Cloning and heterospecific expression of the resistance determinant vanA encoding high-level resistance to glycopeptides in Enterococcus faecium BM4147.

Fragments of plasmid pIP816, which confers high-level glycopeptide resistance in Enterococcus faecium BM4147, were cloned into a conjugative gram-negative-gram-positive shuttle vector. The resulting hybrids were transferred by conjugation from Escherichia coli to Enterococcus faecalis and Bacillus thuringiensis. A 4-kilobase EcoRI fragment from pIP816 was found to confer vancomycin resistance in these hosts but not in E. coli or Bacillus subtilis. Images

We previously established that resistance to high levels of vancomycin and teicoplanin in four clinical isolates of Enterococcus faecium was plasmid mediated and inducible by subinhibitory concentrations of glycopeptide antibiotics (10,11). Plasmids conferring glycopeptide resistance in these strains were either nonconjugative (pIP816 in E. faecium BM4147) or self transferable to E. faecium.
In this report, we describe the cloning of the resistance determinant vanA encoding inducible high-level resistance to glycopeptides in E. faecium BM4147 (10). A particular cloning strategy was developed, which enabled us to study the expression of this gene in several bacterial species.
Cloning strategy. Cloning of the vanA resistance determinant in Escherichia coli on the basis of vancomycin selection was not possible, since E. coli, like most gram-negative organisms, displays a natural resistance to glycopeptides which cannot cross the outer membrane. Although E. coli mutants susceptible to these drugs have been obtained under laboratory conditions (18), these strains exhibit a high reversion rate (A. Brisson-Noel, unpublished data) which precludes their use for cloning. It was therefore necessary to use a gram-positive organism as a host for cloning experiments. Direct cloning in gram-positive bacteria implies tedious techniques such as protoplast transformation or elec-* Corresponding author. troporation, which are often of poor efficacy. We therefore developed a two-stage procedure based on the use of the gram-negative-gram-positive shuttle vector pAT187 (20). This plasmid can replicate in both gram-positive and gramnegative organisms, where it confers kanamycin resistance, and can be mobilized from E. coli to various gram-positive bacteria. In order to allow cloning of EcoRI-generated DNA fragments, a pAT187 derivative with a unique EcoRI site was constructed as follows. Plasmid pAT187, which contains two EcoPJ sites, was linearized by partial digestion with EcoRI, the cohesive ends were filled to generate blunt ends, and the plasmid was recircularized by ligation, giving plasmid pAT187-1.
The strategy of cloning is schematically represented in Fig. 1. In the first step, EcoRI restriction fragments of pIP816-1 were cloned into pAT187-1 and introduced by transformation into E. coli 1M83 (13). Multiple enzyme restriction profiles of recombinant plasmids from transformants resistant to kanamycin were analyzed by agarose gel electrophoresis and were compared with those of pIP816-1. Plasmid pAT211 contained a 10-kb insert that could result from a partial EcoRI digestion of pIP816-1. Plasmids pAT212 and pAT213 contained the 6and 4-kb EcoRI portions of the 10-kb insert, respectively (Fig. 2).
In the second step, pAT211, pAT212, and pAT213 were transformed into E. coli JM83 containing the conjugative plasmid pRK24 (4), which can mobilize the shuttle vector pAT187-1 and its derivatives. The resulting transformants were then used as donors in mating experiments (20) with Enterococcusfaecalis JH2-2 (7) and a Bacillus thuringiensis HD1 cry mutant (12). Kanamycin-resistant transconjugants were tested for resistance to glycopeptides. In parallel experiments, the same plasmids were introduced by transformation into Bacillus subtilis BS168 (19)  mants resistant to kanamycin were tested for glycopeptide resistance. The plasmid content of the transconjugants and of the transformants was studied by agarose gel electrophoresis of crude bacterial lysates after digestion with EcoRI endonuclease.
Expression of glycopeptide resistance in E. faecalis JH2-2. E.faecalis JH2-2 containing pAT211 expressed glycopeptide resistance at a high level, similar to that of E. faecium BM4147 (Table 1) (10). In contrast, pAT213 conferred lowlevel resistance and pAT212 did not confer glycopeptide resistance. These results indicate that a determinant conferring glycopeptide resistance is present on the 4-kb fragment. However, additional sequences in the 6-kb EcoRI fragment appear to be required for full expression of the glycopeptide resistance phenotype mediated by pIP816, pIP816-1, and pAT211. Study of the induction of glycopeptide resistance revealed that E. faecalis JH2-2 containing plasmids pAT211 and pAT213 displayed the same pattern of inducibility as did the original E. faecium clinical isolate BM4147 (10; Fig. 3).
Heterospecific expression of the vanA determinant. The results listed in Table 1 show that the glycopeptide resistance encoded by vanA is expressed in E. faecalis JH2-2 and a B. thuringiensis HD1 cry mutant. However, whereas vanA confers in E. faecalis the same level of resistance as it does in the original E. faecium host (10), the expression appeared to be lower in B. thuringiensis. No expression of resistance was found in B. subtilis BS168 or in E. coli 16. To rule out the possibility that lack of expression was due to deletions, plasmids pAT211 and pAT213 were purified from the B. subtilis and E. coli transformants, transformed into E. coli JM83 containing pRK24, and transferred by conjugation into E. faecalis JH2-2. In all cases, vancomycin resistance was fully expressed in E. faecalis JH2-2. Since most enterococcal promoters function in E. coli (21) and in B. subtilis (14),