Journal of Antimicrobial Chemotherapy

JAC Advance Access originally published online on June 2, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):1-6; doi:10.1093/jac/dkl204

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Leading articles

Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons

Mark A. Toleman, Peter M. Bennett and Timothy R. Walsh^*

Bristol Centre for Antimicrobial Research and Evaluation (BCARE), Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, University Walk Bristol BS8 1TD, UK

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^*Corresponding author. Tel: +44-117-9288819; Fax: +44-117-9287896; E-mail: t.r.walsh{at}bristol.ac.uk

	Abstract

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
The ability of bacteria to procure, sometimes rearrange, and evince acquired DNA continues to impress us—even more so if this genetic plasticity involves the sequestering of antibiotic resistance genes. The acquisition of genes in bacteria is often facilitated by transposons, integrons and archetype insertion elements. Recently however, a new element, ‘orf513’, has been increasingly associated with class 1 integrons. Moreover, these ‘complex’ class 1 integrons can potentially mediate resistance to chloramphenicol, trimethoprim, aminoglycosides and tetracycline and may carry a range of ß-lactamase genes as well as the qnrA gene. Elements such as ‘orf513’ demonstrate IS91-like characteristics and will mobilize adjacent DNA via a process called rolling circle replication, and thus we have renamed them ‘insertion sequence CRs’ (ISCRs) to appropriately reflect their structure–function properties. In this article, we provide a brief description of these new and clinically important mobile elements, and how they are able to mobilize antibiotic resistance genes.

Keywords: mobile elements , insertion elements , rolling circle transposition

	Introduction

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
Something strange is happening in the world of bacterial class 1 integrons. Not only are the integrons evolving as expected, by acquisition and expulsion of gene cassettes, but they are also accreting non-cassette resistance genes and integron fragment duplications. We propose that these unexpected changes reflect the activity of an IS element, ISCR1, which belongs to a family of unusual insertion sequences, typified by IS91.¹

Bacteria are noteworthy for their remarkable abilities to adapt to environmental change, even when it is potentially lethal. Collectively they possess an impressive set of tools with which to change the genetic blueprint of individual cells.² Evidence is now emerging that the ‘genetic tool box’ is more versatile than previously thought.

Historically, transposons were considered to be the main vehicles of gene transfer between DNA molecules. It was then discovered that the structures of some resistance transposons are not fixed; some, such as Tn21-like elements, contain gene integration systems, called integrons, that capture and reassort individual resistance genes.³ Integrons comprise a gene, int, that specifies a site-specific recombinase, integrase, and an adjacent site, attI, at which resistance genes, in the form of gene cassettes, can be specifically inserted. Integrons can assemble impressive resistance gene arrays.² These elements are responsible for much of the spread of antibiotic resistance among Gram-negative bacteria.

When a particular sequence is discovered in a number of different genetic locations the suspicion arises that it is a transposable element. When that sequence is associated with a variety of resistance genes, suspicion hardens that it is responsible for the movements of those genes. A DNA sequence, termed ‘orf513’, has recently been found associated with a number of different antibiotic resistance genes, most of which are closely associated with class 1 integrons (Figure 1). The sequence is an example of a CR (common region) element, a group of putative ‘mobile’ genetic elements that are found in ‘Salmonella genomic islands’ (SGIs) and on the integrative conjugative element SXT from Vibrio cholerae.⁴ Recent insights suggest that CR elements belong to a family of unusual IS elements, typified by IS91, which are distinctive because they use rolling circle replication for transposition (RC transposition).¹ Such a system allows genetic rearrangements that other IS elements cannot easily execute. We propose re-naming CR elements ‘insertion sequence CRs’ (ISCRs) because their IS91-like features indicate that they are a subgroup of IS91-like elements.¹

View larger version (12K): [in this window] [in a new window]   Figure 1. Genetic context of selected ISCR elements (shaded boxes) demonstrating their close proximity to complex class 1 integrons.

