S. BONTRON¹, L. POIREL¹, G. KUEFFER¹, E. SAVOV³, P. NORDMANN<sup>1,2

1</sup>Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Department of Medicine, Faculty of Science, University of Fribourg, Switzerland, ²Hôpital Cantonal HFR, Fribourg, Switzerland. , ³Laboratory of Microbiology, Military Medical Academy, Sofia, Bulgaria

A series of 26 Proteus mirabilis clonally-related clinical isolates recovered during an outbreak in a Bulgarian hospital showed heterogeneous resistance levels to carbapenems. All isolates produced the VIM-1 enzyme known to hydrolyze efficiently carbapenems. MICs of the carbapenem meropenem ranged from 0.25- to >32-mg/L. Resistance levels to carbapenems correlated with the blaVIM-1 copy number, present at 1, 2, or 4 copies per bacterial cell. In all cases, the blaVIM-1 gene was chromosomal, and laid within a class 1 integron, embedded in a transposon delimited by two IS26 elements. In isolates with 2 or 4 blaVIM-1 copies, the transposon was duplicated in tandem, the genetic structure being consistent with non-equal homologous recombination between directly orientated repeats. In-vitro, clones with higher resistance levels to β-lactams, displaying amplification of the blavim-1 gene from 1 copy to 10 to 70 copies, were selected with high concentrations of ceftazidime, a substrate of the VIM-1 carbapenemase. The blaVIM-1 bearing transposon was found in tandem in some but not all clones, suggesting at least two distinct mechanisms of gene amplification. Conversely, exposure to meropenem selected clones with a marked increased resistance to this antibiotic, never in association with amplification of the blavim-1 gene, but rather with permeability defects. This work showed that within a given epidemic, bacterial isolates may evolve towards an increased resistance to carbapenems by amplification of the carbapenemase gene. In addition, it showed that gene amplification can be induced in vitro, preferentially upon exposure to specific antibiotics.