4th Department of Internal Medicine, Molecular Biology Section, University of Athens Medical School, Athens; University General Hospital Attikon, Rimini 1, 124 62 Chaidari, Greece
Received 15 October 2004; returned 10 November 2004; revised 24 January 2005; accepted 24 January 2005
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Abstract |
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Materials and methods: MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of ß-lactamases. PCR assays with primers specific for the blaVIM gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-ß-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the blaVIM-1 gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the blaVIM-1 gene as a probe, amplified by PCR.
Results: The isolate was resistant to extended-spectrum ß-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 108 cfu/mL was used. Isoelectric focusing detected a ß-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the blaVIM-1 gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The blaVIM-1 probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene.
Conclusion: We describe a novel class 1 integron containing blaVIM-1 and aac(6')-IIc genes in an E. cloacae clinical isolate.
Keywords: metallo-ß-lactamases , carbapenemases , E. cloacae
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Introduction |
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Since the introduction of routine use of the imipenem-EDTA-disc synergy test7 in our Infectious Diseases Research Laboratory, we have encountered several strains of Enterobacteriaceae that have shown a positive result although they were not resistant to carbapenems according to the NCCLS.8 In this report, we describe an E. cloacae clinical isolate exhibiting reduced susceptibility to imipenem and ertapenem and a positive EDTA-disc synergy test due to production of VIM-1 metallo-ß-lactamase encoded by a novel class 1 integron.
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Materials and methods |
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E. cloacae isolate 87 was identified by the ATB 32 GN System (bioMérieux SA, Marcy 1'Étoile, France). The MICs of ß-lactams and aminoglycosides were determined by the broth microdilution method according to the NCCLS,9 with the exception of ertapenem for which the Etest (AB Biodisk, Solna, Sweden) was used according to the manufacturer's instructions. The MICs of carbapenems were also determined using a higher inoculum (108 cfu/mL). Susceptibility to other antimicrobial agents was assessed by the disc diffusion method.9 To screen for metallo-ß-lactamase production, a synergy test using imipenem- and EDTA-containing discs was employed.7 Conjugation experiments were carried out by the filter mating method, with Escherichia coli RC85 R K12 strain (rifampicin MIC, > 128 mg/L) as the recipient.10 Rifampicin (128 mg/L) and ceftazidime (16 mg/L)-containing agar plates were used to select for transconjugants. The isoelectric points of ß-lactamases were determined by isoelectric focusing of cell sonicates on precast polyacrylamide gels (PhastGel IEF 39; Amersham Biosciences, Uppsala, Sweden) with a PhastSystem Apparatus (Pharmacia Biotech AB, Uppsala, Sweden). Enzyme bands were visualized with nitrocefin (Oxoid Ltd, Basingstoke, UK). Hydrolysis of imipenem (0.25 mg/L) by the metallo-ß-lactamase band was detected with a microbiological method, as described previously.11
PCR amplification was carried out as described previously with primers VIM-B (5'-ATGGTGTTTGGTCGCATATC-3') and VIM-F (5'-TGGGCCATTCAGCCAGATC-3') amplifying a 510 bp (nucleotides 152661) internal fragment of the blaVIM-1 gene (EMBL/GenBank accession no. AF 191564).12 Detection and mapping of a class 1 integron were carried out using primers specific for the conserved segments 5'-CS (5'-CTTCTAGAAAACCGAGGATGC-3') and 3'-CS (5'-CTCTCAAGATTTTAATGCGGATG-3')13 amplifying the variable region containing the resistance gene cassettes. The PCR amplicon (In87) was ligated to pCR2.1 vector and the ligation product pCR2.1(In87) was used to transform E. coli TOP10F' (TA Cloning Kit; Invitrogen, Carlsbad, CA, USA), as described by the manufacturer. Nucleotide sequences of the cloned PCR product were determined on both strands with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequence analysis and database similarity searches for nucleotide and deduced amino acid sequences were carried out at the NCBI website (http://www.ncbi.nlm.nih.gov/). Alignment of protein sequences was carried out by the CLUSTAL W multiple sequence alignment program available over the Internet (http://www.clustalw.genome.ip/).
Extraction of plasmid DNA was carried out with the QIAprep Miniprep (Qiagen GmbH, Hilden, Germany), as described by the manufacturer and by an alkaline lysis method.14 In order to identify the location of the blaVIM-1 gene, genomic DNA in the form of a plug was digested with SpeI (New England BioLabs (UK) Ltd, Hitchin, Hertfordshire, UK), electrophoresed in Gene Navigator apparatus (Pharmacia Biotech AB, Uppsala, Sweden Dassell, Germany), transferred onto a nylon membrane (Schleicher & Schuell, Dassell, Germany) and hybridized with a PCR-obtained 510 bp internal fragment of the blaVIM-1 gene labelled with a digoxigenin DNA labelling and detection kit (Roche Diagnostics, Mannheim, Germany).
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Results |
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The nucleotide sequence of integron In87 has been assigned the EMBL/GenBank accession no. AY648125.
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Discussion |
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The other question that arises upon isolation of a carbapenem-susceptible metallo-ß-lactamase-producing strain is whether infection control measures are required. Phenotypically, E. cloacae isolate 87 did not exhibit multiple resistances and the same is true for all the Enterobacteriaceae carrying blaVIM genes that we have isolated thus far.8 Nevertheless, an outbreak of VIM-1-producing Klebsiella pneumoniae causing fatal infections in many tertiary care hospitals in Athens has already been described.21 These strains are now endemic in many Intensive Care Units in Athens. The mobile nature of metallo-ß-lactamase genes, the broad hydrolysis profile of the enzymes, and their frequent association with other resistance genes such as those coding for aminoglycoside-modifying enzymes, make them an important potential threat for hospital epidemiology. For these reasons, we advocate barrier precautions (private room, if available, gloves and gowns) for the infected or colonized patient, although consensus guidelines on this issue are also lacking. We have described the presence of the blaVIM-1 gene in an E. cloacae clinical isolate and the association of blaVIM-1 and aac(6')-IIc in the same integron for the first time. The identity of these gene cassettes with those previously described in P. aeruginosa15 and E. coli16 suggests extensive spread of resistance genes between various Gram-negative species.
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References |
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