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

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Abstract
The metallo-β-lactamase determinant of Acinetobacter 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) of bla IMPfound in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla 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 in Escherichia coliresulted 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 an Escherichia 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 of bla IMP alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.