JAC Advance Access originally published online on June 2, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):432-433; doi:10.1093/jac/dkl240
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Molecular characterization of an Escherichia coli clinical isolate that produces both metallo-ß-lactamase VIM-2 and extended-spectrum ß-lactamase GES-7: identification of the In8 integron carrying the blaVIM-2 gene
4th Department of Internal Medicine, Molecular Biology Section, University General Hospital ‘ATTIKON’ Rimini 1, 124 62 Chaidari, Greece
*Corresponding author. Tel: +30-2105326426; Fax: +30-2105326446; E-mail: hgiama{at}ath.forthnet.gr
Received 8 February 2006; returned 7 April 2006; revised 15 May 2006; accepted 15 May 2006
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Abstract |
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Objectives: We have previously reported the isolation of a VIM-2-producing Escherichia coli clinical isolate and the coexistence of blaVIM-2 and blaGES-7 in the same isolate. The aim of this study was to elucidate the genetic environment of these genes.
Methods: PCR and sequence analysis were used to identify and analyse the blaVIM-containing integron. The location of the blaVIM-2 and blaGES-7 genes was identified by hybridization experiments on I-CeuI-digested genomic DNA after PFGE.
Results: Only the blaVIM-2 gene has been found on a class I integron, designated In8 (1474 bp). Hybridization with the blaVIM-2, the blaGES-7 and the 16S–23S rRNA probes confirmed the chromosomal location of both genes. The blaVIM-2 probe hybridized with a 710 kb fragment whereas the blaGES-7 probe hybridized with a 144 kb fragment of genomic DNA of the E. coli isolate.
Conclusions: This report provides evidence for the chromosomal location of both blaVIM-2 and blaGES-7 genes carried by an E. coli clinical isolate. In8, containing blaVIM-2, presents an emerging threat of carbapenem resistance among E. coli isolates in Greece.
Keywords: E. coli , blaVIM-2 , blaGES-7
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Introduction |
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Integrons are genetic structures capable of capturing gene cassettes. Class 1 integrons are the most commonly involved in antibiotic resistance in clinical isolates and contain two conserved segments (5'-CS and 3'-CS) flanking a variable region that contains the integrated genes in the form of cassettes. The 5'-CS is composed of the integrase gene (intI1), the recombination site (attI1), which is responsible for the site-specific recombination, and the promoter region, which directs transcription of the gene cassettes. IntI1 mediates integration of gene cassettes into the attI1 site. The 3'-CS may vary in size but usually includes genes encoding resistance to sulphonamides (sulI) and disinfectants (qacE
1). Gene cassettes in class 1 integrons are composed of a single coding sequence and the 59 base element, which is involved in the mobility of gene cassettes. Class 1 integrons are widely distributed among various Gram-negative organisms conferring resistance to commonly used antibiotics such as ß-lactams and aminoglycosides.1,2 Thus far, blaVIM and blaGES have always been identified on class 1 integrons.3,4 We have previously described an Escherichia coli clinical isolate producing VIM-2 and GES-7 enzymes.5 In the present study we have characterized the location and the genetic environment of these genes. |
Materials and methods |
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Detection and mapping of class 1 integrons were carried out using primers specific for the conserved segments 5'-CS (5'-CTTCTAGAAAACCGAGGATGC-3') and 3'-CS (5'-CTCTCAAGATTTTAATGCGGATG-3') amplifying the variable region containing the resistance gene cassettes. The 1474 bp PCR amplicon, designated In8, was ligated to pCR2.1 vector and the ligation product pCR2.1/In8 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/). As previously reported, transfer experiments using an E. coli recipient were repeatedly unsuccessful for both genes.5 In order to identify the location of the blaVIM-2 and blaGES-7 genes, genomic DNA was incorporated in an agarose plug and digested with 0.04 U/µL of I-CeuI (New England BioLabs Ltd, Hitchin, Hertfordshire, UK) at 37°C for 3 h. DNA fragments were separated by PFGE (pulse times, 20–120 s for 12 h and 60–100 s for 8 h), in a Gene Navigator apparatus (Pharmacia Biotech AB, Uppsala, Sweden). A SmaI-digest of genomic DNA from Staphylococcus aureus ATCC 8325 was used as the molecular weight standard. DNA was then transferred onto a nylon membrane (Schleicher & Schuell, Dassell, Germany). Hybridization of chromosomal DNA fragments (separated by PFGE) as well as of the 1474 bp PCR amplicon (In8) was carried out under stringent conditions with three different probes: the PCR-obtained 510 bp internal fragment of blaVIM-2, the PCR-obtained 400 bp internal fragment of the blaGES-7 gene and the 16S–23S rRNA probe [obtained with primers 5'-GAAGTCGTAACAAGGTA(AG)CCGT-3' and 5'-TGCCAAGGCATCCACC] all labelled with a digoxigenin DNA labelling and detection kit (Roche Diagnostics, Mannheim, Germany).3 The nucleotide sequence of the blaVIM-2-carrying integron (In8) has been assigned the GenBank accession no. AY781413.