 

	ISCR elements and their roles in antibiotic resistance

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
The first CR element (now ISCR1) was discovered and reported in the early 1990s as a sequence of DNA of 2154 bp, incorporating orf513 (a putative gene of unknown function) inserted beside the sul1 genes of class 1 integrons, In6 and In7.⁵ The sequence was termed ‘CR’ to distinguish it from the 5' and 3' conserved sequences (CS) of class 1 integrons. In In6 and In7, and subsequently discovered complex integrons, the sequence is located between truncated and full-length versions of 3'CS, together with a resistance gene (Figure 1). These complex structures differ somewhat from standard class 1 integrons.² A further departure from the paradigm is that the ISCR1-linked antibiotic resistance genes are not associated with 59 base elements (site-specific recombination sequences that define gene cassettes) and therefore cannot have been acquired as gene cassettes.² Since the first CR sequence was identified, several more have been discovered and it is now appreciated that ISCR elements are distributed worldwide on plasmids and bacterial chromosomes in both Gram-negative and Gram-positive bacteria.⁶

	ISCR elements are IS91-like elements

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
Each ISCR accommodates a single gene encoding a putative transposase. These proteins differ in amino acid identity from 53% to 95%, but all lack the hallmark active site amino acid motif, DDE, typical of most other transposases.

ISCRs show homology to each other, but display little similarity to other mobile genetic elements. Amino acid alignments reveal, however, that the proteins encoded by ISCR elements possess the signature motifs, particularly tyrosine 253, of REP proteins involved in rolling circle replication, including the transposase of IS91, an unusual IS element.¹ A notable feature of IS91-like elements is that they lack terminal inverted repeats (IRs); their ends, oriIS and terIS, are quite different, indicating that each has a different role. This contrasts with conventional IS elements where the short terminal IRs have identical roles. A few studies have been published that identify ISCR 3' ends.⁷–⁹ When the nucleotide sequences immediately downstream of the transposase genes of several different ISCR elements are aligned, homology persists for 240–250 bp and is then abruptly lost. The discontinuities precisely identify the 3' ends of each element. The consensus for this end of an ISCR sequence, reading inside-out, is 5'-GCGTTTGAACTTCCTATACXX-3', a sequence strikingly similar to 5'-GxTTTTxAAATTCCTATxCAT-3', the consensus sequence of the origins of transposition (oriIS) for IS91, IS801 and IS1294.¹⁰ Given that ISCR elements are also similar in size to IS91 and encode IS91-like transposase proteins, we suggest that ISCR elements originated from the same ancestral source that evolved to IS91.

RC transposition provides a mechanism whereby a single IS element can mobilize sequences to which it is attached. Tavakoli et al.¹⁰ showed that a proportion of transposition events involving the IS91-like element IS1294 also moved a variety of sequences attached to the terIS end of the element, as a consequence of low-level misreading of terIS. Also, IS91-like elements have been shown to form free circular entities that may be intermediates in the transposition process.¹ We predict that these are involved in the DNA rearrangements that give rise to complex class 1 integrons.

We propose that ISCR elements move resistance genes by transposing next to them and then co-mobilizing them by a second, extended transposition event (Figure 2). If this hypothesis is correct, ISCR elements are powerful genetic tools that can mobilize any gene from any location, without the need for element duplication, as for most IS elements, in which case, ISCR elements are likely to be at least of equal importance to transposons and integrons in the evolution of antibiotic-resistant bacterial pathogens from humans and animals.

View larger version (16K): [in this window] [in a new window]   Figure 2. The mobilization of class 1 complex integrons by ISCR1. (a) oriIS and terIS-1 are denoted as the insertion and termination sites of ISCR1 transposition, respectively. Sequences underlined read 5'3' for each site and the nucleotides in bold match those of the consensus sequence of IS91, IS1294 and IS801 as reported by Tavakoli et al.10 The larger bold horizontal arrow indicates the direction (from right to left)and origin of replication. (b) The first event involves the transposition of ISCR1 next to or near to the 3' end of a class 1integron (denoted as qac/sul). Normally the transposase would recognize the putative termination sequence terIS-1 but misreads the termination sequence and instead terminates at a similar sequence, terIS-2. The small segment of DNA involving the 3' end of the class 1 integron and the 5' end of the intact ISCR1 is deleted truncating the sul gene (now denoted as a grey box) and erasing the normal termination site, terIS-1, and thereby creating an integron-ISCR1 fusion. From this point on ISCR1 is able to mobilize the integron (Int-qac/sul), any antibiotic resistance gene cassettes contained therein (represented by abxr), and any associated 5' composite transposons (Tnp) by an IS91-like RC mechanism. It is possible that ISCR1 might recognize the putative termination sequence terIS-2 or terIS-3 which may or may not be similar to terIS-1. However, the possibility also exists that ISCR1 does not possess an intrinsic termination site and accordingly terminates transposition randomly thereby mobilizing varied lengths of 5' (upstream) DNA.

