett and Walsh, 2006a). These are pocket-size cryptic sequences of sizes similar to those of many IS elements. They are predicted to transpose, like IS elements, hence the IS designation, but past a fundamentally unlike mechanism chosen rolling circumvolve (RC) transposition, which couples RC replication, equally used by some bacterial plasmids and bacteriophages, and recombination to achieve transposition (Tavakoli et al., 2000; Garcillian-Bracia et al., 2002). ISCR elements were outset detected as sequences associated with but distinct from class one resistance integrons (Effigy 9). Considering the same sequence was commonly found it became known as the common region, or CR (Stokes et al., 1993). Later it was establish that the original CR sequence, now designated as ISCR1, is a member of an extended set up of similar elements (Figure 10). When the identities of CR sequences were appreciated, information technology was thought desirable to retain the CR designation for the purpose of continuity, while besides advert their essential nature through use of the prefix IS. To date, more a dozen related elements have been discovered; now designated as ISCR1, ISCR2 and then on (Toleman et al., 2006a, 2006b).

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Examples of complex grade 1 bacterial resistance integrons displaying copies of ISCR1 and duplications of 3′-CS. The broken line indicates the v′-CS and variable regions of the class i integron components of the complex integrons; DHA-ane, Fox-1, CMY-ane,eight, MOX-1, genes encoding β-lactamases that confer resistance to third generation cephalosporins, probably recruited from the chromosomes of the bacterial species indicated; for other abbreviations see legend to previous effigy.

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A phylogenetic tree of ISCR elements. Based on a CLUSTAL alignment with the PAM 250 matrix prepared using Lasergene DNAstar software (for farther information see Toleman et al., 2006a).

ISCR elements are distantly related to a trio of closely related IS elements, IS91, IS801 and IS1294 (Garcillan-Bracia et al., 2002; Toleman et al., 2006a, 2006b). It has been known for some time that these elements differ markedly from most other IS elements in both structure and mode of transposition. Most IS elements are delineated by curt, inverted nucleotide repeats (see Figures two and 3) that functionally are interchangeable and serve to ascertain the ends of the elements and act equally bounden and cleavage sites for the cognate transposase. Nigh, if non all, circuitous transposons also brandish these features (meet Figures 4 and v). In contrast, ISCR elements lack terminal inverted repeats. Rather they possess distinct last sequences designated oriIS and terIS (come across Figure 11) that point the unique sites for the initiation and termination, respectively, of the RC replication stage of RC transposition (Tavakoli et al., 2000). One characteristic of these systems, relevant to the spread of antibiotic resistance genes, is that recognition of terIS shows a degree of inaccuracy, upwards to 10%, allowing replication to proceed across terIS and into the adjacent sequence, where information technology appears to be terminated more or less at random. Sequences several times the size of the element itself can be co-transposed in this fashion (Tavakoli et al., 2000), simply always those adjacent to terIS. The mobilization is intrinsic to the RC transposition machinery and will, in principle, mobilize any DNA post-obit the replication termination indicate. This is predicted to be the central to the construction of complex form i integrons.

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Mobilization of a form ane integron by ISCR1. ISCR1 is shown in (a) delineated by its terminal sequences terIS-1 and oriIS. Information technology is proposed that a re-create of ISCR1 is transposed into a site close to the three′-CS end of a class ane integron. A deletion and then removed part of the 3′-CS (including orf5,half-dozen) to generate the distinctive iii′-CS-ISCR1 arrangement seen in circuitous class i integrons.

Complex class 1 integrons accept 2 notable features; approximately one-half of the structure comprises a typical class 1 integron with 5′-CS, 3′-CS and intervening variable region, followed by a copy of ISCR1 and and so another variable region that accommodates a diverseness of resistance genes, including bla CMY and bla CTX variants, qnrA, dfrA variants and catAII, which between them confer resistance to a range of cephalosporins, fluoroquinolones such equally ciprofloxacin, trimethoprim and chloramphenicol. This variable region is, in plow, followed past a repetition of iii′-CS (Effigy nine) (Toleman et al., 2006b). Accordingly, an explanation of their construction must account for the ubiquitous presence of ISCR1 and its constant location within 3′-CS, a diversity of resistance genes that are non components of factor cassettes and consequently accept not been inserted at an attI site and the three′-CS duplication. The model proposed in Figures 11 and 12 does this. It utilizes the known transposition activities of IS91-similar elements, especially the trend to overshot terIS when engaged in RC transposition and the power to create free circular species, which may exist transposition intermediates (Tavakoli et al., 2000; Garcillan-Bracia et al., 2002).

