Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate

Abstract

Production of a metallo-beta-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-beta-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B beta-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the beta-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to blaIMP, blaVIM was also found to be carried on a gene cassette inserted into a class 1 integron. The blaVIM-containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo-beta-lactamase-encoding determinant was not transferable to E. coli by conjugation. Expression of the integron-borne blaVIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.

FIG. 1

Results of IEF analysis of crude extracts of P. aeruginosa VR-143/97 (lane 1), E. coli DH5α(pACYC184) (lane 2), E. coli DH5α(pAC2AL) (lane 3), and E. coli DH5α(pAC2IL) (lane 4). Approximately 10 μg of total protein was loaded in each lane.

FIG. 2

Physical map of the insert of plasmid pAC2AL and subcloning strategy. Thick lines represent cloned DNA, and thin lines correspond to vector sequences. Plasmids whose names begins with pAC are pACYC184 derivatives, while those whose name begins with pBC are pBC-SK derivatives. Production of metallo-β-lactamase activity (β-lact.) was assayed with crude extracts as described in Materials and Methods. B, BamHI; C, ClaI; H, HindIII; Sa, SalI; Sm, SmaI; S/B, Sau3AI/BamHI junction; V, EcoRV; X, XhoI.

FIG. 3

Nucleotide sequence of the bla_VIM gene cassette and flanking regions. Initiation codons of the various ORFs are indicated, and protein translation is reported below the sequence. The bla_VIM cassette boundaries are indicated by vertical arrows. The conserved recombination core sites located at the cassette boundaries and the inverse core site are boxed. The internal 2L and 2R core sites of the 59-base element (36) are overlined with arrows.

FIG. 4

Comparison of the VIM-1 sequence (VIM-1; boldfaced) with those of other molecular class B β-lactamases. Bc-II, β-lactamase II of B. cereus 569/H (13); BlaB, BlaB enzyme of C. meningosepticum CCUG4310 (32); IMP-1, IMP-1 enzyme encoded by the bla_IMP gene cassette found in various gram-negative bacteria (2, 18, 25, 41); CcrA, CcrA enzyme of B. fragilis TAL3636 (30); CphA, CphA enzyme of A. hydrophila AE036 (21); L1, L1 enzyme of S. maltophilia IID1275 (38). Identical residues are indicated by an asterisk. Conserved amino acid substitutions are indicated by a colon. Secondary structure elements of Bc-II (5) are also indicated above the sequences. The numbering scheme refers to the Bc-II enzyme.