Genetic characterization of a novel metallo-ß-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme

Mark A. Toleman1,*, Doug Biedenbach2, David Bennett1, Ronald N. Jones2,3 and Timothy R. Walsh1

1 Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, UK; 2 The JONES Group/JMI Laboratories, North Liberty, Iowa; 3 Tufts University School of Medicine, Boston, Massachusetts, USA

Received 23 May 2003, returned 13 June 2003, revised 8 July 2003; accepted 9 July 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86–14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection. The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA. The resistance determinant was investigated.

Methods: The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach.

Results: Sequence analysis revealed the presence of a novel IMP-type metallo-ß-lactamase (MBL) gene, blaIMP-13. This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy). The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8. The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron. A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative. This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York. Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland.

Conclusions: The resistance determinant of P. aeruginosa 86–14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.

Keywords: metallo-ß-lactamases, Pseudomonas aeruginosa, integron Tn21, Tn402


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carbapenems are the most potent antimicrobial agents in the treatment of Gram-negative (GN) infections due to their broad-spectrum activity and stability to hydrolysis by class A, C and D ß-lactamases, including those that are extended-spectrum.1,2 The advent of acquired metallo-ß-lactamases (MBLs), singularly found in GN bacilli, further challenges already compromised therapies because of their broad-spectrum hydrolytic profile, including carbapenems, and the fact that there are no clinical inhibitors in existence, nor are there likely to be for another 3–4 years. Since the report of IMP-1 in Japan in the early 1990s3,4, a number of MBLs have been identified in many countries and in several different nosocomial GN pathogens.5 These can be divided broadly into three types: IMP,4 VIM6 and SPM,7 based on sequence differences. The genes encoding MBLs can be plasmid or chromosomally mediated and most are associated with integrons; usually these are class 1 integrons, although IMP MBLs are also found on class 3 integrons.8,9 Integrons are able to capture genes that are part of gene cassettes via a site-specific recombination event between two sites, one in the integron and one in the gene cassette.10 Integrons consist of three regions: the 5' conserved region (5'CS); the 3' conserved region (3'CS) and a variable region. The 5'CS consists of an integrase gene (intI), its adjacent recombination site (attI) and a promoter, which drives the expression of captured gene cassettes in the variable region. The 3'CS usually consists of a partially deleted qac gene (qacE{Delta}1) fused to a sul1 gene. These confer antiseptic and sulphonamide resistance, respectively. MBL genes are often associated with multiple antibiotic resistance gene cassettes, particularly those conferring resistance to aminoglycosides.

Horizontal and vertical transfer of antibiotic resistance genes in members of the Enterobacteriaceae and Pseudomonas families are mostly due to large broad-host-range plasmids and to the transposons they carry and share with other replicons.11 Many transposons encoding multiple antibiotic resistances in GN pathogens belong to the Tn21 subgroup of the Tn3 family of transposable elements.12 Here we describe a novel blaIMP variant, blaIMP-13, found on the chromosome of Pseudomonas aeruginosa strain 86–14571A. We also describe its genetic context and its association with a novel Tn21-like composite transposon.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical isolates, bacterial strains and plasmids

In 2001, P. aeruginosa strain 86–14571A was isolated at a hospital in Rome, Italy, from the blood of a 45-year-old female patient suffering from cancer. Escherichia coli strain DH5{alpha} [endA1 gyrA96 hsdR17 {Delta}lacU169 ({phi}80lacZ{Delta}M15) recA1 relA1 supE44 thi-1] was used as the host strain for transformation experiments. The genomic library was generated in the cloning vector pK18, as described previously.7 P. aeruginosa strains 48–501 and 75–3677 (clinical isolates from Sao Paulo and Genoa and containing blaIMP-1 and blaVIM-1, respectively) were used as positive controls for blaIMP and blaVIM in PCR reactions.