An external file that holds a picture, illustration, etc.  Object name is 0707607f12.jpg

Model of ISCR1-mediated generation of a circuitous class i integron. See text for an explanation of the steps involved in generation of circuitous class 1 integrons.

The model proposes that ISCR1 became associated with the three′-CS of a class i integron, as a chance outcome (Effigy 11). A series of abnormal transpositions then placed ISCR1, together with dissimilar lengths of the class 1 integron alongside a multifariousness of resistance genes (Figure 12, step A). From these constructs complimentary, circular species containing ISCR1, whole or part of 3′-CS and a non-gene cassette resistance gene(southward), were generated by the RC transposition machinery (Figure 12, step B) and subsequently rescued by homologous recombination into another class 1 integron, using the common three′-CS sequence as point of crossover (Figure 12, pace C). This model accommodates successfully all complex class 1 integron structures identified (Toleman et al., 2006a, 2006b) and a number of resistance gene arrays assembled circular other ISCR elements (Toleman et al., 2006a).

For several years, IS91 and its like were curiosities in the earth of IS elements (Garcillan-Bracia et al., 2002); a rather esoteric area of study, which was of only minor interest beyond IS aficionados. The discovery of their intimate interest in the motility of resistance genes that challenge the use of some of the latest and about potent of currently used antibiotics has brought them centre-stage. Farther, their ability to mobilize, at least in principle, whatever bacterial DNA sequence ways they pose a considerable threat in the future, their activities being limited only by their distributions. That several, if not all, ISCR elements have migrated on to plasmids means that their potential for movement between bacterial species is considerable. Accordingly, a large proportion of the entire bacterial gene pool on the planet is potentially accessible for transposition on to bacterial plasmids. It is to be expected that this is happening all the time, simply that only a small minority of such rearrangements are fixed into the populations of cells where they occur because the appropriate selective forces needed to set up and amplify particular rearrangements are missing, at the fourth dimension. Our intensive use of antibiotics establishes the necessary selective pressures to ensure survival of a detail subset of rearrangements, namely, those involving genes conferring antibiotic resistance to the host jail cell. Whether these occur in bacteria able to crusade infection is immaterial. Information technology is sufficient, in the offset instance, merely to preserve and dilate the rearrangement. It will afterwards be spread horizontally by plasmids to other leaner, among which will exist human pathogens. The route may exist straightforward or somewhat circuitous, but I have no dubiousness that the journeying will be completed. The only question is 'How long will information technology take?', a question that cannot exist answered with any degree of confidence.

Ramifications of MGE activities

Thus, at least three fundamentally dissimilar recombination systems human activity to get together and re-assort resistance genes on bacterial plasmids. Any combination may be involved in the generation of a particular resistance plasmid, but the operation of all three mechanisms provides bacteria with an extremely powerful and flexible genetic tool box that makes up with prolific activity what it lacks in management. It is the divergence betwixt a hacker and a computer searching for a password; whereas the former brings to bear intelligence and insight to the task, the latter uses its ability to address all possible permutations. So it is with the global bacterial population. What is defective in sentient ability is more than than compensated for past sheer numbers. One gauge of the global bacterial population is put at x31 individual cells, so one would gauge that even if an event occurs only at very depression frequency, the sheer size of the global bacterial population means that it is likely to occur and re-occur in many places and on many occasions.

