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ISCR Elements: Novel Gene-Capturing Systems of the 21st Century?

Bristol Centre for Antimicrobial Research and Evaluation, Department of Cellular and Molecular Medicine, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, United Kingdom

SUMMARY

INTRODUCTION

CRs AND THEIR DISCOVERY

CHARACTERIZATION OF CRs

CRs ARE IS91-LIKE TRANSPOSABLE ELEMENTS

ISCR ELEMENTS AND ASSOCIATED GENES

ISCR1

ISCR1 association with trimethoprim resistance genes.

ISCR1 association with plasmid-mediated quinolone resistance.

ISCR1 association with aminoglycoside resistance genes.

ISCR1 association with class A ß-lactamases.

ISCR1 association with blaCTX-M genes.

ISCR1 association with other class A ß-lactamase genes.

ISCR1 association with class C ß-lactamase genes.

ISCR2

Involvement of ISCR2 with SXT.

ISCR2 association with other resistance mechanisms.

ISCR3

Involvement of ISCR3 and ISCR1 with the Salmonella genomic island 1 genetic element.

ISCR4

ISCR5

ISCR6

ISCR7 and ISCR8

Other ISCR Elements

ISCRs MOBILIZE AND CONSTRUCT COMPLEX CLASS 1 INTEGRONS

SIMILARITIES AND DIFFERENCES BETWEEN ISCR AND IS91-LIKE TRANSPOSASES

SPECIFICITY OF INSERTION OF IS91 AND ISCR ELEMENTS

ISCR ELEMENTS AND PROMOTER SEQUENCES

ORIGINS OF CR ELEMENTS

IS91 AND VIRULENCE FACTORS

CONCLUSIONS

REFERENCES

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INTRODUCTION

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Bacterial antibiotic resistance is, once again, high on the clinical and scientific agenda. The ability of bacteria to evolve antibiotic-resistant forms is impressive, as attested to by a continuing concern that untreatable "superbugs" will continue to emerge in clinical medicine. Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments, not least for their adaptability in reassorting and redistributing genetic information. Bacteria possess an impressive set of tools with which to adjust the blueprint of the cell, the bacterial genome, enabling the cell to alter, add, or lose genetic information. Nowhere is this potential for change better illustrated than the bacterial riposte to antibiotics, antiseptics, disinfectants, and heavy metals used in clinical medicine. Although mutation has an important role to play in the evolution of antibiotic resistance, the predominant factor for the escalation of antibiotic resistance in more than half a century is the acquisition of antibiotic resistance genes.

The acquisition and spread of antibiotic resistance genes among bacteria that are intimately associated with humans and their domesticated animals are well documented. Data from studies examining this horizontal gene transfer indicate that it is driven by multiple systems that involve both cell-to-cell transfer and gene transfer from one DNA molecule to another. The latter can take place regardless of similarity between the donating and recipient molecules (38, 73) and includes systems such as transposons and site-specific recombination systems termed integrons (8, 9, 19). Recently it has become clear that while these systems can account for much of the movement of resistance genes between DNA molecules, they fail to explain the spread of a substantial and growing subset of resistance genes. These resistance genes are linked to sequences termed "common regions" (CRs), which are often found beyond but close to the 3' conserved sequences of class 1 integrons. A comparative analysis of these CRs has revealed that they are related to each other and resemble an atypical class of insertion sequences (ISs), designated IS91-like. IS91 and like elements differ from the insertion sequence paradigm (59) in that they lack terminal inverted repeats (IRs) and are thought to transpose by a mechanism termed rolling-circle (RC) transposition (because the mechanism involves rolling-circle replication). As a consequence, they can transpose adjacent DNA sequences, mediated by a single copy of the element (96). This is unlike most IS elements, where two copies (one of which must be intact) need to flank the mobilized gene (6), as in the mobilization of the bla_CTX-M genes by ISEcp1 (75, 76). We will argue that CRs are members of an extended family of IS91-like elements that transpose by RC transposition and are responsible for the mobilization of virtually every class of antibiotic resistance genes, including those encoding extended-spectrum ß-lactamases (ESBLs), carbapenemases, and enzymes conferring broad-spectrum aminoglycoside resistance, florfenicol/chloramphenicol resistance, and resistance to trimethoprim and quinolones.

CRs AND THEIR DISCOVERY

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Common regions were first discovered and reported in the early 1990s as a DNA sequence of 2,154 bp that was found in two complex class 1 integrons, In6 and In7 (93). These arrays possess partial duplications of the 3' conserved sequence of the class 1 integrons (Fig. 1), between which are located the CR and antibiotic resistance genes. The sequence was initially described as a common region to distinguish it from the 5' and 3' conserved sequences (CS) of class 1 integrons and was found immediately downstream of a shortened 3' CS (Fig. 1A).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Genetic context of ISCR1 elements found in association with antibiotic resistance genes as part of complex class 1 integrons. ISCRs are represented by yellow boxes. The qac/sul 3' ends of the class 1 complex integrons are denoted by gray and black boxes, respectively. The blue box denoting the int gene (encoding the integrase) represents the 5' end of the class 1 integron. Gene cassettes forming parts of class 1 integrons are represented by white boxes with aqua vertical lines. Genes that are not part of cassettes have been given their own individual color as indicated by the key in Fig. 4. Open reading frames are indicated with open boxes and the direction of their transcription indicated with arrows. See the text for a detailed description of class 1 integrons. (A) Complex class 1 integrons In6 and In7, in which ISCR1 elements were first observed. (B to F) Genetic context of ISCR1 associated with trimethoprim resistance dfr genes, ciprofloxacin resistance qnrA genes, aminoglycoside resistance genes, class C ß-lactamase genes, and class A ß-lactamase genes, respectively. Accession numbers of the nucleotide sequences of the various gene arrays are included on the right.