Antibiotics and reagents

ß-Lactam antibiotics used in this study were: ceftazidime, clavulanic acid (GlaxoSmithKline, Worthing, UK); benzyl penicillin, ampicillin, oxacillin, cefotaxime, aztreonam (Sigma Chemicals Co., St. Louis, MO, USA); piperacillin (Lederle, Carolinas, Puerto Rico); cefoxitin, imipenem (Merck Sharp & Dohme, West Point, PA, USA); meropenem (Zeneca Pharmaceuticals, Macclesfield, UK); nitrocefin (Becton Dickinson, Cockeysville, MD, USA). Reagents used for DNA manipulation were obtained from Gibco BRL (Life Technology Ltd, Paisley, UK). Other general reagents were from Sigma Chemical Co. or BDH (both Poole, UK).

MIC determination

MICs were determined according to NCCLS guidelines by agar dilution using Mueller–Hinton agar.13

Detection of MBLs

Strains suspected of possessing MBLs were plated onto nutrient agar containing imipenem 10 mg/L and incubated overnight. The following day, several colonies were suspended into sterile deionized water and plated onto Mueller–Hinton agar (Becton Dickinson), such that the final inoculum was equivalent to a density of a 1 McFarland Standard. An Etest strip (AB Biodisk, Solna, Sweden) was applied that incorporates imipenem with and without EDTA (an inhibitor of MBLs), formulated for the specific detection of MBLs.14 If the presence of EDTA reduced the imipenem MIC by >=8-fold then the organism was categorized as MBL-positive.

Confirmatory test for the presence of MBLs

Bacterial colonies from a nutrient agar plate (containing imipenem 10 mg/L) were used to inoculate 10 mL of nutrient broth, and grown overnight at 37°C. The following day the cells were harvested at 13 000g by centrifugation, resuspended in 1.5 mL of assay buffer (30 mM cacodylate buffer, pH 7.0, 100 mg/L ZnCl2) and sonicated (Sonics Vibra Cell, Basingstoke, UK). The sonicated cells were centrifuged at 13 000g for 30 min at 4°C. The crude cell extract was examined by spectroscopy at 298 nm for its ability to hydrolyse imipenem. The enzyme activity was expressed as nmol substrate hydrolysed/min/mg protein. The protein concentration of each bacterial extract was determined using the Bio- Rad protein assay reagent (Bio-Rad, Munich, Germany), following the method described by the manufacturers.

Recombinant DNA methodology

DNA procedures were performed as described previously.15 Genomic DNA was isolated from P. aeruginosa strain 86–14571A by the cetryl-tri-ammonium bromide method. Plasmids were purified by alkaline lysis using a Qiagen miniprep kit. To construct the genomic library, Sau3A1 genomic fragments were purified after gel electrophoresis using a Qiagen gel purification kit (Qiagen). Five micrograms of purified size-fractionated genomic fragments (>1 kb) were ligated to 1 µg of pK18 that had been linearized and dephosphorylated previously using BamHI and calf intestinal alkaline phosphatase, respectively. The ligation mixture was subsequently dialysed and used to transform E. coli DH5{alpha} by electroporation using a Bio-Rad Gene Pulser. Plating of the library on to X-gal (30 mg/L) and kanamycin (25 mg/L) plates yielded several hundred recombinants. The library was amplified by purifying the plasmids from the recombinants. This amplified library was used to transform E. coli DH5{alpha}, and cloned MBL genes were selected on media containing ceftazidime 10 mg/L.

DNA sequencing and sequence analysis

Three clones, pMATRI-1–3, containing overlapping inserts from the P. aeruginosa strain 86–14571A library, of 1.6, 2.1 and 3.1 kb, respectively, were sequenced on both strands. Sequencing was performed by the dideoxynucleotide chain termination method using a Perkin Elmer Biosystems 377 DNA Sequencer. Sequence analysis was performed using the Lasergene DNASTAR software package. Alignments and phylogenetic analysis were obtained using the Clustal W and PAM 250 matrix. The putative cleavage site of the signal sequence was identified by computer analysis using the Center for Biological Sequence Analysis website (http://www.cbs.dtu.dk/services/SignalP/submission).

