Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamaseand its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France

Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year -old-woman treated,vith imipenem in Marseilles, France, in 1996. This strai n was resistant to beta-lactams, including ureidopenicillins, ticarcillin-c lavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remaine d susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing beta- lactamase gene of P. aeruginosa COL I was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B beta-lactamase, with amino acid identities of 32% with E-II from B acillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, in cluding P. aeruginosa; 27%,vith CcrA from Bacteroides fragilis; 24% with Bl aB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacter ium indologenes; 21% with CphA-l from Aeromonas hydrophila; and 11% with L- 1 from Stenotrophomonas maltophilia. It was most closely related to VIM-I b eta-lactamase recently reported from Italian P. aeruginosa clinical isolate s (90% amino acid identity). Purified VIM-2 beta-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis ra nge, including penicillins, cephalosporins, cephamycins, oxacephamycins, an d carbapenems, but not monobactams. As a metallo-beta-lactamase, its activi ty was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 mu M). VIM-2 conferred a resistance pattern to beta-lactams in E. coli D H10B that paralleled its in vitro hydrolytic properties, except for suscept ibility to ureidopenicillins, carbapenems, and cefepime. bla(VIM-2), was lo cated on a ca. 45-kb plasmid that in addition conferred resistance to sulfa mides and that was not self-transmissible either from P. aeruginosa to E. c oli or from E. coli to E. coli. bla(VIM-2), was the only gene cassette loca ted,within the variable region of a novel class I integron, In56, that was weakly related to the bla(VIM-1)-containing integron. VIM-2 is the second c arbapenem-hydrolyzing metalloenzyme characterized from a P. aeruginosa isol ate outside Japan.