However, the gene arrays of In6 and In7 are therefore somewhat different from the more common form of class 1 integrons, which contain only one copy of the 3' CS and no CR, and are often called complex class 1 integrons in the literature (9). These common regions were also interesting in that they are intimately associated with different resistance genes carried on each integron. In the case of In6 the CR is found immediately upstream of a chloramphenicol resistance gene (catAII) (Fig. 1A), while the CR in In7 is associated with a novel trimethoprim resistance gene (dfrA10) (Fig. 1A). The particularly striking aspect of these two CR-associated resistance genes is the fact that neither is linked to 59bes, as is typical for the overwhelming majority of resistance genes embedded in class 1 integrons. Integrons generally evolve by integration and excision of a variety of gene cassettes. Accordingly, it was unexpected to find noncassette resistance genes closely associated with integron structures. Searches of available databases in 1993 detected only one other sequence with high identity to the CR, i.e., a 349-bp section of the small nonconjugative plasmid, RSF1010 (93). This plasmid is a broad-host-range (incQ) plasmid that confers resistance to sulfonamide and streptomycin and has been the basis of numerous broad-host-range vectors for recombinant DNA manipulation. This fragment of the CR is the result of truncation at both ends relative to the sequence found in In6 and In7. The sequence is related but not identical to the CR and is now known to be a degenerate copy of a closely related variant of the original CR sequence (now designated CR2 and CR1, respectively). The truncated CR in RSF1010 is also associated with a gene (sul2) encoding resistance to sulfonamide (Fig. 2) (accession no. D37825). Integrons In6 and In7 were discovered on plasmids pSa and pDG0100, respectively, with pDG0100 being a 120-kb incC group plasmid and pSa a 39-kb incW group plasmid (93). Recently, a retrospective study found an integron that is virtually identical to In6 on the incU group plasmid pAR-32, which confers resistance to sulfonamide, chloramphenicol, and streptomycin. This plasmid, which is globally distributed and identical to the incU reference plasmid RA3, was found in a resistant Aeromonas salmonicida isolate that came from Japan in 1970 (92) (Fig. 1A).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Genetic context of ISCR2 elements. Open reading frames are indicated with open boxes and the direction of their transcription indicated with arrows. The ISCR2 elements are colored yellow, and all other genes are colored as per the key in Fig. 4. Accession numbers of the nucleotide sequences of the various gene arrays are included on the right.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Genetic context of ISCR4 to -8 elements. Open reading frames are indicated with open boxes and the direction of their transcription indicated with arrows. The ISCR2 elements are colored yellow, and all other genes are colored as per the key. Accession numbers of the nucleotide sequences of the various gene arrays are included on the right.

CHARACTERIZATION OF CRs

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CRs comprise a set of related sequences. Each CR identified so far accommodates a single open reading frame (ORF). The predicted products of these putative genes, all approximately 500 amino acids long, are closely related to each other, differing by 4 to 88% in amino acid identity (Fig. 5 and 6). However, CR identity extends beyond the ORFs to include nucleotide sequences in both directions. To the left-hand end of the CR ORFs, the sequences show some homology for 240 to 250 bp before ending in a short section of conserved sequence (Fig. 7). This end of a CR sequence will be referred to as the 3' end of the CR because it is 3' to the ORF. The other end, therefore, is the 5' end.

View larger version (25K): [in this window] [in a new window]   FIG. 5. Phylogenetic tree (A) and sequence identity comparisons (B) of all extant ISCR elements, based on a CLUSTAL alignment with the PAM 250 matrix prepared using Lasergene DNAstar software. Alignments were undertaken with partial amino acid sequences (250 amino acids) encompassing a central section of the putative ISCR transposases of ISCR1 to -12 (complete protein sequences are available only for ISCR1 to -5). Comparative identities of ISCR elements are given below the phylogenetic tree, and sequences of individual ISCR elements displaying most and least identity are highlighted.

View larger version (101K): [in this window] [in a new window]   FIG. 6. Alignment of the transposase genes of ISCR1 to -5, based on a CLUSTAL alignment with the PAM 250 matrix prepared using Lasergene DNAstar software. Residues found in the majority of the sequences are highlighted, and the consensus sequence is given at the top of the alignment.

View larger version (44K): [in this window] [in a new window]   FIG. 7. Alignment of the 3'-terminal DNA sequences of the various ISCR elements. The stop codons of the various ISCR elements are indicated with asterisks. Sequences found in the majority of the ISCR elements are highlighted and the consensus sequence given above. The 29-bp sequence found at the 3' ends of the various ISCR elements is underlined.

