1 Department of Microbiology, University Hospital of Thessaly, Mezourlo, Larissa 412 22; 2 Department of Microbiology, Medical School and 3 Department of Microbiology, Faculty of Nursing, School of Health Sciences, University of Athens, Athens 115 27, Greece
Received 4 January 2003; returned 10 February 2003; revised 6 March 2003; accepted 6 March 2003
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Abstract |
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Materials and methods: All carbapenem-resistant (imipenem- and/or meropenem-resistant) P. aeruginosa isolates recovered from separate patients during a 1 year period in the Clinical Microbiology Laboratory at the University Hospital of Thessaly, Larissa, Greece, were studied for metallo-ß-lactamases. They were tested by Etest MBL, PCR analysis and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA.
Results: A blaVIM gene was detected in 47 of the 53 (88.7%) carbapenem-resistant P. aeruginosa isolates. PFGE grouped the blaVIM-positive isolates in six unrelated genotypes; one type included two subtypes. Nucleotide sequencing of the PCR amplicons of a randomly selected isolate from each one of the seven subtypes, detected the variant sequences blaVIM-2 in four and blaVIM-4 in three cases, respectively. They were carried as single gene cassettes or along with an aminoglycoside resistance gene (aacA29a) in class 1 integrons.
Conclusions: These findings suggest that different strains of P. aeruginosa carrying unrelated metallo-ß-lactamase gene variants predominate in our hospital environment.
Keywords: carbapenemases, polyclonal, outbreak, Greece
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Introduction |
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Materials and methods |
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The susceptibility of the isolates to a range of anti-pseudomonal antimicrobials was also determined by the Vitek system according to the recommendations of the manufacturer. The MICs of imipenem and meropenem for the isolates were determined in addition by an agar dilution method using MuellerHinton agar (Oxoid, Basingstoke, UK) and a final inoculum of 104 cfu/mL. The possible presence of an MBL among carbapenem-resistant P. aeruginosa was tested by the Etest MBL (AB Biodisk, Solna, Sweden).
Pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA of P. aeruginosa isolates was carried out with a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UK), as described elsewhere.4 Banding patterns of the strains were compared visually. The possible carriage of the carbapenemase-encoding genes blaIMP and blaVIM was tested by PCR using primers and amplification conditions that have been described previously.4,8 Primers specific for blaVIM-1 and blaVIM-2 genes were also used.6 PCR assays combining primers specific for conserved 5'-CS and 3'-CS sequences5 with the blaVIM-1- and blaVIM-2-specific primers were also carried out to investigate the possible association of the MBL gene with a class 1 integron. Positive controls for detecting blaIMP and blaVIM genes were, respectively, the strains P. aeruginosa 101/1477 (kindly provided by Dr N. Woodford, PHLS, UK), and P. aeruginosa 174.5
Nucleotide sequencing of both strands of the PCR products was performed on amplicons derived using the primers 5'-CS and blaVIM-1 (reverse), and 5'-CS and blaVIM-2 (reverse). The amplicons were sequenced on an ABI PRISM 377 DNA sequence analyser (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA).
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Results |
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The PCR for the blaVIM gene was repeatedly positive (PCR products of 261 bp) and for the gene blaIMP, negative, in all 47 isolates that were resistant to both carbapenems, and negative for both genes in the six strains that were resistant to imipenem or meropenem. The blaVIM gene was not detected in any of the seven carbapenem-sensitive isolates that were used as negative controls in the PCR experiments. Six unrelated genotypes, each containing three to 16 isolates, were detected among the 47 blaVIM-positive isolates by PFGE (Table 1 and Figure 1). Only one clone (PFGE type I) was relatively more common, including 16 isolates. One genotype (PFGE type III) contained two subtypes with three-band differences from each other, whereas isolates that belonged to the other genotypes were indistinguishable. Four distinct genotypes were also identified among the six isolates that were resistant to imipenem or meropenem and these genotypes were distinct from those detected among the blaVIM-positive isolates by more than five bands (data not shown).
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Discussion |
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It is noteworthy that the MBL found in our region is not only VIM-2 type, which is scattered in several European countries,3,5 but also VIM-4. The identification of P. aeruginosa strains that carried either of the variant genes blaVIM-2 or blaVIM-4, in the same setting, taken together with the finding that the host pseudomonads were genetically distinct, possibly indicates a wide distribution of VIM-type genes in the hospital environment. This is further supported by the fact that several integrons of different phylogeny have been described to carry the blaVIM genes.2,9 Our carbapenemase-producing P. aeruginosa isolates carried blaVIM genes mostly as a sole gene cassette in a class 1 integron as reported previously for In56 of the strain COL-1,3 unlike most integrons containing blaVIM genes that usually carry two or more gene cassettes.2,9 In our study, blaVIM was detected only once together with another gene (aacA29a); the latter gene cassette was previously reported only in In59, where it was followed at its 3' end by gene cassettes blaVIM-2 and aacA29b.9
It is of particular concern that VIM enzymes have been detected in widely separated Eurasian countries. The results of this study suggest that if effective infection control approaches are not stringently applied at this stage, horizontal but also clonal spread of VIM-producing Pseudomonas might dramatically increase in our region.
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Footnotes |
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References |
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2
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