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Antimicrobial Agents and Chemotherapy, April 2007, p. 1304-1309, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01058-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evolution of TEM-Type Enzymes: Biochemical and Genetic Characterization of Two New Complex Mutant TEM Enzymes, TEM-151 and TEM-152, from a Single Patient{triangledown} ,{dagger}

Frédéric Robin,1,2* Julien Delmas,1,2 Cédric Schweitzer,1,2 Olivier Tournilhac,3 Olivier Lesens,4 Catherine Chanal,1,2 and Richard Bonnet1,2

CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Bactériologie Clinique, Clermont-Ferrand F-63003, France,1 Université Clermont 1, UFR Médecine, Laboratoire de Bactériologie, EA3844, Clermont-Ferrand F-63001, France,2 CHU Clermont-Ferrand, Hôtel-Dieu, Service d'Hématologie Clinique, Clermont-Ferrand F-63003, France,3 CHU Clermont-Ferrand, Hôtel-Dieu, Service de Maladies Infectieuses et Tropicales, Clermont-Ferrand F-63003, France4

Received 22 August 2006/ Returned for modification 26 October 2006/ Accepted 2 January 2007

Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel blaTEM genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel ß-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (kcat, 5 and 16 s–1, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 µM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.

* Corresponding author. Mailing address: CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Bactériologie Clinique, Clermont-Ferrand F-63003, France. Phone: 33-473178150. Fax: 33-473277494. E-mail: frobin{at}chu-clermontferrand.fr

{triangledown} Published ahead of print on 12 January 2007.

{dagger} This work is dedicated to the memory of Catherine Chanal.

Antimicrobial Agents and Chemotherapy, April 2007, p. 1304-1309, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01058-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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