CRs ARE IS91-LIKE TRANSPOSABLE ELEMENTS

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The CR embedded in the complex class 1 integrons In6 and In7 is 2,154 bp long and accommodates an ORF, Orf513, encoding a putative product of 513 amino acids. This was originally misidentified as an ORF of 341 codons and accordingly designated Orf341. The mistake was corrected when it was discovered (71). When the CR sequence is used to interrogate the nucleotide databases, the only sequences that show significant degrees of homology are CRs. The search provides no convincing clues as to CR identity. This is also the case when the amino acid sequence of Orf513 is used to interrogate the protein databases. No convincing identity is revealed, although Cloeckaert et al. noted that Orf513 displays some identity with the transposition protein of IS801 but that the identity is insufficient to be confident of the match in the absence of other data (17). The putative identity of CRs was also suggested by other studies after the publication of that by Cloeckaert et al. (17, 29, 60, 70, 71). Significant identity is seen only between Orf513 and the putative products of other CR ORFs. Given that CRs are frequently associated with antibiotic resistance genes that clearly are mobile, and given their sizes, it is possible that CRs are atypical transposable elements. However, there is a trio of somewhat unusual IS elements, typified by IS91 and including IS801, that employ transposition proteins and lack the DDE motifs of standard transposases (34). Instead, the activities of these proteins are tyrosine based, and they are related to the Rep proteins of Staphylococcus aureus plasmids and to the replication protein of coliphage X174 (64). When the amino acid sequences of the putative products of the CR ORFs are aligned with those of the transposition proteins of IS91 and its relatives, IS801 and IS1294, although overall amino acid identity between the CR-encoded proteins and the transposition proteins of IS91, IS801, and IS1294 is very low, key amino acid motifs of the transposition proteins can be seen to be present in the CR gene products (Fig. 8), indicating they also may indeed be transposition proteins, consistent with the idea that CRs are transposable elements (34, 96).

View larger version (28K): [in this window] [in a new window]   FIG. 8. Alignment of motif regions of IS91 group protein sequences with the transposase sequences of ISCR1 to -4 elements. All ISCR elements have the five motifs found within IS91 group elements, and all residues conserved throughout the various IS91 elements are found within ISCR1 to -4 elements. Residues conserved in all IS91 group elements are highlighted.

With these elements, transposition is a processive system in which one end of the element, oriIS, serves as an origin of replication while the other, terIS, is a replication terminator (34, 96). The oriIS sequence is 5'GxTTTTxAAATTCCTATxCAT3' and is located approximately 300 bp downstream from the transposase gene in IS91 and IS1294 but only 179 bp downstream from the transposase gene in IS801. Sequences in a similar position relative to CR ORFs, i.e., 240 to 250 bp downstream and at the 3' end of the CR sequence, have the consensus sequence 5'GCGTTTGAACTTCCTATACxx3' (Fig. 7). This sequence is strikingly similar to the oriIS consensus sequence (identity between the nucleotide sequence of the consensus of the 3' end of CRs and that of the oriIS of IS91-like elements is indicated in boldface), to the extent that, given the likely identity of CR-encoded proteins, it is not unreasonable to conclude that this end of a CR sequence is also an origin for RC replication (34, 65, 96). Hence, CR sequences have two key features of IS91-like elements that are similarly located. We conclude, therefore, that these sequences are IS91-like transposable elements. Accordingly, we propose that they be designated ISCR elements, and will so refer to them hereafter, so as to highlight both their identities as insertion sequences and the history of their discovery. Further, the numbers presently used to distinguish CRs are retained to distinguish different ISCRs. Therefore, CR1 becomes ISCR1, CR2 becomes ISCR2, etc.

The third feature of IS91 that is strongly conserved both in IS801 and in IS1294 is terIS (96), located approximately 300 bp upstream from the start of the transposase gene in IS91 and IS1294 but only 101 bp from the start of the transposase gene in IS801 (33, 34, 65, 96). The replication terminator sequence, terIS, is a 25- or 26-bp sequence that contains a perfect 6-nucleotide punctuated inverted repeat and a final terminating tetranucleotide, 5'GTTC3' (96). Unfortunately, in the cases of ISCR elements there is, in general, too little information to determine with confidence the 5' ends of the elements. In the case of ISCR1, although there are many examples of this element (Fig. 1), the sequence at the 5' end is identical in virtually every case. Therefore, it is not possible to determine precisely where the junction between ISCR1 and the carrier sequence is. However, there are four instances where ISCR2 has different sequences on the 5' side of its ORF (Fig. 9). As with the 3' end, the marked differences between these sequences can be used to determine the likely 5' end of ISCR2, which is approximately 120 bp upstream from the start of the transposase gene (Fig. 9). A comparison between this end of ISCR2 and the terIS sequence consensus for IS91, IS801, and IS1294 shows differences but also similarities (34, 96). The terIS of ISCR2, like the terIS of IS91 group elements, has a short punctuated inverted repeat, but it is only 4 bp, in comparison with 6 bp for IS91 (Fig. 10). The overall sequence identity is about 50% and is accompanied by the nucleotide pair GA instead of the tetranucleotide GAAC for IS91 and IS1294 (Fig. 9 and 10). Whether the difference represents sequence drift or replacement with a sequence similar to terIS that can perform the same function is difficult to judge, given the paucity of relevant data.

View larger version (27K): [in this window] [in a new window]   FIG. 9. Alignment of DNA sequences found upstream of ISCR2 elements, depicting the 5' end of the ISCR2 element. Nucleotide bases found in the majority of sequences are highlighted and consensus sequence given above the alignment. The start codon of the ISCR2 transposase gene is indicated with asterisks, and the palindromic terIS sequence is underlined. Accession numbers are given at the ends of the sequences.

View larger version (35K): [in this window] [in a new window]   FIG. 10. Alignment of the oriIS and terIS of IS91 family elements and ISCR elements. (Top panel) Alignment of the complement of the last 21 bp of the various ISCR elements with the first 21 bp of IS91, IS1294, and IS801, showing oriIS. Nucleic acids found in the majority of sequences are highlighted. (Bottom panel) Alignment of terIS of IS91 family elements with the sequences found at the equivalent termini of ISCR2 and ISCR4. Inverted repeats are underlined. The direction of rolling-circle replication is indicated with arrows. Accession numbers are given on the right.