 

	ISCR1 mediates formation of complex class 1 integrons

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
ISCR1 appears to be unusual in the sense that it is almost always found in complex class 1 integrons beside the same truncated version of 3'CS. This suggests a single deletion event that was then translocated to a variety of other sites. Given that ISCR1 is somewhat shorter than other ISCR and IS91-like elements, but retains oriIS and an intact transposase gene, it is likely that the terIS end of the element has been lost. The truncation of 3'CS and of the ISCR1 progenitor probably reflect the same molecular event, namely a deletion that encompassed both components.

At some time in the past, possibly in the first half of the twentieth century, the ISCR1 progenitor transposed into a site close to the 3'CS of a class 1 integron. This was followed by a deletion that started within the 3'CS region of the integron, removing orfs 5 and 6, and extended a short way into the ISCR element, creating ISCR1. ISCR1 then mediated a series of secondary transposition events that transposed ISCR1 and various lengths of the attached class 1 integron into sites next to a variety of other resistance genes, such as catA2, dfrA, qnr and various bla_CMY genes (Figure 1). Once in these locations, the ability of IS91-like elements to generate free circular forms was manifested. Circular entities carrying ISCR1, the truncated 3'CS and linked resistance genes were generated. These were then rescued by homologous recombination into the 3'CS sequence of other class 1 integrons (Figure 3), in two ways. First, recombination into standard class 1 integrons created complex integrons, such as In6 and In7, which carry single copies of ISCR1 beside an internal truncated copy of 3'CS. Alternatively, recombination into a class 1 integron already attached to a copy of ISCR1 would generate a direct duplication of the ISCR1 element following the duplication of the 3'CS sequence, a particular arrangement that has been reported by Mammeri et al.¹¹

View larger version (17K): [in this window] [in a new window]   Figure 3. Model of ISCR1-mediated construction of complex class 1 integrons. The construction of complex class 1 integrons can be explained by a three step mechanism. (A) Aberrant RC replication of the ISCR1 element (fused to 3'CS) generates transposition intermediates of different length. These intermediates then transpose adjacent to an antibiotic resistance gene (e.g. catA or qnr), in another location. (B) A second aberrant RC replication event produces circular intermediates which now include catA or qnr. (C) These circular intermediates can then be rescued by recombination events between 3'CS on another ‘normal’ class 1 integron (D and E) producing the complex integrons G and H, or they can be rescued by (F) a class 1 integron already including a copy of ISCR1 generating the complex integron I. Such aberrant RC transposition and recombination rescue events provide an explanation for the spectrum of complex class 1 integrons observed in nature. Boxes represent the open reading frames of the various genes with arrows indicating the direction of their transcription. The open reading frame of the ISCR1 elements is shaded in grey and the resistance genes that lack a 59 base element are patterned.

 
According to this model, the terminal full-length copy of 3'CS on a complex class 1 integron was originally contiguous with the int-gene cassette array of the complex integron (Figure 2). The internal, shortened version of 3'CS together with ISCR1 and associated resistance gene(s) comprise the added sequences.

