IBC-1, a novel integron-associated class A beta-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain

Abstract

A transferable beta-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. The bla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including aac(6')-Ib. The encoded beta-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with beta-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica, a carbenicillinase from Proteus mirabilis GN79, the species-specific beta-lactamases of Klebsiella oxytoca, and the carbapenemase Sme-1. However, a phylogenetic association with established beta-lactamase clusters could not be conclusively shown.

FIG. 1

IEF of β-lactamases produced by E. cloacae HT9, E. coli 14R519(pHT9), and E. coli DH5α(pHT9-2) (lanes 1 to 3, respectively). β-Lactamases with known pIs (TEM-1, PSE-2, SHV-1, SHV-5, and LAT-1) are shown in lanes M. Isoelectric points are indicated on the right.

FIG. 2

Schematic presentation of the IBC-1-encoding integron. Part of the variable region and the 3′ conserved sequence of the integron (dashed lines) were postulated from the results of the PCR assays.

FIG. 3

Nucleotide sequence of the IBC-1 gene cassette. The boundaries of the cassette are indicated with vertical arrows. The putative ribosome binding site (RBS) and the start and stop codons of the bla_IBC-1 are underlined. The core sites for recombination are double underlined. The deduced amino acid sequence of IBC-1 is also shown. The exclamation point indicates the cleavage site of the putative signal peptide.

FIG. 4

Amino acid sequence alignment of IBC-1 and six of the β-lactamases exhibiting the highest similarity scores with it. The sequence of TEM-1 is included for comparison. The dashes indicate gaps introduced to optimize alignment. Identical amino acids are indicated by asterisks. The dots indicate conservative amino acid substitutions corresponding to the exchange groups described in reference . Residues that are strictly conserved in class A β-lactamases are shown in boldface type. The putative Ω loop of IBC-1 is underlined. Other amino acid residues of potential importance are shaded. Ambler's numbering scheme was followed.