ISCR ELEMENTS AND ASSOCIATED GENES

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ISCR elements are notable for their close association with a wide variety of antibiotic resistance genes.

ISCR1 association with plasmid-mediated quinolone resistance. Plasmid-mediated decreased susceptibility to quinolones, which was subsequently associated with the gene qnrA, was first detected in 1994 in a K. pneumoniae isolate collected in Birmingham, Alabama (43, 61). However, it has recently emerged in China, Korea, and Europe, suggesting that it is likely to become much more common in the future (47, 50, 60, 68, 107). In all cases tested, the qnrA gene was found immediately downstream of ISCR1 as part of a complex class 1 integron that included the 3' CS duplication (Fig. 1C). The qnrA genes isolated in the different geographical locations are virtually identical: i.e., the qnrA gene isolated in Europe is the same as the one identified in the United States, and these have only one silent nucleotide difference from those of the Chinese isolates (60). This suggests a common source for this gene irrespective of where it is isolated, which has recently been identified as Shewanella algae (70). The genetic contexts of the qnrA gene in several different isolates suggest that it has been mobilized on more than one occasion. The association of ISCR1 with the qnrA gene in unrelated isolates is also striking. The gene assemblies are found on plasmids of different sizes and have been seen to move from one plasmid to another during conjugation experiments (107). Several ß-lactamase genes have also been found on qnrA-carrying plasmids, such as bla_VEB-1, bla_FOX-5, bla_SHV-7, bla_CTX-M9, and bla_PSE-1 (107). It is interesting to note that some of these genes also have been shown to be associated with ISCR1 (Fig. 1E and F).

ISCR1 association with aminoglycoside resistance genes. The screen of the EMBL prokaryote databases with the ISCR1 sequence identified ISCR1 in close association with the 16S rRNA methylase gene armA in four separate sequences. This important aminoglycoside resistance gene was first identified in a Pseudomonas aeruginosa isolate from Japan (112). Aminoglycosides are among the most commonly used broad-spectrum antibiotics for the treatment of infectious diseases caused by gram-negative bacteria. They cause bacterial cell death by binding irreversibly to the 30S ribosomal subunit (54). The 16S rRNA methylases are particularly powerful resistance mechanisms, because by altering the ribosome they can confer high-level resistance to almost all clinically important aminoglycosides (31). This is in contrast to the more commonly found aminoglycoside resistance genes that encode aminoglycoside-modifying enzymes that individually confer resistance to a much more limited range of drugs (78).

The ISCR1 element was found upstream of armA in isolates of C. freundii from Poland, Escherichia coli and Serratia marcescens from Japan, and K. pneumoniae from France. The isolates from France (31) and Poland (accession no. AF550415) were shown to harbor the gene array on large plasmids. The C. freundii plasmid was sequenced in its entirety, and this revealed the ISCR1 element as part of a complex class 1 integron but without the second copy of the 3' CS (accession no. AF550415) (35). The armA gene was the second gene beyond ISCR1 and distal to the integron and was separated from ISCR1 by a gene with high identity (76%) to a transposase gene from Providencia rettgeri (Fig. 1D). Recently, a number of studies have found the armA resistance determinant on plasmids in several clinical isolates, including isolates of C. freundii, E. coli, K. pneumoniae, Enterobacter cloacae, S. enterica, Shigella flexneri, and S. marcescens collected in France, Bulgaria, and Poland (32) and isolates of E. coli and K. pneumoniae collected in Taiwan (111), suggesting mobility. It has also been found on an incN R-46-like plasmid in E. coli isolated from pigs in Spain (35, 36).

ISCR1 association with class A ß-lactamases. ISCR1 has also been found to be associated with several class A ß-lactamase genes, including those belonging to two of the four groups of extended-spectrum bla_CTX-M genes (2, 87), the extended-spectrum class A ß-lactamase gene bla_VEB-3 from China (48), and the extended-spectrum bla_PER-3 gene isolated from Aeromonas punctata in France (accession no. AY740681) (Fig. 1E).

ISCR1 association with bla_CTX-M genes. CTX-M ß-lactamases initially emerged as resistance determinants in Europe and South America at the end of the 1980s and have become the dominant ESBLs (39, 73, 97). For example, in Argentina, the bla_CTX-M-2 enzyme alone now accounts for almost 69% of all ESBLs in Enterobacteriaceae that are analyzed in Buenos Aires (30). In both instances, the resistance genes are closely associated with ISCR1 (13, 89).

