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Antimicrobial Agents and Chemotherapy, June 2000, p. 1470-1478, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Biochemical-Genetic Characterization and Regulation of Expression of an ACC-1-Like Chromosome-Borne Cephalosporinase from Hafnia alvei

Delphine Girlich, Thierry Naas, Samuel Bellais, Laurent Poirel, Amal Karim, and Patrice Nordmann*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre cedex, France

Received 14 September 1999/Returned for modification 24 January 2000/Accepted 17 March 2000

A naturally occurring AmpC beta -lactamase (cephalosporinase) gene was cloned from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coli. The deduced AmpC beta -lactamase (ACC-2) had a pI of 8 and a relative molecular mass of 37 kDa and showed 50 and 47% amino acid identity with the chromosome-encoded AmpCs from Serratia marcescens and Providentia stuartii, respectively. It had 94% amino acid identity with the recently described plasmid-borne cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting the chromosomal origin of ACC-1. The hydrolysis constants (kcat and Km) showed that ACC-2 was a peculiar cephalosporinase, since it significantly hydrolyzed cefpirome. Once its gene was cloned and expressed in E. coli (pDEL-1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an uncommon reduced susceptibility to cefpirome. A divergently transcribed ampR gene with an overlapping promoter compared with ampC (blaACC-2) was identified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid identity with the closest AmpR protein from P. stuartii. beta -Lactamase induction experiments showed that the ampC gene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H. alvei 1 cultures that expressed an inducible-cephalosporinase phenotype, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plates, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced cephalosporinase. Transformation of H. alvei 1 DER or E. coli JRG582 (ampDE mutant) harboring ampC and ampR from H. alvei 1 with a recombinant plasmid containing ampD from E. coli resulted in a decrease in the MIC of beta -lactam and recovery of an inducible phenotype for H. alvei 1 DER. Thus, AmpR and AmpD proteins may regulate biosynthesis of the H. alvei cephalosporinase similarly to other enterobacterial cephalosporinases.

* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78, rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45 21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr.

Antimicrobial Agents and Chemotherapy, June 2000, p. 1470-1478, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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