1 Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, UK; 2 Central Research Laboratory, Warsaw, Poland; 3 The JONES Group/JMI Laboratories, North Liberty, IA; 4 Tufts University School of Medicine, Boston, MA, USA
Received 22 November 2002; returned 18 December 2002; revised 14 April 2003; accepted 16 April 2003
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Abstract |
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Keywords: metallo-ß-lactamases, Poland, Pseudomonas aeruginosa
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Introduction |
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As part of the SENTRY Antimicrobial Surveillance Program, an imipenem-resistant isolate of P. aeruginosa (strain 81-11963A) was recovered from a blood culture of a female neonate from the local childrens hospital in Warsaw, Poland. Here we report the genetic characterization of the carbapenem resistance determinant from P. aeruginosa 81-11963A. We also offer an explanation as to how this new integron may have arisen from In58. The isolation of P. aeruginosa 81-11963A represents the first reported appearance of the metallo-ß-lactamase VIM-2 in Eastern Europe.
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Materials and methods |
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P. aeruginosa 81-11963A was a clinical isolate from Warsaw, Poland. Escherichia coli strain DH5 [supE44
lacU169 (
80lacZ
M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used as the host strain to express the cloned ß-lactamase gene. Positive controls for the IMP- and VIM-type metallo-ß-lactamases were P. aeruginosa strains carrying the respective genes. The genomic library was generated in the cloning vector pK18 as previously described.1
Materials
Antimicrobial agents used were ceftazidime (GlaxoSmithKline, Worthing, UK) and kanamycin (Sigma Chemical Co., St Louis, MO, USA). PCR primers were purchased from Sigma-Genosys Ltd (Pampisford, UK). General reagents for DNA manipulation were obtained from Invitrogen (Groningen, The Netherlands). All other reagents were obtained from Sigma Chemicals Co. or BDH (both of Poole, UK).
Determination of MICs
Mid-log phase grown cultures (optical density of 0.6 at 600 nm) were diluted to 1/104 in water. Ten microlitres from each dilution was spotted onto MuellerHinton agar (BBL; Becton Dickinson, Oxford, UK), containing serial dilutions of the appropriate agent, using a multipoint inoculator. After 24 h incubation at 37°C, the MIC was noted as the lowest concentration of antimicrobial that inhibited the growth in those dilutions which, when inoculated onto nutrient agar containing no drug, gave rise to single colonies.
PCR screening for blaVIM and blaIMP metallo-ß-lactamase genes
For amplification using primers based on the conserved regions of the imp and vim genes, PCR was performed using AB-gene Expand Hi-Fidelity master mix containing a mix of Pfu non-proof reading Taq polymerases and dNTPs. Primers used to detect vim/imp genes were: VIM forward, TTATGGAGCAGCAAGCAGTG; VIM reverse, CGAATGCGCAGCACCAGG; IMP forward, ATGAGCAAGTTATCCTTATTC; and IMP reverse, GCTGCAACGACTTGTTAG. 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 of 95°C for 1 min, annealing at 45°C for 1 min and extension 68°C for 1 min, and ending with a 5 min incubation at 68°C.
Recombinant DNA methodology and DNA sequencing analysis
Genomic DNA was isolated from P. aeruginosa strain 81-11963A by the cetyl-trimethyl-ammonium bromide method and gene libraries were created using size fractionated DNA as described previously.1 The ligation mixture was subsequently dialysed and used to transform E. coli DH5 to ceftazidime resistance by electroporation. The clone containing the metallo-ß-lactamase gene was recovered by plating the gene library on to plates containing kanamycin (25 mg/L) and ceftazidime (6 mg/L). Sequencing was carried out on both strands by the dideoxy-chain termination method with a Perkin Elmer Biosystems 377 DNA sequencer. Sequence analysis was performed using the Lasergene DNASTAR software package. Sequence alignments were performed using Clustal_W with a PAM 250 matrix.
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Results |
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By NCCLS criteria, P. aeruginosa 81-11963A was susceptible only to polymyxin (MIC 2 mg/L). The isolate was intermediately susceptible to aztreonam (MIC 16 mg/L), amikacin (MIC, 32 mg/L) and ciprofloxacin (MIC 2 mg/L) and highly resistant (MIC > 256 mg/L) to all other common antimicrobial agents tested, including penicillins, cephalosporins and carbapenems. When the isolate was screened with the imipenem-EDTA Etest strip (AB Biodisk, Solna, Sweden), the MIC of imipenem decreased from >256 to 4 mg/L in the presence of the metal chelator, EDTA, indicating production of a zinc-dependent ß-lactamase. To determine which type of enzyme was produced, the strain was investigated for carriage of blaIMP- or blaVIM-type genes. Two PCR primer pairs, one targeted to conserved regions of blaIMP-type genes and the other to blaVIM-type genes, were used in low stringency PCRs (annealing temperature 45°C). Metallo-ß-lactamase positive controls were strains of P. aeruginosa carrying blaIMP-1 or blaVIM-1. Using genomic DNA from P. aeruginosa 81-11963A, a PCR amplicon of the expected size was obtained using the primer pair to detect blaVIM-type genes, but not with the blaIMP-type primers (data not shown).
