IMP-12, a new plasmid-encoded metallo-beta-lactamase from a Pseudomonas putida clinical isolate

Abstract

A Pseudomonas putida strain showing broad-spectrum resistance to beta-lactams, including expanded-spectrum cephalosporins and carbapenems, was isolated from a patient with a urinary tract infection at the University Hospital of Varese in northern Italy. The isolate was found to produce metallo-beta-lactamase activity and to harbor a 50-kb plasmid, named pVA758, carrying a new bla(IMP) determinant, named bla(IMP-12). Plasmid pVA758 was not self-transferable by conjugation to either Escherichia coli or Pseudomonas aeruginosa but could be introduced by electroporation and maintained in the latter host, where it conferred resistance or decreased susceptibility to various beta-lactams. The IMP-12 enzyme is quite divergent from other IMP variants: its closest relatives are IMP-8 and IMP-2 (89 and 88% sequence identity, respectively), and IMP-1 is 85% identical to IMP-12. The bla(IMP-12) determinant is carried on an integron-borne gene cassette whose attC recombination site is related to those present in cassettes containing bla(IMP-1), bla(IMP-6), bla(IMP-7), bla(IMP-10), and bla(IMP-11) and unrelated to that present in cassettes containing bla(IMP-2) and bla(IMP-8). IMP-12 was overproduced in E. coli by using a T7-based expression system and was purified by cation-exchange chromatography followed by gel filtration. Kinetic analysis revealed that, like other IMP variants, IMP-12 exhibits an overall preference for cephalosporins and carbapenems rather than for penicillins and does not hydrolyze temocillin and aztreonam. However, IMP-12 also exhibits some notable functional differences from other IMP variants, including uniformly poor activity toward penicillins (k(cat)/K(m) values, around 10(4) M(-1). s(-1)) and a remarkably high K(m) (around 900 micro M) for imipenem.

FIG. 1.

(A) Agarose gel electrophoresis of a plasmid DNA preparation from P. putida VA-758/00, either undigested (lane U) or digested with EcoRI (lane RI). Identical plasmid profiles (not shown) were observed with β-lactam-resistant transformants obtained by electroporation of the plasmid preparation into P. aeruginosa PAO1. (B) Results of Southern blot analysis of the digested plasmid preparation by using a bla_IMP-1/2 probe mix. DNA size standards (in kilobases) are shown on the left.

FIG. 2.

Sequence alignment of the attC recombination sites of bla_IMP-containing gene cassettes. Descriptions and sources of the sequences are as follows: IMP-1, attC site of the bla_IMP-1-containing cassette of integron In31 from P. aeruginosa 101/1477 (20); IMP-1/6/10, attC site of the bla_IMP-1-containing cassette from Serratia marcescens AK9373 (1), which is identical to that of the bla_IMP-6-containing cassette from S. marcescens KU3838 (37) and to those of the bla_IMP-10-containing cassettes from P. aeruginosa PAI97 and Alcaligenes xylosoxidans AXI2 (13); IMP-11, attC site of the bla_IMP-11-containing cassette from P. aeruginosa PAI112 (EMBL/GenBank accession no. AB074437); IMP-5, attC site of the bla_IMP-5-containing cassette from Acinetobacter baumannii 65FFC (4); IMP-7, attC site of the bla_IMP-7-containing cassette from P. aeruginosa 98/P/6327 (8); IMP-12, attC site of the bla_IMP-12-containing cassette from P. putida VA-758/00 (this report); IMP-2/8, attC site of the bla_IMP-2-containing cassette of In42 from A. baumannii AC-54/97 (27), which is identical to that of the bla_IMP-8-containing cassette from Klebsiella pneumoniae KPO787 (36). The inverse core site is boxed; the positions of the 2L and 2R core sites (32) are indicated by arrows. Conserved residues in the first group of sequences are shaded; in the IMP-2/8 sequence, residues identical to those conserved within the first group are shaded.

FIG. 3.

Amino acid alignment of the sequence of the IMP-12 protein with those of other IMP-type enzymes. Stars indicate residues involved in the coordination of zinc ions. Substitutions observed in other variants, compared to IMP-1, are shown on a solid background. Secondary-structure elements (H, helices; S, strands) of IMP-1 (3) are also indicated below the sequences. Numbering is according to the BBL scheme (7). References for the various sequences are as follows: references (IMP-1), (IMP-2), (IMP-3), (IMP-4), (IMP-5), (IMP-6), (IMP-7), (IMP-8), and (IMP-10); EMBL/GenBank accession no. AY033653 (IMP-9) and AB074437 (IMP-11); and this study (IMP-12).