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
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Abstract |
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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 8614571A 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
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Introduction |
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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 8614571A. We also describe its genetic context and its association with a novel Tn21-like composite transposon.
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Materials and methods |
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In 2001, P. aeruginosa strain 8614571A 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 [endA1 gyrA96 hsdR17
lacU169 (
80lacZ
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 48501 and 753677 (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 MuellerHinton 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 MuellerHinton 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 8614571A 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 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
, and cloned MBL genes were selected on media containing ceftazidime 10 mg/L.
DNA sequencing and sequence analysis
Three clones, pMATRI-13, containing overlapping inserts from the P. aeruginosa strain 8614571A 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-13 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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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
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Results |
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P. aeruginosa 8614571A 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 8614571A incubated with and without EDTA demonstrated that meropenem hydrolysis was inhibited with 25 mM EDTA (data not shown).
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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-13) encoding the MBL were isolated from the genomic library of P. aeruginosa strain 8614571A that conferred ceftazidime resistance to E. coli DH5. In addition to conferring ceftazidime resistance, the E. coli DH5
(pMATRI-13) 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 8614571A 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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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 8111963A, 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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The nucleotide sequence for the blaIMP-13 locus will appear in the GenBank databases under accession number AJ550807.
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Discussion |
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Footnotes |
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References |
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