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Antimicrobial Agents and Chemotherapy, March 2008, p. 915-919, Vol. 52, No. 3
0066-4804/08/$08.00+0     doi:10.1128/AAC.01028-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

E240V Substitution Increases Catalytic Efficiency toward Ceftazidime in a New Natural TEM-Type Extended-Spectrum β-Lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens Clinical Isolates{triangledown}

Mariagrazia Perilli,1 Giuseppe Celenza,1 Francesca De Santis,1 Cristina Pellegrini,1 Chiara Forcella,1 Gian Maria Rossolini,2 Stefania Stefani,3 and Gianfranco Amicosante1*

Department of Sciences and Biomedical Technologies, University of L'Aquila, L'Aquila,1 Department of Molecular Biology, University of Siena, Siena,2 Laboratory of Microbiology, University of Catania, Catania, Italy3

Received 6 August 2007/ Returned for modification 8 October 2007/ Accepted 14 December 2007

The aim of this study was to characterize a novel extended-spectrum β-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149T182M, in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The blaTEM-149 and blaTEM-149/T182M genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant Escherichia coli HB101 by the same procedure. Both enzymes hydrolyzed all β-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149T182M enzymes, a reduction of the catalytic efficiency for the TEM-149T182M mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 β-lactamase.

* Corresponding author. Mailing address: Department of Biomedical Sciences and Technologies, University of L'Aquila, Clinical Biochemistry and Molecular Biology, Loc. Coppito, I-67100 L'Aquila, Italy. Phone: 39 0862 433455. Fax: 39 0862 433433. E-mail: amicosante{at}cc.univaq.it

{triangledown} Published ahead of print on 26 December 2007.

Antimicrobial Agents and Chemotherapy, March 2008, p. 915-919, Vol. 52, No. 3
0066-4804/08/$08.00+0     doi:10.1128/AAC.01028-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Falcone, M., Mezzatesta, M. L., Perilli, M., Forcella, C., Giordano, A., Cafiso, V., Amicosante, G., Stefani, S., Venditti, M. (2009). Infections with VIM-1 Metallo-{beta}-Lactamase-Producing Enterobacter cloacae and Their Correlation with Clinical Outcome. J. Clin. Microbiol. 47: 3514-3519 [Abstract] [Full Text]  
  • Celenza, G., Luzi, C., Aschi, M., Segatore, B., Setacci, D., Pellegrini, C., Forcella, C., Amicosante, G., Perilli, M. (2008). Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43. J Antimicrob Chemother 62: 991-997 [Abstract] [Full Text]  


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