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

Laurent Poirel,Thierry Naas,El Bachir Chaibi,Michele Guibert,Roger Labia,Patrice Nordmann

doi:10.1128/aac.43.3.573

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.

Highlights

Naturally susceptible to extended-spectrum cephalosporins, Escherichia coli strains may become resistant to these ␤-lactams by several mechanisms, mainly alterations in outer membrane proteins, overproduction of cephalosporinase, or production of an extended-spectrum ␤-lactamase (ESBL)
In addition to these ESBLs, non-SHV non-TEM derivative enzymes have been detected in E. coli: FEC-1 [27], CTX-M1 (MEN-1) [5, 6], CTX-M2 [6], PER-2 [7], and TOHO-1 [19]
Preliminary hybridization experiments indicated that E. coli MG-1 harbored a TEM-derived resistance gene as indicated by a positive hybridization signal detected by a blaTEM- probe. blaSHV-3, oxa18, and blaPER-1 probes failed to give positive hybridization signals

Summary

Introduction

Naturally susceptible to extended-spectrum cephalosporins, Escherichia coli strains may become resistant to these ␤-lactams by several mechanisms, mainly alterations in outer membrane proteins, overproduction of cephalosporinase (chromosomal or plasmid mediated), or production of an extended-spectrum ␤-lactamase (ESBL). Most of the ESBLs found so far in Enterobacteriaceae are Ambler class A ␤-lactamases They are plasmid encoded, and the enzymes most commonly observed in E. coli are TEM derivatives and to a lesser extent SHV derivatives [28]. The extension of their hydrolysis properties results from single amino acid changes within their catalytic sites. The integrons most commonly isolated from antibiotic-resistant clinical isolates from members of the family Enterobacteriaceae and Pseudomonads belong to class 1 These class 1 integrons possess two conserved regions located on either side of the integrated gene cassettes. Resistance phenotype: ESBL, Kan, Tm, AN, Net, Tet, Cm, Sul Natural plasmid from E. coli MG-1 containing blaTEM-1. Resistance phenotype: ESBL, Kan, Tm, AN, Net, Tet, Cm, Sul Recombinant plasmid containing 1.4-kb genomic Sau3A fragment containing blaTEM-1

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Conclusion