Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene

Abstract

A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.

FIG. 1

Schematic map of the recombinant plasmids pRLT1, encoding VEB-1, (A) and pRLT-50, encoding TEM-1 (B). The thin line represents the cloned inserts from E. coli MG-1 while the dotted lines indicate the vector pBK-CMV. The open boxes represent the genes, and the arrows indicate their translational orientation. The designations of the gene names are provided in the text. The core sites (GTTRRRY) along with the inverse core sites (RYYYAAC) are indicated along with the composite 59-BEs. The boundaries of veb1 gene cassette are indicated in bold, while the surrounding sequence is in gray.

FIG. 2

Nucleotide sequence of the 1,282-bp fragment of pRLT1 containing the VEB-1 β-lactamase coding region. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. Conserved motifs for class A enzymes are underlined. The start and stop codons of the various genes are shown in boldface and underlined. RBS, putative ribosomal binding site of bla_VEB-1. The core sites (GTTRRRY) are double underlined, while the inverse core sites (RYYYAAC) are underscored with a dashed line. The composite 59-be’s are italicized.

FIG. 3

Alignment of the amino acid sequence of VEB-1 with those of the closely related class A β-lactamases PER-1 (34), PER-2 (7), CBLA (43), CEPA (39), and CFXA (35) and to that of TEM-3. Dashes indicate gaps within the alignment. Standard numbering for class A enzymes according to Ambler (1) is indicated. The Roman numerals below the shaded boxes indicate conserved regions within the class A β-lactamases according to Joris et al. (21). The two facing arrows near the top of the figure indicate the cleavage site of the PER-1 leader peptide (34).

FIG. 4

Dendrogram obtained for 20 class A β-lactamases according to the parsimony method (49). Branch lengths are drawn to scale and are proportional to the number of amino acid changes. The percentages at branching points (underlined) refer to the number of times a particular node was found in 100 bootstrap replications (the stars indicate uncertainty of nodes with bootstrap values of less than 50%). The distance along the vertical axis has no significance. Abbreviations for β-lactamases are given in Materials and Method section. Percentages in parentheses are amino acid identities to VEB-1.