Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC beta-lactamase in Acinetobacter baumannii

Abstract

A clinical strain of Acinetobacter baumannii (strain Ab RYC 52763/97) that was isolated during an outbreak in our hospital and that was resistant to all beta-lactam antibiotics tested produced three beta-lactamases: a TEM-1-type (pI, 5.4) plasmid-mediated beta-lactamase, a chromosomally mediated OXA-derived (pI, 9.0) beta-lactamase, and a presumptive chromosomal cephalosporinase (pI, 9.4). The nucleotide sequence of the chromosomal cephalosporinase gene shows for the first time the gene encoding an AmpC beta-lactamase in A. baumannii. In addition, we report here the biochemical properties of this A. baumannii AmpC beta-lactamase.

FIG. 1

Nucleotide sequence of the ampC gene. The deduced amino acid sequence of AmpC β-lactamase is shown below the nucleotide triplets. The boldface ATG and TAA represent the initiation and termination codons, respectively. A putative Shine-Dalgarno (S.D.) ribosomal recognition site (underlined) is indicated. The positions of the primers used to sequence the gene are indicated by underlining with arrowheads. The β-lactamase active site S-V-S-K, the conserved triad K-T-G, and the class C typical motif Y-X-N are presented in boldface. A putative terminator sequence is underlined.

FIG. 2

Detection of the ampC gene in different A. baumannii isolates. The amplified products were resolved by 1.5% agarose gel electrophoresis and stained with ethidium bromide (50 μg/ml). Lanes 1 and 11, DNA molecular weight markers III and V, respectively (Boehringer-Mannheim); lane 2, negative control (genomic DNA of Listeria monocytogenes amplified with primers P1 and P2); lanes 3 to 8, A. baumannii strains (Ab 1 to 6); lane 9, A. junii strain; lane 10, positive control (strain Ab RYC 52763/97).