Genetic analysis of the bla_CTX-M genes in pathogens in Argentina began with the bla_CTX-M-2 gene in a Proteus mirabilis strain isolated in 1993 (1). The bla_CTX-M-2 gene was found on a plasmid (pMAR-12) in an integron, In35, containing the 3' CS duplication typical of complex class 1 integrons but not as part of a gene cassette. In this array the bla_CTX-M-2 gene is immediately downstream of a copy of ISCR1 (Fig. 1E). Interestingly, this copy of bla_CTX-M-2 is preceded by 266 bp of DNA displaying 96% identity to bla_KLUA-1, encoding the class A ß-lactamase of Kluyvera ascorbata. In addition, 1,043 bp of sequence downstream of bla_CTX-M-2 also displays high identity to Kluyvera DNA, a finding that provides evidence that the bla_CTX-M-2 gene almost certainly originates from K. ascorbata (42). It therefore seems likely that the associated ISCR1 element was involved in sequestering this section of DNA from the chromosome of K. ascorbata or a near relative into a plasmid carried by the host cell (42). A similar situation was found with an isolate of S. enterica serovar Infantis that was collected in 1997. Here the bla_CTX-M-2 gene was again found on a plasmid (73 kb) together with 266 bp of Kluyvera DNA upstream of the ß-lactamase gene and adjacent to a copy of ISCR1 in a complex class 1 integron, InS21, but associated with gene cassettes different from those in In35 (22). In addition, a screen of 130 clinical isolates, which included gram-positive and gram-negative isolates collected between 1993 and 2000 from various hospitals in Buenos Aires, identified ISCR1 next to bla_CTX-M-2 in all bla_CTX-M-2-containing isolates, strongly implicating ISCR1 in the emergence and dissemination of this particular resistance gene in South America (2). The bla_CTX-M-2 gene has been found on plasmids of different sizes and associated with different class 1 integrons. The association of bla_CTX-M-2 and ISCR1 has been identified in 10 different gram-negative pathogens (including Acinetobacter spp., Enterobacter cloacae, E. coli, P. mirabilis, Pseudomonas aeruginosa, Salmonella spp., and Serratia marcescens) (2), in K. pneumoniae (62), and in Morganella morganii (78). In addition, bla_CTX-M2 has also been found in isolates of the gram-positive species Enterococcus faecium and serogroup G Streptococcus agalactiae (2), again in close association with ISCR1.

In bacteria isolated in Europe, the bla_CTX-M variant most commonly associated with ISCR1 elements is bla_CTX-M-9. The different structural subgroups probably reflect the different origin of each subgroup from source organisms of different Kluyvera spp. Thus, ISCR1 would appear to have been involved in sequestering and mobilizing ß-lactamase genes intrinsic to bacterial chromosomes from different species of Kluyvera.

The only incidence of bla_CTX-M-9 that has been fully analyzed with respect to its genetic context is from an E. coli isolate collected in 1996 in Spain (85). In this instance, the bla_CTX-M allele is linked to sequences displaying significant identity to Kluyvera spp. The bla_CTX-M-9 gene and its downstream sequence share 81% and 78% identity, respectively, with the bla_KLUA-1 ß-lactamase gene and an adjacent sequence, orf3, from the chromosome of K. ascorbata (86). Further, the bla_CTX-M-9 allele is next to a copy of ISCR1, which together comprise part of a complex class 1 integron, In60 (Fig. 1E). This same arrangement has been found in 30 other Spanish E. coli isolates and in 4 S. enterica isolates collected between 1996 and 1999. The bla_CTX-M-9 allele has also been found associated with ISCR1 in bacterial isolates from hospitals in Paris and southwestern France. However, in the former location bla_CTX-M-9 was found to be mostly associated with the insertion element ISEcp1 rather than ISCR1, although evidence of ISCR1 activity in the form of short ISCR1 terminal sequences was identified upstream of bla_CTX-M-9 in an E. coli isolate, and similar sequences were found in two P. mirabilis isolates harboring either bla_CTX-M-2 or bla_CTX-M-20 (88). Further analysis should elucidate whether these short sequences reflect intact copies of ISCR1 or simply fragments. The bla_CTX-M-9-harboring isolates from southwestern France are of particular interest because they were isolated from both human and poultry sources, suggesting an animal reservoir of ISCR1 and bla_CTX-M alleles. The bla_CTX-M-9 alleles were again found adjacent to copies of ISCR1 in nine isolates of S. enterica originating from a single hatchery supplying chicks to six farms (108). The integron structure in these isolates was 99% identical to that of In60, found in Spain, and in all isolates was on a large plasmid (>126 kb). Since this integron was first reported in Spain, it has also been found in France, Turkey, Brazil, and China (108).

ISCR1 association with other class A ß-lactamase genes. Other class A ß-lactamase genes that are associated with a copy of ISCR1 are bla_VEB-3 (48) and bla_PER-3 (accession no. AY740681). The bla_VEB-3 gene has been found in numerous E. cloacae isolates in Shanghai, China, and has features that suggest that it is part of a gene cassette. However, it could not be linked to the class 1 integrons that were found in these strains; rather, it was shown to be linked to the 3' end of a fragment of ISCR1 found upstream of the bla_VEB-3 gene. The sequence following this ISCR1 fragment displays high identity to IS6100, and therefore it seems likely that bla_VEB-3 was initially mobilized by ISCR1, which was then fragmented by an IS6100 insertion. The bla_PER-3 gene was identified in an isolate of Aeromonas punctata in France adjacent to a copy of ISCR1, as part of a complex class 1 integron. Again, the gene bla_PER-3 does not appear to have the associated 59-base element that is characteristic of a gene cassette.

ISCR1 association with class C ß-lactamase genes. Class C ß-lactamases, unlike the extended-spectrum class A ß-lactamases, mediate resistance to cephamycins as well as broad-spectrum cephalosporins (39, 97). However, unlike those of class A ESBLs, the activities of class C ß-lactamases are not generally inhibited by clavulanic acid or tazobactam. Genes encoding class C ß-lactamases are found on the chromosomes of many enteric bacteria and also are intrinsic to some nonfermenting gram-negative bacteria such as P. aeruginosa and Acinetobacter spp., where they may be silent and under tight transcriptional control (58, 91). However, recently resistance to cephamycins and cephalosporins has been acquired by Klebsiella spp. and E. coli, mediated by plasmids carrying genes encoding enzymes closely related to chromosomal AmpC ß-lactamases. It appears likely that ISCR1 has also played a major role in the mobilization of these genes into human bacterial pathogens via plasmids and other mobile genetic elements (T. R. Walsh, unpublished data).

