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Antimicrobial Agents and Chemotherapy, May 2005, p. 1973-1980, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1973-1980.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

OXA-46, a New Class D ß-Lactamase of Narrow Substrate Specificity Encoded by a blaVIM-1-Containing Integron from a Pseudomonas aeruginosa Clinical Isolate

Francesco Giuliani,1 Jean-Denis Docquier,1,2 Maria Letizia Riccio,1 Laura Pagani,3 and Gian Maria Rossolini1*

Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100 Siena,1 Dipartimento di Scienze Morfologiche, Eidologiche e Cliniche, Sezione di Microbiologia, Università di Pavia, I-27100 Pavia, Italy,3 Centre d'Ingénierie des Protéines & Laboratoire d'Enzymologie, Université de Liège, B-4000 Liège, Belgium2

Received 19 August 2004/ Returned for modification 31 October 2004/ Accepted 19 January 2005

A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a blaVIM-1 metallo-ß-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D ß-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of blaOXA-46 in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D ß-lactamases.

* Corresponding author. Mailing address: Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, Policlinico Santa Maria alle Scotte, 53100 Siena, Italy. Phone: 39 0577 233455. Fax: 39 0577 233334. E-mail: rossolini{at}unisi.it.

Antimicrobial Agents and Chemotherapy, May 2005, p. 1973-1980, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1973-1980.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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