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Antimicrobial Agents and Chemotherapy, October 2008, p. 3725-3736, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00163-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic and Structural Insights into the Dissemination Potential of the Extremely Broad-Spectrum Class A β-Lactamase KPC-2 Identified in an Escherichia coli Strain and an Enterobacter cloacae Strain Isolated from the Same Patient in France{triangledown}

Stephanie Petrella,1 Nathalie Ziental-Gelus,2 Claudine Mayer,2 Murielle Renard,3 Vincent Jarlier,1,3 and Wladimir Sougakoff2,3*

UPMC Univ Paris 06, EA1541, Bacteriologie-Hygiène,1 Centre de Recherche des Cordeliers, LRMA, INSERM, UMRS 872-12,2 AP-HP, Hôpital Pitié-Salpêtrière, Bactériologie-Hygiène, F-75013 Paris, France3

Received 5 February 2008/ Returned for modification 13 April 2008/ Accepted 4 July 2008

Two clinical strains of Escherichia coli (2138) and Enterobacter cloacae (7506) isolated from the same patient in France and showing resistance to extended-spectrum cephalosporins and low susceptibility to imipenem were investigated. Both strains harbored the plasmid-contained blaTEM-1 and blaKPC-2 genes. blaKLUC-2, encoding a mutant of the chromosomal β-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of blaKLUC from the chromosome. Determination of the KPC-2 structure at 1.6 Å revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 β-lactamase by the inhibitory protein BLIP. Determination of the 1.23-Å structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6{alpha}-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand β3.

* Corresponding author. Mailing address: LRMA, UMRS 872-12, Laboratoire de Recherche Moléculaire sur les Antibiotiques, Faculté de médecine Pitié-Salpêtrière, 91 boulevard de l'hôpital, F-75634 Paris Cedex 13, France. Phone: 33 (1) 40 77 97 46. Fax: 33 (1) 45 82 75 77. E-mail: sougakof{at}chups.jussieu.fr

{triangledown} Published ahead of print on 14 July 2008.

Antimicrobial Agents and Chemotherapy, October 2008, p. 3725-3736, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00163-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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