Oligonucleotide primers for sequencing/PCR

The primers used to sequence the DNA inserted in the pK18 multiple cloning site in the clones pMATRI-1–3 were firstly pK18 and pK18R, designed to hybridize at either end of the multiple cloning site of pK18. Further primers were then designed to extend these sequences through the insert (Table 1). Oligonucleotide primers designed to conserved regions of blaVIM and blaIMP, genes as well as primers specific to Tn5051 tnpA, the aacA4 gene and the sul1 region of the 3'CS are listed in Table 1.


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Table 1. Nucleotide sequences of primers used for PCR and sequencing
 
PCR conditions

For amplification using IMP and VIM primers, PCR was performed using AB-gene Expand Hi-fidelity master mix containing a mix of Pfu/non-proof-reading Taq polymerases and dNTPs (ABGENE house, Surrey, UK). Primers were used at 10 pM concentrations, and 1 µL of bacterial culture at density OD 1 at 600 nm was used as template. Cycling parameters were: 95°C for 5 min, followed by 30 cycles at 95°C for 1 min, annealing at 45°C for 1 min, an extension at 68°C for 1 min, ending with incubation for 5 min at 68°C. PCR products were visualized by electrophoresis on 0.8% agarose gels in TBA/EDTA buffer (pH 7.0), and stained with 1% ethidium bromide, as described previously.7


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phenotypic expression of an MBL from P. aeruginosa 86–14571A

P. aeruginosa 86–14571A was resistant to all ß-lactam antibiotics including aztreonam (Table 2). The strain was also resistant to gentamicin, tobramicin, trimethoprim and sulphonamides. The presence of an MBL was detected using an Etest strip, comparing the MIC of the isolate against imipenem with and without the MBL inhibitor EDTA. The MIC dropped from >64 mg/L to 8 mg/L in the presence of EDTA. Further experiments using cellular extracts of P. aeruginosa 86–14571A incubated with and without EDTA demonstrated that meropenem hydrolysis was inhibited with 25 mM EDTA (data not shown).


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Table 2. MIC values (mg/L) for P. aeruginosa 86–14571A, E. coli DH5{alpha} and E. coli DH5{alpha} (pMATRI-1)
 
Screening for known mobile MBL genes

Initial screening of this strain with primers designed against conserved regions of blaIMP and blaVIM MBL genes in a low stringency (45°C annealing) PCR screen failed to amplify any products, whereas blaIMP- and blaVIM-containing controls were positive. A shotgun cloning approach was therefore adopted to clone the resistance determinant.

Cloning and sequence analysis of IMP-13

Three different clones (pMATRI-1–3) encoding the MBL were isolated from the genomic library of P. aeruginosa strain 86–14571A that conferred ceftazidime resistance to E. coli DH5{alpha}. In addition to conferring ceftazidime resistance, the E. coli DH5{alpha} (pMATRI-1–3) also conferred resistance to cefotaxime, ceftriaxone, amoxicillin and co-amoxiclav acid, but not to aztreonam or carbapenems. The clones, sequenced on both strands, were found to contain overlapping sequences. The sequences were assembled to produce a contig of 3219 bp. This contained an open reading frame (ORF) of 741 nucleotides encoding a putative protein of 246 amino acids. The putative protein showed high identity to IMP-type MBLs and was designated blaIMP-13 (Figure 1). The blaIMP-13 gene had a GC content of 38% and was surrounded by features typical of a gene cassette. These were 5', a core site GTTAGGC, and 3', a 59 base element, which, in the case of blaIMP-13, is 133 bp long (Figure 1), and similar to the 59 base elements from IMP-7, -4 and -10 (AJ420864) displaying 93.5 %, 92 % and 91% identities, respectively. A phylogenetic tree has been constructed to compare the relatedness between the deduced protein and the other sequenced IMP MBLs (Figure 2). Overall, IMP-13 displays identities to IMP-8 and IMP-2 (identified in Italy) of 93% and 92.3%, respectively, and has 19 amino acid changes from IMP-2 and 17 from IMP-8. Thirteen of these amino acid substitutions were not found in any of the other sequenced IMPs. None of the substitutions were the same as those found in IMP-3 and IMP-6, i.e. glycine for serine at amino acid position 196, which is known to modulate the hydrolysis profile of these enzymes.16,17 The active site HFHSD was identical to all the other IMP enzymes. The N terminus of the protein shows features typical of bacterial signal peptides that target proteins to the periplasmic space, and the most likely cleavage site identified by computer analysis was between amino acids 20 and 21 (AGA-AL). This produced a mature protein of 25 076 Da with a theoretical pI of 7.5. Repeated attempts at isolating plasmids from P. aeruginosa 86–14571A were unsuccessful, as were attempts to transfer resistance to P. aeruginosa PA01 by conjugation. This indicates that blaIMP-13 is probably chromosomally encoded.



