Characterization of the Metallo-β-Lactamase Determinant of Acinetobacter baumannii AC-54/97 Reveals the Existence of bla IMP Allelic Variants Carried by Gene Cassettes of Different Phylogeny

The metallo-β-lactamase determinant ofAcinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (namedbla IMP-2 ) ofbla IMP found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar tobla IMP ,bla IMP-2 was also carried by an integron-borne gene cassette. However, the 59-base element of thebla IMP-2 cassette was unrelated to those of thebla IMP cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-bornebla IMP-2 gene inEscherichia coli resulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from anEscherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several β-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some β-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir ofbla IMP alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.