ISCR1 elements have been found adjacent to a number of cephalosporinase genes, including bla_DHA-1 (originating from the chromosome of M. morganii) (103), bla_CMY-1 and bla_CMY-8 to bla_CMY-11 (3, 23, 55, 110) (thought to originate from an Aeromonas sp.), and a variant of bla_CMY-type genes, bla_MOX-1 (41) (Fig. 1F). Of these class C ß-lactamase genes, the genetic loci of only the bla_CMY-9 and bla_DHA-1 genes have been sequenced and analyzed in any detail. In the case investigated, bla_CMY-9 was plasmid encoded in an E. coli isolate, HKYM68, collected in Japan in 1995 and displaying high-level resistance to ceftazidime, cefpirome, and moxalactam (23). Cloning and sequencing of 8 kb around the bla_CMY-9 gene (Fig. 1F) revealed that the gene is adjacent to a copy of ISCR1 and downstream of a class 1 integron but that there is no second copy of the 3' CS, as is usual when ISCR1 is linked to a class 1 integron. The bla_CMY-9 allele shows 78% identity to cepH of Aeromonas hydrophila. Comparison of the nucleotide sequence upstream of bla_CMY-9 and those of the other related cephalosporinase genes bla_CMY-10 and bla_CMY-11 with the corresponding region of the A. hydrophila cepH locus reveals 64% identity, again suggesting that the origins of these particular ß-lactamase genes are probably A. hydrophila or closely related strains (23).

It is interesting that all ISCR1-associated bla_CMY genes identified belong to the CMY-1 subgroup and that no ISCR CMY-2 subgroup genes have been found. These genes are, instead, invariably associated with the insertion element ISEcp1. This may reflect the environmental niche of ISCR1 elements or may simply indicate that there are insufficient data concerning the genetic environments of mobile bla_CMY-2 genes.

Involvement of ISCR2 with SXT. Before 1992 the major cause of cholera on the Indian subcontinent was Vibrio cholerae El Tor. However, in 1992, V. cholerae El Tor was replaced by V. cholerae serogroup 0139 as the predominant cause of cholera (40). This strain was also different from V. cholerae El Tor in being resistant to several antibiotics, i.e., sulfamethoxazole, trimethoprim, chloramphenicol, florfenicol, and streptomycin. The resistance genes were located on a novel genetic element of the integrative conjugative element group named SXT. This element is now present in virtually all clinical V. cholerae serovars (40). SXT is a conjugative, self-transmissible element that integrates into the prfC gene on the chromosome of V. cholerae and has also been found on the chromosome of Providencia alcalifaciens (4). Integration is necessary for replication of the element, and SXT can be transferred into several other gram-negative organisms, including E. coli, by conjugation. Of interest and concern is the fact that challenge with antibiotics actually promotes mobilization (5). The resistance genes harbored by SXT are embedded in a composite transposon-like structure and were probably acquired recently (4). Within this antibiotic resistance region, a copy of ISCR2 is found beside the trimethoprim resistance gene dhfR18. In addition to this intact copy of the element, there are two truncated copies located close to the florfenicol resistance gene, floR, and genes encoding streptomycin and sulfonamide resistance (4) (Fig. 2). It seems likely, therefore, that ISCR2 has been involved at some time in the past in the construction of the antibiotic resistance region of SXT. Furthermore, it has been shown that homologous recombination events between the various copies of the ISCR2 element, leading to deletions, have given rise to SXT variants that differ with respect to their resistance gene complements (4; compare Fig. 2, accession no. AY055428 and AB114188).

ISCR2 association with other resistance mechanisms. The ISCR2 element has also been found next to resistance mechanisms in other gram-negative organisms. ISCR2 is present on several plasmids, often in truncated form, including plasmids which have been studied for several decades. As previously stated, it is closely associated with the sulfonamide resistance gene sul2 in the conjugative plasmid RSF1010 (81). ISCR2 has also been found associated with a florfenicol resistance gene found on plasmids in E. coli isolated from cattle, poultry, and pigs (12). This has spread to the major target bacterium for cattle, Pasteurella multocida (52). Florfenicol was licensed in Europe for the control of bacterial respiratory tract infections in cattle and pigs in 1995 and 2000, respectively, and is active against chloramphenicol-resistant isolates with chloramphenicol acetyltransferases and efflux systems (12). The first florfenicol resistance gene was detected in 1996 on a plasmid in the fish pathogen Pasteurella damsalae subsp. piscida (formerly known as Pasteurella piscida) (46) and has recently been found in the Salmonella genomic island SGI1 in S. enterica serovar Typhimurium DT104 (14) and in the SXT element of V. cholerae (18) (Fig. 2). The close association between ISCR2 and sul2 prompted us to study Stenotrophomonas maltophilia isolates that were resistant to trimethoprim-sulfamethoxazole (TMP-SMX). Out of 25 TMP-SMX-resistant isolates collected from various different geographic regions, the ISCR2 element was found in 6 isolates (M. A. Toleman, unpublished data). In each case, the ISCR2 element was found adjacent to a truncated phosphoglucosamine mutase gene, glmM, and the sul2 gene. This arrangement is identical to that found in the previously mentioned E. coli strains isolated from cattle in France and Germany, together with on plasmid pRVS1 isolated from V. salmonicida in Norway, on a plasmid from an S. enterica strain isolated in Japan, and on the chromosome of an S. flexneri strain isolated in the United States. The arrangement of glmM and sul2 has also been found in plasmids isolated from marine psychrotrophic bacteria from Norway (accession numbers AJ306553 and AJ306554), but in these cases the ISCR2 element was absent. Two of the six S. maltophilia isolates also carried a copy of a floR gene immediately upstream of ISCR2 and showed the arrangement incorporating ISCR2 seen in the E. coli ISCR2-carrying strains from cattle in Germany and France. The two S. maltophilia isolates came from Turkey and the United States, indicating widespread dissemination of the floR resistance gene on plasmids in association with ISCR2 (Toleman, unpublished data).

