Molecular and biochemical characterization of Ambler class A extended-spectrum beta-lactamase CGA-1 from Chryseobacterium gleum

Abstract

Antibiotic susceptibility testing by disk diffusion of a Chryseobacterium gleum isolate, strain CIP 103039, showed a typical synergy image between clavulanic acid and expanded-spectrum cephalosporins. Shotgun cloning gave a recombinant plasmid in Escherichia coli that produced a beta-lactamase, CGA-1, with a pI value of 8.9 that conferred resistance to most penicillins (except ureidopenicillins) and narrow-spectrum cephalosporins and an intermediate susceptibility to expanded-spectrum cephalosporins and aztreonam. The CGA-1 amino acid sequence shared only 60% amino acid identity with CME-1 and CME-2 from Chryseobacterium meningosepticum, the most closely related beta-lactamases. CGA-1 was very likely chromosome encoded. It is a novel member of the PER subgroup of Ambler class A beta-lactamases (Bush functional group 2be).

FIG. 1.

Amino acid sequence comparison of 12 representative class A ESBLs. The amino acid residues highlighted in grey are specific to the β-lactamases of the PER subgroup suggested by Tranier et al. (21), while those of the Ω-loop sequence are boxed. Asterisks indicate the conserved motifs SXXK, SDN, and KTG of Ambler class A β-lactamases (1). Dashes indicate gaps introduced to optimize the alignment. The putative cleavage site for the leader peptide of CGA-1 is indicated by a vertical arrow.

FIG. 2.

Dendrogram obtained for 18 Ambler class A ESBLs by the parsimony method (20). Branch lengths are drawn to scale and are proportional to the number of amino acid changes. The percentages at the branching points (underlined) refer to the number of times that a particular node was found in 100 bootstrap replications. The distance along the vertical axis has no significance. The percentages in parentheses are amino acid identities to CGA-1.