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Results and discussion |
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Only the blaVIM-2 gene has been found on a class I integron. Sequencing of pCR2.1/In8 revealed only the blaVIM-2 gene cassette array between the 5'- and 3'-CS of a class I integron. The sequence of the 5'-CS of In8 was identical to that of In105 reported in Acinetobacter baumannii and of the integron harboured by an environmental isolate of Pseudomonas pseudoalcaligenes.6,7 The 5'-CS and the blaVIM-2 gene cassette (including the 72 nt of the 59 base element) of In8 had G to T and G to A transitions in the promoter region that lies within the integrase structural gene in the In8 integron. These mutations have led to a strong version of the P1 promoter as compared with In56 of the first Pseudomonas aeruginosa isolate reported to produce VIM-2.8
Both blaVIM-2 and blaGES-7 genes were located on the chromosome of the E. coli isolate, as was suggested by hybridization of blaVIM-2, blaGES-7 and 16S–23S rRNA probes on I-CeuI fragments of chromosomal DNA. This restriction endonouclease cleaves DNA in a 26 bp site located exclusively upstream of the V domain of 23S rRNA in the rrl gene. Digestion with I-CeuI revealed seven fragments (sizes 3.3 kb, 710 bp, 670 bp, 580 bp, 144 bp, 96 bp and 41 bp, approximately), as many as the rrn copies of E. coli. All fragments hybridized with the 16S–23S rRNA probe confirming the chromosomal origin of all fragments. The blaVIM-2 probe hybridized with the 710 kb fragment whereas the blaGES-7 probe hybridized with the 144 kb fragment.
In conclusion, this report provides evidence for the chromosomal location of both blaVIM-2 and blaGES-7 genes carried by an E. coli clinical isolate. Previous reports on the blaGES-7 gene described its location on transferable plasmids always inserted into class 1 integrons.3 This is the first identification of that gene on the chromosome, not associated with a class 1 integron. blaVIM genes are mainly plasmid-mediated but there are a few reports confirming the chromosomal location of blaVIM-1 and blaVIM-3 in P. aeruginosa and blaVIM-2 in P. pseudoalcaligenes.4,7,9 However, this does not preclude integration of blaVIM-2 in conjugative plasmids in Enterobacteriaceae in the future, similarly to what happened with blaVIM-1, considering the mobile nature of gene cassettes.10–12
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None to declare.
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References |
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1 Collis CM and Hall RM. (1995) Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother 39:155–62.[Abstract]
2 Fluit AC and Schmitz FC. (1999) Class 1 integrons, gene cassettes, mobility and epidemiology. Eur J Clin Microbiol Infect Dis 18:761–70.[CrossRef][ISI][Medline]
3
Kartali G, Tzelepi E, Pournaras S, et al. (2002) Outbreak of infections caused by Enterobacter cloacae producing the integron-associated ß-lactamase IBC-1 in a neonatal intensive care unit of a Greek hospital. Antimicrob Agents Chemother 46:1577–80.
4
Lauretti L, Riccio ML, Mazzariol A, et al. (1999) Cloning and characterization of blaVIM, a new integron-borne metallo-ß-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother 43:1584–90.
5 Galani I, Souli M, Chryssouli Z, et al. (2004) First identification of an Escherichia coli clinical isolate producing both metallo-ß-lactamase VIM-2 and extended-spectrum ß-lactamase IBC-1. Clin Microbiol Infect 10:757–60.[CrossRef][ISI][Medline]
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Yum JH, Yi K, Lee H, et al. (2002) Molecular characterization of metallo-ß-lactamase-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korea: identification of two new integrons carrying the blaVIM-2 gene cassettes. J Antimicrob Chemother 49:837–40.
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Quinteira S, Ferreira H, Peixe L. (2005) First isolation of blaVIM-2 in an environmental isolate of Pseudomonas pseudoalcaligenes. Antimicrob Agents Chemother 49:2140–1.
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Poirel L, Naas T, Nicolas D, et al. (2000) Characterization of VIM-2, a carbapenem-hydrolyzing metallo-ß-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother 44:891–7.
9 Yan J-J, Hsueh P-R, Ko W-C, et al. (2001) Metallo-ß-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrob Agents Chemother 5:2224–8.
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Miriagou V, Tzelepi E, Gianneli D, et al. (2003) Escherichia coli with a self-transferable, multiresistant plasmid coding for metallo-ß-lactamase VIM-1. Antimicrob Agents Chemother 47:395–7.
11 Scoulica EV, Neonakis IK, Gikas AI, et al. (2004) Spread of blaVIM-1-producing E. coli in a university hospital in Greece. Genetic analysis of the integron carrying the blaVIM-1 metallo-ß-lactamase gene. Diagn Microbiol Infect Dis 48:167–72.[CrossRef][ISI][Medline]
12 Recchia GD and Hall RM. (1995) Gene cassettes: a new class of mobile element. Microbiology 141:3015–27.[ISI][Medline]
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