Molecular characterization of a beta-lactamase gene, blaGIM-1, encoding a new subclass of metallo-beta-lactamase - PubMed (original) (raw)

Molecular characterization of a beta-lactamase gene, blaGIM-1, encoding a new subclass of metallo-beta-lactamase

Mariana Castanheira et al. Antimicrob Agents Chemother. 2004 Dec.

Abstract

As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant Pseudomonas aeruginosa clinical isolates were detected with metallo-beta-lactamase (MbetaL) activity. The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany. The resistant determinant was isolated amplifying the region between the integrase and the aacA4 gene cassette. Sequencing revealed a novel MbetaL gene, designated bla(GIM-1). Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively. The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MbetaL family. Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-beta-lactamase inhibitors. bla(GIM-1) was found on a 22-kb nontransferable plasmid. The new MbetaL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an aacA4 cassette downstream of the MbetaL gene that appeared to be truncated with bla(GIM-1). The aacA4 was followed by an aadA1 gene cassette that was interrupted by a copy of the IS1394. This integron also carried an oxacillinase gene, bla(OXA-2), before the 3'-CS region. GIM-1 appears to be a unique MbetaL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MbetaL enzymes to be characterized.

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Figures

FIG. 1.

FIG. 1.

Phylogenetic tree obtained for most of the mobile MβLs. The alignment used was prepared with CLUSTALW.

FIG. 2.

FIG. 2.

Alignment of the amino acid sequence of GIM-1 with three representatives of the MβL groups IMP-1, VIM-1, and SPM-1. Differences in the amino acid sequences are noted by a single letter representing the amino acid change within a particular sequence. Residues involved in the coordination of the zinc iron are underlined and numbered according to the BBL system (in italics).

FIG. 3.

FIG. 3.

(a) Agarose gel electrophoresis. Lanes: M, 1-kb plus ladder; P, probe; TD, genomic DNA; p5671, plasmid from strain 73-5671. (b) Results of Southern blot analysis showing the hybridization of intl1F/GIMR marked probe with the probe fragment and the 22-kb plasmid from the index isolate harboring _bla_GIM-1.

FIG. 4.

FIG. 4.

Schematic representation of integron ln77 carrying bla_GIM-1 (the arrows in the gene boxes indicating the direction of transcription). The black dots indicate 59-bes. In the third cassette position the aadA1 is interrupted by a copy of the IS_1394. Block arrows beneath the gene map indicate the positions of the primers used for PCRs and sequence analyses.

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