Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

ABSTRACT The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) β-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of β-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 β-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 β-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low Km values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type β-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

[1]  J. Campos,et al.  Emergence of imipenem resistance in clinical Escherichia coli during therapy. , 2008, International journal of antimicrobial agents.

[2]  M. Galleni,et al.  Role of the Ser-287-Asn Replacement in the Hydrolysis Spectrum Extension of AmpC β-Lactamases in Escherichia coli , 2008, Antimicrobial Agents and Chemotherapy.

[3]  P. Nordmann,et al.  Contribution of extended-spectrum AmpC (ESAC) beta-lactamases to carbapenem resistance in Escherichia coli. , 2008, FEMS microbiology letters.

[4]  M. Galleni,et al.  Structure of the plasmid-mediated class C beta-lactamase ACT-1. , 2008, Acta crystallographica. Section F, Structural biology and crystallization communications.

[5]  P. Nordmann,et al.  Extension of the hydrolysis spectrum of AmpC β-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix—authors' response , 2008 .

[6]  P. Nordmann,et al.  Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered in a French hospital. , 2008, The Journal of antimicrobial chemotherapy.

[7]  P. Nordmann,et al.  Extension of the hydrolysis spectrum of AmpC beta-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. , 2007, The Journal of antimicrobial chemotherapy.

[8]  N. Woodford,et al.  Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 beta-lactamases co-mediated by porin loss. , 2007, International journal of antimicrobial agents.

[9]  Jung Hun Lee,et al.  Structural basis for the extended substrate spectrum of CMY‐10, a plasmid‐encoded class C β‐lactamase , 2006, Molecular microbiology.

[10]  J. Frère,et al.  Kinetic Properties of Four Plasmid-Mediated AmpC β-Lactamases , 2005, Antimicrobial Agents and Chemotherapy.

[11]  N. Hanson,et al.  Promoter Sequences Necessary for High-Level Expression of the Plasmid-Associated ampC β-Lactamase Gene blaMIR-1 , 2004, Antimicrobial Agents and Chemotherapy.

[12]  P. Nordmann,et al.  In Vivo Acquisition of High-Level Resistance to Imipenem in Escherichia coli , 2004, Journal of Clinical Microbiology.

[13]  R. Labia,et al.  Sequence analysis and biochemical characterisation of chromosomal CAV-1 (Aeromonas caviae), the parental cephalosporinase of plasmid-mediated AmpC 'FOX' cluster. , 2003, FEMS microbiology letters.

[14]  B. Shoichet,et al.  Structural Basis for Imipenem Inhibition of Class C β-Lactamases , 2002, Antimicrobial Agents and Chemotherapy.

[15]  Brian K Shoichet,et al.  Structure-based approach for binding site identification on AmpC beta-lactamase. , 2002, Journal of medicinal chemistry.

[16]  C. Bizet,et al.  Chromosomal ampC genes in Enterobacter species other than Enterobacter cloacae, and ancestral association of the ACT-1 plasmid-encoded cephalosporinase to Enterobacter asburiae. , 2002, FEMS microbiology letters.

[17]  George A. Jacoby,et al.  Plasmid-Determined AmpC-Type β-Lactamases , 2002, Antimicrobial Agents and Chemotherapy.

[18]  B K Shoichet,et al.  Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design. , 2001, Biochemistry.

[19]  P. Courvalin,et al.  Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. , 2000, The Journal of antimicrobial chemotherapy.

[20]  P. Nordmann,et al.  Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in Chryseobacterium meningosepticum , 2000, Antimicrobial Agents and Chemotherapy.

[21]  G. French,et al.  Carbapenem Resistance in Escherichia coli Associated with Plasmid-Determined CMY-4 β-Lactamase Production and Loss of an Outer Membrane Protein , 1999, Antimicrobial Agents and Chemotherapy.

[22]  P. Nordmann,et al.  Cloning, Sequence Analyses, Expression, and Distribution of ampC-ampR from Morganella morganii Clinical Isolates , 1999, Antimicrobial Agents and Chemotherapy.

[23]  J. Frère,et al.  When drug inactivation renders the target irrelevant to antibiotic resistance: a case story with β‐lactams , 1999, Molecular microbiology.

[24]  P. Lagrange,et al.  Characterisation of CMY-4, an AmpC-type plasmid-mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis. , 1998, FEMS microbiology letters.

[25]  K. Bush,et al.  Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein , 1997, Antimicrobial agents and chemotherapy.

[26]  M C Peitsch,et al.  ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. , 1996, Biochemical Society transactions.

[27]  Manuel C. Peitsch,et al.  Protein Modeling by E-mail , 1995, Bio/Technology.

[28]  F. Baquero,et al.  Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated beta-lactamase with two molecular variants , 1994, Antimicrobial Agents and Chemotherapy.

[29]  J. Frère,et al.  Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[30]  U. K. Laemmli,et al.  Maturation of the head of bacteriophage T4. I. DNA packaging events. , 1973, Journal of molecular biology.

[31]  U. K. Laemmli,et al.  Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles. , 1973, Journal of molecular biology.

[32]  Kristiina Takkinen,et al.  Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. , 2008 .

[33]  M. Ferraro.  Performance standards for antimicrobial susceptibility testing , 2001 .

[34]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[35]  J. R. Paulson,et al.  Maturation of the head of bacteriophage T4. V. A possible DNA packaging mechanism: in vitro cleavage of the head proteins and the structure of the core of the polyhead. , 1974, Journal of supramolecular structure.