It has been suggested recently that a section of DNA encompassing the floR gene and an intact copy of ISCR2 is part of a novel transposon, TnfloR (28), that transposes via a circular intermediate by a mechanism similar to that proposed for the staphylococcal transposon Tn554 (28). The authors of that paper correctly recognize that the presence of the floR gene on the chromosome or on different plasmids in a number of unrelated Salmonella enterica and E. coli isolates is characteristic of movement by transposition. The evidence provided to substantiate the claim that floR is part of a transposon is production of a PCR product that contains both ends of the proposed element, using primers that direct replication out of the linear form of the sequence, indicating a circular form of the sequence in the cell. This is interpreted to indicate formation of a circular intermediate in the transposition process. However, no account has been taken of the fact that the floR gene in this particular strain is flanked by a complete copy of ISCR2 on one side and a truncated version on the other. Accordingly, homologous recombination between the repeated sequence would generate a circular structure of the form reported. Given that the strain used in the experiment was a wild-type E. coli strain and not a recA mutant deficient for homologous recombination, generation of free "ISCR2-floR circles" is to be expected. Strikingly, the circular "intermediate" form of the proposed transposon was detected in only one of six florfenicol-resistant E. coli strains and, specifically, in the only one in which the floR gene is flanked by a sequence duplication. Furthermore, transposition of the novel "transposon" was not actually demonstrated. Accordingly, definitive proof of the existence of TnfloR was not presented. We would argue that the floR gene has indeed been transposed on several occasions, mediated by ISCR2 via RC transposition, but that the combination ISCR2-floR does not constitute a transposable element.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Genetic context of ISCR3 elements. Open reading frames are indicated with open boxes and the direction of their transcription indicated with arrows. The ISCR2 elements are colored yellow, and all other genes are colored as per the key in Fig. 4. Accession numbers of the nucleotide sequences of the various gene arrays are included on the right.

Since its initial discovery in S. enterica serovar Typhimurium DT104, SGI1 has also been found in other S. enterica serovar Typhimurium phage types, i.e., DT120, DT12, DT1, and U302, and in other serovars such as Agona, Paratyphi B, Albany, Meleagridis, Newport, Emek, Cerro, Derby, Dusseldorf, Infantis, and Kiambo (15, 26, 56). These observations are indicative of SGI1 transfer, and transduction of SGI1 between S. enterica serovar Typhimurium DT104 isolates has been demonstrated (11). However, attempted transfer to other S. enterica serovars in vitro was not successful, and SGI1 excision has not been detected experimentally (14). It has been suggested, therefore, that the presence of this element in different serovars of S. enterica and in different phage types of S. enterica serovar Typhimurium is due to separate emergence events rather than horizontal movement between S. enterica strains, but the source of the genomic island is as yet unknown. SGI1 is found at the same place on the chromosomes of all of these organisms, inserted between thdF and yidY (26). DT104 isolates also have the addition of another element, described as a cryptic retronphage inserted between thdF and the left end of SGI1 (14). Whether this element is involved in SGI1 movement is not known.

SGI1 contains a resistance module of approximately 14 kb at one end. This consists of floR and tetR bracketed by two class 1 integrons, one carrying the gene cassette aadA2 and the other carrying the bla_PSE-1 cassette (Fig. 3). Recently, seven variants of SGI1 in isolates originating from Belgium and Scotland (SGI1A to -G) (15) to Australia (SGIA I and J) (56) have been described. These variants differ mostly in the resistance genes that they carry and in the presence or absence of CR elements (15, 56).

ISCR3 is the ISCR variant most often found in SGI1. It is present in SGI1 variants SGI1A, SGI1E, SGI1-I, and SGI1-J, as well as in SGI1 (Fig. 3). However, recently ISCR1 has also been detected in SGI1 variants in isolates originating from poultry in Belgium (27), specifically, in variants SGI1A, SGI1D, and SGI1G. Hence, SGI1A harbors both ISCR1 and ISCR3, whereas variants SGI1B and SGI1C lack an ISCR element (Fig. 3). In all variants containing ISCR1, the element is found immediately upstream of the trimethoprim resistance gene dfrA10, in a complex class 1 integron and downstream of a class 1 integrase/groEL gene fusion (Fig. 3); i.e., it is found with dfrA10 between repeat copies of the 3' CS of class 1 integrons. The ISCR1 element has also been found in deletion versions of SGI1 where it is not associated with any antibiotic resistance gene (27). In these cases, the entire SGI1 sequence normally found downstream of ISCR1 has been deleted, bringing the chromosomal gene yieE or yieF into juxtaposition with ISCR1 (Fig. 3, accession numbers AY434091 to -3). Such a deletion has occurred on at least three occasions, involving deletion of sequences of 7.4 kb, 7.7 kb, and 8.5 kb. Such deletions are characteristic of recombination events between direct copies of insertion elements and are likely to have been ISCR1 mediated (27).