Thus gene cassettes can insert into integrons that incorporate part of a transposon that is carried on a plasmid. The integron conveying transposon Tn21, found on a plasmid in one of the first multiresistant strains of E. coli in the 1960s is a prime example. ISCR activeness can add further genes to the system via interaction with integron sequences and the whole associates can so be transferred from 1 bacterial prison cell to another. So these various recombination systems provide bacterial cells with the necessary mechanisms with which to capture and re-assort a whole diversity of resistance genes that they demand at present, and in the futurity. The movement is random, and generally independent of drug use, but drug utilise provides a powerful choice for these events in one case they have happened. The consequence is the rising of the 'superbug', bacteria that are largely unaffected by many different antibiotics, a few being resistant to most anything in the pharmaceutical armoury that would commonly be deployed against them. Notable amid them currently are multiresistant strains of P. aeruginosa and Acinetobacter baumannii. Between them, these recombination systems take the capacity to recruit on to plasmids any cistron from any bacterial cell. Thus, as noted to a higher place, the entire global bacterial gene pool is, in principle, available as a source of resistance genes. This has major implications for antibiotic therapy. Given that all antibiotics are necessarily selective and none is effective against all bacteria, then for each antibiotic, there is a subset of bacteria that are innately resistant. If the basis of resistance is genetically discrete, that is dependent on a single gene or a few linked genes, rather than the interaction of complex systems, then there is no reason, in principle, why these cannot be mobilized on to plasmids and transferred to other leaner; witness the somewhat complex genetic make-up of the resistance component of the vancomycin resistance transposon, Tn1546, found in the enterococci (Arthur and Courvalin, 1993; Courvalin, 2006). The logical extension of the argument is that, unless an antibody acts in a fundamentally different manner from those discovered and adult in the by, resistance is likely to arise to any new antibiotic introduced into clinical practise in the futurity. The only question to exist answered is not 'Will resistance ascend?' but rather, as noted to a higher place, 'When will resistance emerge?'. It is not possible to reply this question. Past experience suggests that clinical resistance will emerge in the curt term rather than the long term, that is in 4–5 years rather than decades. The driving force will be the use of the antibody and the more constructive it is the more it will be used, and then 'ratchetting upwardly' the selective pressure for the emergence of resistant bacteria. Initially this volition lead to an increase in the environment of the numbers of innately resistant microbes, about of which are unlikely to cause infection, and a subset with acquired resistance generated by the activities of mobile genetic elements, but their increment in numbers will raise the supply of potential resistance genes from which leaner able to crusade man infection will recruit those that are suitable for them. Which genes are transferred, how often and to what volition be random processes, but some of the results will, in turn, be subject to the selective pressure of exposure to the antibiotic. Some, if non all, of these genetic constructions will survive and will be passed to other bacteria. Eventually, the resistance genes will find their way into bacteria that are intimately associated with man and domestic animals. It is then a short step to their conquering by potential human pathogens. This is predictable; as noted, the precise time calibration is not.

Conclusions

Given that resistance has arisen to all antibiotics introduced into general clinical practice in the past and is likely to arise to any antibiotic introduced in the future, information technology would exist sensible to consider what tin can be done to minimize the impact of these developments. Modern clinical practise relies on antibiotic use, not simply to gainsay many bacterial infections but also to preclude infection in the first place. If antibody use becomes fatally compromised, there may exist footling nosotros tin can do when someone is infected apart from palliative care, but thought should exist given to what strategies, autonomously from prophylactic use of antibiotics, can be developed and deployed to avoid bacterial infections.

Despite the high cost of new antibiotic discovery, it is conspicuously desirable that new antibiotics continue to be developed for clinical use, since the time will come when currently used compounds are compromised by resistance to the point where effective use can no longer exist guaranteed. To postpone the emergence of resistance, the level of employ of all antibiotics should exist reduced to the minimum compatible with clinical imperative. This has to exist on a global footing, every bit resistant strains of bacteria tin sally anywhere and travel rapidly to all parts of the world, courtesy of the world's airlines and mass air travel. Appropriately, earth-broad education is needed to emphasize the consequences of overuse and misuse of antibiotics. The emergence of resistance may likewise exist delayed by antibiotic cycling, if this is feasible, or by combination therapy to make treatment more than potent; however, the combination of resistance to aminoglycosides and β-lactams is quite common and has probably arisen from the widespread apply of aminoglycoside-β-lactam combination therapy.

Given that antibiotic utilize volition lead to the emergence of bacteria resistant to the antibiotic, it is sensible to investigate procedures that will minimize their impact. Accordingly, best exercise in hospital infection control systems should be established in all institutions where antibiotics are used routinely, including thorough, regular cleaning and disinfection of infirmary wards and surrounds. In the future, this may be facilitated past improve pattern of hospital facilities that minimizes hard-to-access aspects and areas (curves are better than angles) and use materials that clean easily. A science fiction scenario would be the self-cleaning ward. Coupled to this, to ensure bacterial cleanliness, cleaned areas should be subject field to bacterial surveillance. Indeed, it may be desirable to subject patients to bacterial surveillance prior to admission to clean areas and to undertake decontamination procedures if contamination is detected prior to access.

If antibody use does get fatally compromised, alternative effective prophylactic procedures will exist needed for complex surgical processes, given that it is near impossible to guarantee hygienic atmospheric condition for prolonged recovery periods. One possibility, the evolution of vaccines that would protect against the most common nosocomial infections, is an obvious suggestion.

Glossary

CS common sequence
CR common region
IS insertion sequence
RC rolling circumvolve

Footnotes

Conflict of interest

The authors state no disharmonize of interest.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268074/

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