	Association of ISCR elements with antibiotic resistance genes

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
ISCRs are closely associated with many antibiotic resistance determinants and not necessarily within an integron context. ISCR1 is associated with genes encoding resistance to chloramphenicol (catAII), trimethoprim (dfrA10, dfrA23, dfrA3b, dfrA19) and aminoglycosides (armA), and also with class A ß-lactamases (bla_CTX-M-2, bla_CTX-M-9, bla_CTX-M-20, bla_PER-3, bla_VEB-3) and class C ß-lactamases (bla_DHA-1, bla_CMY-1, bla_CMY-8, bla_CMY-9, bla_CMY-10, bla_MOX-1). The recent discovered gene, qnr, which confers resistance to quinolones and reduced susceptibility to fluoroquinolones, is also closely linked to ISCR1.¹¹ ISCR2 is linked to genes encoding resistance to trimethoprim (dfrA18, dfrIX, dfrA20), tetracycline (tetR), chloramphenicol (floR) and sulphonamides (sulII) (Table 1). ISCR3 is linked to qac, dfrA10, ereB, yieE and yieF; ISCR4 to bla_SPM-1; and ISCR5 to both bla_OXA-45 and ant4'IIb. The association of ISCR elements with genes encoding extended spectrum ß-lactamases (ESBLs), mobile AmpC-type ß-lactamases and metallo-ß-lactamases (SPM-1) is of particular concern regarding further spread of these resistance genes.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the ISCR subgroups and their association with antibiotic resistance genes

 

	Conclusions

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
The increasing number of database submissions concerned with CR elements linked to antibiotic resistance genes indicates that this situation is evolving and poses a potential clinical problem that could get worse. The most worrying aspect is that ISCR elements are increasingly linked with resistance genes that can pose clinical difficulties, e.g. those encoding metallo-ß-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia (M. A. Toleman, unpublished data).⁹ In 1999, Bennett hypothesized that bacteria would surprise us by extending their ‘genetic construction kit’ to facilitate capture of additional antibiotic-resistant genes and warned us to ‘watch this space’.² It would appear that ISCR elements have now firmly established themselves as part of that space.

	Transparency declarations

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
We confirm no conflicting financial interests.

	Acknowledgements

 
M. A. T. is funded by the EC COBRA project—contract LSHM-CT-2003-503335.

	References

Top Abstract Introduction ISCR elements and their... ISCR elements are IS91-like... ISCR1 mediates formation of... Association of ISCR elements... Conclusions Transparency declarations References

 
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2 Bennett PM. (1999) Integrons and gene cassettes: a genetic construction kit for bacteria. J Antimicrob Chemother 43:1–4.[Medline]

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4 Beaber JW, Hochhut B, Waldor MK. (2002) Genomic and functional analyses of SXT, an integrating antibiotic resistance gene transfer element derived from Vibrio cholerae. J Bacteriol 184:4259–69.[Abstract/Free Full Text]

5 Stokes HW, Tomaras C, Parsons Y, et al. (1993) The partial 3'-conserved segment duplications in the integrons In6 from pSa and In7 from pDGO100 have a common origin. Plasmid 30:39–50.[CrossRef][Web of Science][Medline]

6 Arduino SM, Catalano M, Orman BE, et al. (2003) Molecular epidemiology of orf513-bearing class 1 integrons in multiresistant clinical isolates from Argentinean hospitals. Antimicrob Agents Chemother 47:3945–9.[Abstract/Free Full Text]

7 Doi Y, Shibata N, Shibayama K, et al. (2002) Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate. Antimicrob Agents Chemother 46:2427–34.[Abstract/Free Full Text]

8 Partridge SR and Hall RM. (2003) In 34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob Agents Chemother 47:342–9.[Abstract/Free Full Text]

9 Poirel L, Magalhaes M, Lopes M, et al. (2004) Molecular analysis of metallo-ß-lactamase gene bla_SPM-1-surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil. Antimicrob Agents Chemother 48:1406–9.[Abstract/Free Full Text]

10 Tavakoli N, Comanducci A, Dodd HM, et al. (2000) IS1294, a DNA element that transposes by RC transposition. Plasmid 44:66–84.[CrossRef][Web of Science][Medline]

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12 Boyd D, Cloeckaert A, Chaslus-Dancla E, et al. (2002) Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46:1714–22.[Abstract/Free Full Text]

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