lactamase activity evolution of Aeromonas hydrophila CphA metallo-lactamase by design of a chimeric CphA-VIM1 enzyme
Abstract number: P1507
Perilli M., Di Lisio C., Rainaldi S., Celenza G., Galleni M., Mercuri P.S., Pellegrini C., Forcella C., Bellio P., Amicosante G.
Objectives: Metallo-b-lactamases are zinc enzymes belonging to molecular class B. They are able to hydrolyze b-lactam antibiotics in particular carbapenems. Among subclass B2 b-lactamases, Aeromonas hydrophila CphA enzyme efficiently hydrolyses only carbapenems while VIM-1, belonging to subclass B1, hydrolyses a broad array of b-lactam antibiotics including penicillins and cephalosporins. CphA (254 aa, 25kDa, pI 8.0) and VIM-1 (266 aa, 26kDa, pI 5.2) contain a-b-b-a sandwich structure with one and two zinc ions, respectively, essential to the hydrolysis reaction. The goal of this study was to design and produce a new chimeric enzyme from CphA and VIM-1 in order to improve the catalytic efficiency of CphA against non-carbapenem b-lactam antibiotics.
Methods: The construction of chimeric enzyme was performed by overlapping three different DNA segments obtained from PCR amplification of blaCphA and blaVIM-1 genes. Automatic DNA sequencing was performed on PCR fragments and recombinant plasmid using an automatic sequencer ABI-PRISM 310. blaCphA-VIM-1 gene was generated by a PCR-overlap using blaCphA and blaVIM-1 genes as template. blaCphA-VIM-1 gene was cloned into pBC-SK vector and the recombinant strain was inserted by transformation into E. coli JM109. The determination of MICs was performed by the conventional macrodilution broth procedure as recommended by CLSI.
Results: The CphA-VIM-1 chimera was made using CphA enzyme as scaffold. A domain of 92 amino acid residues, including Asn116, Asn118 and Asp120, was substituted in CphA metallo-b-lactamase by the corresponded domain of 78 amino acid from VIM-1. In vitro susceptibility was tested on E. coli pBC-CphA-VIM-1, E. coli pBC-CphA and E. coli pBC-VIM-1 versus a large pattern of antibiotics. E. coli pBC-CphA-VIM-1 recombinant strain showed an higher MIC value for piperacillin (MIC value, 32 mg/L) with respect to E. coli pBC-CphA (8 mg/L) and E. coli pBC-VIM-1 (8 mg/L). Concerning cephalosporins, MIC values for cefazolin was 64 mg/L for E. coli pBCCphA-VIM and 2 mg/L for E. coli pBC-CphA and E. coli pBC-VIM-1. The molecular weight and pI was evaluated on pure enzyme by SDS-Page and isoelectrofocusing and seem to be 24 kDa and 6.2, respectively.
Conclusion: Starting with CphA enzyme, that hydrolyzes only carbapenems, we have obtained a new chimeric enzyme that evolves versus a much broader spectrum activity.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
|Back to top|