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Figure 1. The nucleotide sequence of the blaIMP-13 locus. The nucleotide sequence is the contig formed from three overlapping clones, pMATRI-1–3. The 25 bp IRi sequence located at the 5'end of the class 1 integron-containing blaIMP-13 is underlined and the attI1 site immediately 5' to the blaIMP-13 cassette is highlighted in bold. The start codons of the various ORFs are indicated by horizontal arrows and the stop codons by asterisks. The corresponding protein translation is reported below the nucleotide sequence. The signal peptide for secretion of the IMP-13 protein is underlined. The –35 and –10 hexamers of the intermediate strength Pant promoter are overlined. The conserved core sites at the extreme ends of the blaIMP-13 cassette and cassette boundaries are boxed, and recombination cross-over sites are indicated by vertical arrows. Conserved sequences within the 59 base element are highlighted in bold and labelled 1L, 2L, 2R and 1R. The nucleotide differences and corresponding amino acid changes to the intI1 allele of In31 are boxed.

 


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Figure 2. Phylogenetic tree displaying the relationship between members of the IMP family of MBLs. The tree is based on a CLUSTALW alignment of IMP MBLs generated using the PAM 250 matrix.

 
Genetic context of blaIMP-13

Sequence analysis of the partial blaIMP-13 integron indicates that it contains two gene cassettes. The sequence immediately downstream of blaIMP-13 shows 100% identity with the first 106 bp of the aac(6')1b gene cassette coding for the aminoglycoside acetyltransferase enzyme aacA4. This cassette was truncated by the cloning process, but is intact in the original isolate and confirmed by PCR and sequencing using the primers aacA4F and Sul1R as the only other gene cassette in this integron. These primers amplify the 1046 bp region between the beginning of the aacA4 and sul1 genes of this integron, which is identical in sequence to the respective region of the Polish blaVIM-2-containing integron (AJ515707). The blaIMP-13 gene is in the first gene cassette position and is located immediately downstream of the 5'-CS of a class 1 integron. The 5'-CS of the blaIMP-13-containing integron comprises an intI1 allele, is identical to that of the class 1 integron containing blaGES-1 and only differs from the intI1 allele of the IMP-1-containing integron In3118 by two nucleotides that change threonine to serine at amino acid position 187 (Figure 1). The Pant promoter found towards the 5' end of the integrase gene is identical to the one found in In31, contains a TGGACA (–35) hexamer and a TAAACT (–10) hexamer spaced by 17 bp, and is consistent with a promoter of intermediate strength.18 A 25 bp sequence IRi was identified 174 bp upstream of the stop codon of the class I integrase gene. This sequence represents one of the terminal inverted repeats of Tn402 (Tn5090)-like elements and marks the left-hand end of the blaIMP-13 integron. The sequence immediately preceding this repeat sequence was used to search the GenBank databases for similar insertion sites. An identical insertion site was found for the integron containing blaVIM-2 resident in P. aeruginosa strain 81–11963A, a clinical isolate from Poland (Genbank accession number AJ515707) (Figure 3). A near identical insertion site was also found for another Tn402-like element carrying integron In60, which only differs by a single nucleotide deletion (Figure 3) from the sequence harbouring blaIMP-13. This integron carries the extended- spectrum ß-lactamase blaGES-1, which was identified in a P. aeruginosa isolate from France. Further upstream of the blaIMP-13 integron there is a sequence with features characteristic of the tnp region of transposon Tn21, namely, tnpR and tnpM separated by a resolution site (res). These genes are found in the same orientation as those in Tn21. The tnpR sequence is terminated by the cloning process at the codon for the last amino acid. The presence of tnpA adjacent to tnpR was confirmed in the original isolate by PCR using specific primers (data not shown). The tnpM-like sequence has been truncated by the insertion of the blaIMP-13 integron such that only the sequence coding for the last 61 amino acids is present. The tnpM-like gene sequence shares 81.4% identity with tnpM from Tn21, and 98.4% identity with urf2 from Tn5051. The tnpR-like sequence codes for a putative protein displaying 98.9% identity to the TnpR protein of Tn5051, previously characterized from P. putida isolated in New York,19 and 87% and 86% identity to the TnpR proteins of transposons Tn21 and Tn501, respectively. This sequence information indicates that the blaIMP-13 gene is located on an integron harboured by a Tn402-like transposon, which in turn is harboured by a Tn21 subfamily transposon (Figure 4). Furthermore, the sequence of the tnpR gene and tnpM genes are identical to the transposon harbouring the blaVIM-2 integron previously identified in Poland (accession number AJ515707).



