Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes

Abstract

Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a beta-lactamase with a pI of > or = 9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this beta-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 microg/ml versus 0.06 microg/ml). The kinetic characteristics of this beta-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K(i) < 40 microM). The amino acid compositions of this novel enzyme and other class C beta-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 beta-lactamase. The coalescence of Acinetobacter ampC beta-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.

FIG. 1.

Nucleotide sequence of the 1,152-bp amplification product of the ADC-7 β-lactamase. The deduced amino acid sequence of the ADC-7 β-lactamase is shown in single letter code below the nucleotide triplets. ATG and TAA represent the initiation and termination codons, respectively. The positions of the primers used to amplify bla_ADC-7 are indicated by arrowheads. We have represented the ADC-7 β-lactamase active site as S-V-S-K in bold, the conserved triad K-T-G in bold, and the class C typical motif Y-X-N in bold. The predicted signal peptide cleavage site is between amino acids 23 and 24 (www.expasy.org). The GenBank accession numbers for each of the comparison cephalosporinase genes found in Acinetobacter spp. and O. urethralis are listed.

FIG. 2.

Phylogenetic analysis of the ADC-7 β-lactamase. This phylogram, inferred through Bayesian analysis, represents an estimate of the relationships that exist among the class C β-lactamases and their homologs. Branch lengths are representative of the number of nucleotide mutations that have occurred since the divergence of the genes represented in this tree. Posterior probabilities for groupings that occurred in less than 80% of the trees sampled are indicated by a circle that contains the percentage of time during which that grouping did occur. Plasmidic resistance genes are indicated in boldface italics. A single asterisk represents a known penicillin binding protein, and a double asterisk represents putative penicillin binding proteins. The phylogram was based upon sequence data generated from AmpC β-lactamases in the supplemental material.