Genetic and biochemical characterization of the first extended-spectrum CARB-type beta-lactamase, RTG-4, from Acinetobacter baumannii

Abstract

Acinetobacter baumannii isolate KAR was uncommonly more resistant to cefepime and cefpirome than to ceftazidime and cefotaxime. Cloning and expression of the beta-lactamase gene content of this isolate into Escherichia coli TOP10 identified ss-lactamase RTG-4 (or CARB-10), which corresponds to the first reported extended-spectrum CARB-type enzyme. RTG-4 is a plasmid-encoded Ambler class A beta-lactamase whose sequence differs by 4 amino acid substitutions from the narrow-spectrum beta-lactamase RTG-3. RTG-4 hydrolyzes cefepime and cefpirome and weakly hydrolyzes ceftazidime due to the single Ser-to-Thr substitution at Ambler position 69. RTG-4 is less susceptible to inhibition by tazobactam and sulbactam than RTG-3. Expression of beta-lactamase RTG-4 in a wild-type A. baumannii reference strain showed that it conferred resistance to cefepime and cefpirome. The genetic environment of the bla(RTG-4) gene was made of a peculiar transposon located on a ca. 50-kb plasmid. ISAba9, located upstream of bla(RTG-4), may be responsible for its acquisition by recognizing a secondary right inverted repeat sequence, thus acting by a one-ended transposition process.

FIG. 1.

Alignment of the amino acid sequences of the RTG-4 and RTG-3 β-lactamases. Dashes represent identical amino acids. Amino acids with gray shading represent conserved residues across multiple β-lactamases, according to Joris et al. (12).

FIG. 2.

Schematic map of transposon Tn2014 harboring the bla_RTG-4 gene. The nucleotide sequences of regions identified upstream of the bla_RTG-4 gene in A. baumannii KAR are indicated. The genes and their corresponding transcriptional orientations are indicated by horizontal arrows. The +1 transcription start site and the −35 and −10 sequences of the promoter are indicated in boldface and are boxed. The IRR of ISAba9 is shaded in gray.