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Figure 3. Comparison of the IRi 25 bp inverted repeat sequences and flanking regions of the blaGES-1 -containing integron, In60, the integron containing blaIMP-13 and the blaVIM-2-containing integron from Poland. The identical 25 bp repeats are underlined and identical residues in the flanking sequences are indicated by vertical lines.

 


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Figure 4. Schematic representation of the blaIMP-13 and blaVIM-2 genetic loci, and comparison with the 5' end of the composite mercury resistance transposon Tn21, which includes the transposition (tnp) and integron In2 region (GenBank accession no. AF071413). The tnp regions consist of genes for the transposase (tnpA), the resolvase (tnpR), the putative transposition regulator (tnpM) and the resolution site (res). The class 1 integron In2 harboured by Tn21 consists of a class 1 integrase gene (intI1), its recombination site attI1 and the aminoglycoside resistance gene aadA1. The blaIMP-13-containing integron similarly consists of a class 1 integrase (intI1) and its recombination site, together with at least two gene cassettes, blaIMP-13 and an aacA4 gene cassette, as does the blaVIM-2-containing integron. These integrons are also harboured by Tn21-like transposons displaying most identity to Tn5051 and are inserted in tnpM, truncating this transposon gene. Arrows indicate the direction of transcription of the various genes, solid ellipses represent the position of 59 base elements, broken lines indicate vector sequence. Vertical lines represent the positions of 25 bp imperfect inverted repeats at the ends of the In2 integron, the left hand equivalent inverted repeat in the integrons containing blaIMP-13 and blaVIM-2 and the 38 bp inverted repeat at the left hand end of Tn21. The positions of the two insertion sequences harboured by Tn21 are also shown.