Cloning of blaVIM-2 and adjacent DNA from P. aeruginosa 81-11963A
The gene encoding the metallo-ß-lactamase was isolated from a size-fractionated genomic library, as described previously.1 Approximately 20 colonies were isolated and subsequent analysis determined ceftazidime MICs were >128 mg/L. In the presence of EDTA (10 mM) the ceftazidime MICs decreased from 256 to <4 mg/L, confirming that the recombinant plasmids carried a metallo-ß-lactamase gene. E. coli carrying the recombinant plasmid gave imipenem and meropenem MICs of 1 and 0.125 mg/L, respectively. One clone, pMATWS1, containing an insert of 5 kb was further analysed by sequencing to assess the genetic context of the blaVIM-2 derivative. The insert of P. aeruginosa DNA in this plasmid was sequenced. The sequence has been deposited in the EMBL database under accession number AJ515707.
Sequence analysis of the P. aeruginosa 81-11963A DNA insert in pMATWS1
Analysis of the nucleotide sequence of the DNA from P. aeruginosa 81-11963A carried on recombinant plasmid pMATWS1 revealed the presence of blaVIM-2 (Figure 1). Adjacent to and downstream from blaVIM-2 was aacA4, a gene that encodes AAC(6')-Ib, which confers resistance to the aminoglycosides, kanamycin and netilmicin. Beyond aacA4 was the 3'-CS region typical of class 1 integrons. On the other side of the blaVIM-2 gene was int1, encoding the integrase of a class 1 integron. Between int1 and blaVIM-2 was an attI1 site. Thus, blaVIM-2 from P. aeruginosa 81-11963A was a component of a previously undescribed class 1 integron. While the aacA4 gene on the integron was followed by a typical 59 base element (be), as previously reported,4 the blaVIM-2 gene was not. Instead, there was a truncated version of only 19 base pairs (bp) (Figure 2), comprising an inverse core site and a core site separated by only 5 bp.
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Discussion |
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The blaVIM-2 gene from strain 81-11963A could not be transferred to a susceptible P. aeruginosa recipient, and was shown by PCR to be chromosomally encoded as judged by blaVIM amplicons obtained from genomic DNA preparations but not plasmid DNA preparations. The blaVIM-2-containing integron from P. aeruginosa 81-11963A was very similar to the blaVIM-2-containing integron, In58, found in France.4 In58 also carries the aacA4 gene cassette, but is separated from the blaVIM-2 gene cassette by an aacC1 gene cassette. These gene cassette assortments are somewhat different from other blaVIM-2-containing integrons (Figure 1). The blaVIM-2 gene recovered from P. aeruginosa 81-11963A was unusual in that it is not accompanied by a full-size 59 be. Instead, it was followed by what appears to be a deleted 59 be of 19 bp. The deletion appears to have removed all but 5 bp of the sequence between the inverse core site and the core site of the 59 be (Figure 2). When the sequences were analysed further, it seems likely that the deleted element was the result of integrase-mediated excision. Indeed, the integron found in P. aeruginosa 81-11963A can be derived readily from In58 by two integrase-mediated gene cassette excisions. The first would remove the aacA7 gene cassette to leave the blaVIM-2 gene cassette at the attI1 site. The second excision would remove the aacC1 gene cassette and part of the blaVIM-2 gene cassette 59 be as the result of integrase mistaking the 2L sequence (GTTGCAG) of the blaVIM-2 gene cassette 59 be for the core sequence (GTTAGAT) of the 59 be, separating the blaVIM-2 and aacC1 genes (Figure 2), where recombination would normally occur. Such an event would fuse the first 13 bp of the blaVIM-2 gene cassette 59 be, GCATAACATGAAG, to the terminal 6 bp, TTAGGC, of the core site of the 59 be separating the aacC1 and aacA4 genes, in the process creating the 19 bp hybrid element found (Figure 2). Database searches for other examples of shortened 59 be revealed two blaVIM-2 genes in Italy7 and Korea.8 The 59 be of the blaVIM-2 gene cassette may be prone to a particular aberrant integrase-mediated recombination. An alternative, but perhaps less likely, explanation is that integrase mediates recombination between the 2L site and the core site of the hybrid 59 be that follow the blaVIM-2 gene in different integrons, as suggested by Pallecchi et al.7 One consequence of the misdirected integrase-mediated recombination is the possibility that the blaVIM-2 gene cassettes might become fused to other gene cassettes, in the case of the integron from P. aeruginosa 81-11963A encoding resistance to ß-lactams and aminoglycosides, and become permanently linked and to move genetically as a pair. Consequently, if one was selected by use of one agent so will be the other.
The enzymes encoded by these mobile cassettes can hydrolyse almost all clinically useful ß-lactams, irrespective of class, and that they are resistant to the effects of serine ß-lactamase inhibitors makes such reports particularly alarming. We suggest that monitoring of multidrug-resistant, non-fermenting Gram-negative bacilli for production of metallo-ß-lactamases should become a standard aspect of any local or global surveillance systems.
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Acknowledgements |
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Footnotes |
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References |
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