In SGI1 ISCR3 is located between the two class 1 integrons found on the genomic island in association with floR and tetA/R (Fig. 3). An exception to this is found in variant SGI1E, which has a copy of ISCR3 located at one end of the genomic island, downstream from the second class 1 integron (Fig. 3). This may reflect an inversion event involving the insertion element IS6100, copies of which are found at the ends of the inverted section of DNA. It is therefore very likely that both ISCR1 and ISCR3 elements have been involved individually and/or corporately in the construction of SGI1 and its variant types, as well as being instrumental in incorporating further resistance genes.

ISCR3 has also been found adjacent to the 16S rRNA methylase gene rmtB in a clinical strain of S. marcescens (23) (Fig. 3). This methylase gene confers high-level resistance to various aminoglycosides, including kanamycin, tobramycin, amikacin, arbekacin, gentamicin, sisomicin, and isepamicin, but not neomycin, streptomycin, and hygromycin B. The rmtB gene is likely to have been mobilized to S. marcescens from an environmental microorganism by ISCR3. The deduced amino acid sequence of RmtB is 29% identical to that of the ArmA 16S rRNA methylase found associated with ISCR1 (Fig. 1D). Therefore, similar broad-spectrum aminoglycoside genes have been found adjacent to ISCR1 and ISCR3.

In all cases investigated so far, the left end of the ISCR3 element is flanked by the groEL gene, displaying 78% identity with Xanthomonas campestris. This same association is seen in both the SGI1 of S. enterica and the rmtB locus of S. marcescens. Interestingly, the same groEL gene is found upstream of the bla_SPM-1 gene found in close association with ISCR4.

Using the same generic method, we have also detected ISCR elements in several strains of P. aeruginosa and Acinetobacter baumannii that harbor MBL genes. ISCR2 was discovered in a P. aeruginosa isolate isolated in Brazil that also harbored the MBL gene bla_IMP-1, while ISCR3 was discovered in two strains of P. aeruginosa isolated in Italy that harbored the MBL bla_VIM-1. ISCR11 was discovered in two A. baumannii isolates from Germany that have the MBL bla_VIM-2 and in a P. aeruginosa isolate from Greece that produces the MBL VIM-1. This new ISCR family member encodes a putative transposase that shows highest identity (79%) to the ISCR3 transposase. Finally, a strain of P. aeruginosa that produces the MBL SPM-1 carries two ISCR elements, ISCR4 and a new element, ISCR12, that encodes a putative transposase with 89% identity to the ISCR3 transposase and 75% identity to the ISCR4 transposase (100). We have also recently detected >50 ISCR5-like copies in the A. hydrophila genome (TIGR). The sequences in the A. hydrophila genome display approximately 54% nucleotide identity to ISCR5 and probably represent at least one further family member. ISCR13, an ISCR-like sequence in this array, is found adjacent to a gene with high identity to an aminoglycoside resistance gene (unpublished results).

ISCRs MOBILIZE AND CONSTRUCT COMPLEX CLASS 1 INTEGRONS

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ISCR1 appears to be unusual in the sense that it is almost always found in a complex class 1 integron structure. The sequence upstream of ISCR1 is always the same: i.e., the junction between ISCR1 and the sequence adjacent to the 3' CS of the class 1 integrons is invariant. This suggests that a single event has been responsible for this feature of all complex class 1 integrons. Given that ISCR1 is significantly shorter than other ISCR and IS91-like elements, it is also likely that the terIS end of the element has been deleted following transposition (Fig. 11). This would fuse the remainder of the ISCR1 element (the transposase gene, orf513, and oriIS) to the 3' CS end of the class 1 integron and provide the integron with the basis for genetic mobilization, since the oriIS and transposase gene remain intact. This ISCR1-class 1 integron fusion would also require that in the event of transposition being initiated, an alternative termination site would have to be utilized due to the fact that the original termination site, terIS-1, has now been deleted (Fig. 11). One consequence of the generation of ISCR1 by deletion of its terIS is that it would be expected to cotranspose the adjacent sequence, i.e., class 1 integron sequences, more often. This, then, allows the construction of a model in which ISCR1 has mediated the assembly of a variety of complex class 1 integrons (Fig. 12).

View larger version (25K): [in this window] [in a new window]   FIG. 11. Mobilization of class 1 complex integrons by ISCR1. (A) oriIS and terIS-1 are the initiation and termination sites of ISCR1 transposition, respectively. Underlined sequences read 5' to 3' for each site, and the nucleotides in boldface match those of the consensus sequence of IS91, IS1294, and IS801 as reported by Tavakoli et al. (96). The solid horizontal arrow indicates the direction of transcription. The broken line denotes the direction and origin of replication. The 3' end of the class 1 integron is represented as a crosshatched box. (B) The first event involves the transposition of ISCR1 next to or near the 3' end of a class 1 integron. The black ball prior to the 3' end of the integron denotes the 59be of the gene cassette represented by "abxr." Normally the transposase would recognize the putative termination sequence terIS-1, but it 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 gray 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 and any antibiotic resistance gene cassettes therein by an IS91-like RC mechanism possibly recognizing the putative termination sequence terIS-2 or terIS-3, which may or may not be similar to terIS-1. The possibility also exists that ISCR1 does not posses an intrinsic termination site and accordingly terminates transposition randomly, thereby mobilizing varied lengths of 5' (upstream) DNA.

View larger version (27K): [in this window] [in a new window]   FIG. 12. 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 a 3' CS) generates transposition intermediates of different lengths. These intermediates then transpose adjacent to an antibiotic resistance gene (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 a 3' CS on another "normal" class 1 integron (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 frames of the ISCR1 elements are shaded in gray, and the resistance genes that lack a 59-base element are patterned.