 
Nucleotide sequence accession number

The nucleotide sequence for the blaIMP-13 locus will appear in the GenBank databases under accession number AJ550807.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Investigation of the genetic basis of carbapenem resistance in the P. aeruginosa strain 86–14571A demonstrated that this strain harbours a new IMP-type MBL gene, blaIMP-13. Like all other clinically relevant MBLs so far discovered, with the exception of SPM-1,7 blaIMP-13 is located on a mobile gene cassette inserted into an integron. The identification of blaIMP-13 in P. aeruginosa strain 86–14571A in Rome raises the number of IMP variants now found among nosocomial pathogens in Italy to three, and includes IMP-220 and IMP-12 (AJ420864), and the number of different clinically relevant MBLs identified in Italy with VIM-1 and VIM-2 to five. The blaIMP-13 gene was found in the first position of the integron, which suggests it was the most recently acquired gene cassette, and next to an aminoglycoside modifying gene. This is a common occurrence among integrons carrying MBLs and may reflect the co-administration of ß-lactams with an aminoglycoside. The 3'CS of the blaIMP-13 integron was not represented in any of the sequenced clones. However, its presence was confirmed by PCR and the fact that the parent organism is resistant to sulfa drugs, which can be coded for by the sul1 gene present in the 3'CS of most integrons. P. aeruginosa strain 86–14571A was also resistant to gentamicin and tobramicin, but susceptible to amikacin, which is consistent with the presence of a functional aacA4 gene. The resistance to aztreonam is not a characteristic of MBLs; it may have been encoded by an additional oxa-type gene or have resulted from the over-expression of a chromosomal ampC gene. The GC ratio of the blaIMP-13 was 38%, which falls into the same GC ratio as the other blaIMP genes already sequenced. These range from 36.14% GC for blaIMP-12 (AJ420864) to 39.27% GC for blaIMP-5 (AF290912), and indicates that this gene is not of pseudomonal origin, which in general has a GC percentage of 66%, with the exception of pilin genes (http://www.sanger.ac.uk/Projects/P_fluorescens/). The IMP-type MBLs have now been found in nosocomial pathogens from many countries, ranging from East Asia to Brazil.21 They have been found exclusively in integrons, yet they are rarely identical in integron structure or actual sequence between countries, or, as in the case of Italy, within countries. This suggests a worldwide source for the IMP-type MBLs with low GC content—possibly a Gram-positive organism—and infers that the overuse of carbapenems and/or perhaps third-generation cephalosporins will produce this type of resistance mechanism in the locality where they are used. The finding of a 25 bp inverted repeat upstream of the class 1 integrase gene of the blaIMP-13-containing integron is characteristic of transposons, including Tn402 and Tn5053. These are part of a superfamily of transposable elements that include: retroviruses; IS3 family insertion sequences; phage Mu and the transposons Tn552 and Tn7. They are characterized by the presence of the dinucleotide TG at the transposon 5' ends, and the D,D(35)E motif in their transposases.22 Generally these Tn402-like elements have truncated tni modules and are incapable of self transposition. Future work will determine whether this is the case with the element harbouring blaIMP-13. These elements have been named res site hunters22 because they preferentially transpose into the res sites of other transposons. A study of Tn402 insertion sites demonstrated that most insertion sites were in or near res sites. In addition, experiments with a similar transposon, Tn5053, demonstrated that Tn5053 displays striking insertional preference for the res regions of Tn1721 or Tn5044. Seventy percent of Tn5053 insertion events occur in clusters inside the target res regions, whereas most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. The insertion of the elements containing both blaIMP-13 from Italy and blaVIM-2 from Poland are in an identical site about 200 bases away from the res site of a Tn5051-like transposon. This insertion site is also shared by the element harbouring blaGES-1. Furthermore, the tnpR genes of the elements harbouring blaIMP-13 and blaVIM-2 are identical in sequence, which suggests the same transposon is responsible for the dissemination of both these MBLs. Both P. aeruginosa parent strains had no evidence of containing plasmids and had different ribotypes, which suggests that at least two events differentiate these isolates: (a) transposition of the original Tn5051-type transposon and (b) acquisition of different MBL genes, although these events need not be in this order. It is also likely that the transposition of the Tn402-like transposon into the Tn5051-like transposon occurred before these events and probably gave rise to the transposons now carrying these gene cassettes. Interestingly, it has been shown that all that is needed for insertion of Tn5053 into the res regions of Tn1721 or Tn5044 is the resolvase gene on the target transposon. Since Tn402-like transposons are of the same family as Tn5053, and analysis has shown that the insertion sites of these transposons are similarly in the res regions of Tn21-like transposons, it may be that although most of the studied Tn402-like transposons are defective in their transposition module they may still be mobile by virtue of the tnpR gene of the resident Tn21-like transposon. The tnpR gene on the composite transposon harbouring blaIMP-13 is complete, apart from the last amino acid truncated by the cloning process. It has a perfect Shine Delgarno sequence and promoter and therefore is likely to be active. Additionally, the presence of tnpA upstream from tnpR in the Polish and Italian P. aeruginosa isolates may mean that these Tn5051-like transposons are transposition competent. Future work will determine whether the Tn5051-like transposons harbouring these MBL genes are capable of transposition. Certainly, the mobility of gene cassettes, combined with the mobility of composite transposons harbouring these genes, would confer enormous potential for the dissemination of these genes into other nosocomial pathogens via transposition onto conjugative plasmids.


    Footnotes
 
* Corresponding author. Tel: +44-117-9287522; Fax: +44-117-9287896; E-mail: Mark.Toleman{at}bristol.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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