Association between the vanB2 glycopeptide resistance operon and Tn1549 in enterococci from France

A. Umeda*, F. Garnier§, P. Courvalin and M. Galimand

Unité des Agents Antibactériens, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France

Received 22 January 2002; returned 17 April 2002; revised 24 April 2002; accepted 30 April 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Linkage between the vanB2 gene cluster and transposon Tn1549 was found in clonally unrelated VanB-type vancomycin-resistant Enterococcus spp. strains isolated in France. The transposon was chromosomally located or plasmid borne.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The genus Enterococcus includes at least 19 species. Enterococcus faecalis and Enterococcus faecium are the most common species causing human disease, and represent c. 90% of clinical enterococcal isolates. Enterococci are naturally resistant to low levels of aminoglycosides, relatively resistant to the penicillins and can also become resistant to other drug classes by acquisition of foreign DNA. Acquired resistance to vancomycin is most often due to the presence of the vanA and vanB operons.1 Based on sequence differences within the vanB gene and the vanSBvanYB intergenic region, the vanB operon has been divided into three subtypes, vanB1, vanB2 and vanB3.2 Dissemination of the vanB gene cluster appears to result from the transfer of large genetic elements integrated into the host chromosome,1,3 from plasmid transfer1,4 or from vertical inheritance.1 Conjugative transposon Tn5382 containing the vanB2 gene cluster has been partially sequenced.3 Intercellular transfer of Tn5382, as an integral part of larger chromosomal elements also conferring ampicillin resistance, has been documented.2,3 Vancomycin resistance in unrelated clinical E. faecalis and E. faecium isolates has been shown to be associated with the presence of the vanB gene cluster positioned on conjugative plasmids.4,5 In these plasmids, the vanB operon is carried by the 34 kb transposon Tn1549, which has been entirely sequenced.5 Sequence comparison indicated that Tn1549 and Tn5382 are nearly identical at both ends, and that their right extremity exhibits significant identity with the genes coding for the excisase and the integrase of Tn1545 and related conjugative transposons.3,5 For the sake of simplicity, the Tn5382–Tn1549 family of elements will be collectively designated Tn1549.

The present work was undertaken to assess the contribution of Tn1549 to VanB-type glycopeptide resistance in French isolates of enterococci and to study the genetic localization of the transposon.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains, culture conditions and antibiotic susceptibility tests

Eighteen VanB-type enterococci isolated from humans in France and representing a wide range of geographical sources were studied (Table 1). E. faecalis 268-104 harbouring Tn1549 that carries the vanB2 gene,5 and E. faecalis V583 containing the vanB1 subtype,1 were used as controls. All strains were grown at 37°C in brain–heart infusion broth and agar (Difco, Detroit, MI, USA). MICs were determined by dilution in Mueller–Hinton agar (Bio-Rad, Marnes-la-Coquette, France) with ampicillin at concentrations ranging from 0.5 to 64 mg/L, teicoplanin from 0.125 to 4 mg/L and vancomycin from 0.25 to 128 mg/L.


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Table 1.  VanB-type Enterococcus strains studied
 
PCR amplification

Amplification of DNA was performed in a 2400 thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) with Taq DNA polymerase (Amersham, Little Chalfont, UK) as recommended by the manufacturer. PCR elongation times and temperatures were adjusted according to the expected size of the PCR product and to the nucleotide sequence of the primers, respectively. PCR primer positions and sequences are listed in Table 2.


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Table 2.  PCR primers used in this study
 
Pulsed-field gel electrophoresis and DNA hybridization

Total DNA from enterococci digested with SmaI or I-CeuI (New England Biolabs, Beverly, MA, USA), an intron-encoded endonuclease specific for rRNA genes,8 was fractionated by 0.8% agarose gel electrophoresis in 0.5x Tris–borate buffer with a contour-clamped homogeneous electric field gel electrophoresis apparatus (CHEF-DRIII system; Bio-Rad, Hercules, CA, USA). The DNA fragments were transferred to a nitrocellulose sheet (Nytran; Schleicher & Schuell, Dassel, Germany) using the technique of Southern and hybridized sequentially under stringent conditions with 32P-labelled (Amersham Radiochemical Centre, Amersham, UK) specific probes (Table 2).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Characterization of the VanB-resistant enterococci

All strains expressed resistance to vancomycin (MIC >= 32 mg/L) and susceptibility to teicoplanin (MIC <= 1 mg/L) (Table 1). Detection of glycopeptide resistance genotypes and identification to the species level of vancomycin-resistant enterococci were determined by multiplex PCR.6 The VanB-type isolates (Table 1) were identified as E. faecium (14 strains) or E. faecalis (4 strains) and were associated with the presence of a 635 bp PCR product, as expected for the vanB genotype (data not shown). A 593 bp PCR product corresponding to the vanSBvanYB intergenic region was digested by TaqI into two fragments (237 and 355 bp), which indicated that all the strains were of the vanB2 subtype; the vanB1 and vanB3 subtypes2 do not possess recognition sites for this enzyme (data not shown).

The four E. faecalis strains were isolated in the same hospital between 1996 and 1998. Pulsed-field gel electrophoresis analysis, after DNA digestion with SmaI, revealed that three of the isolates might be clonally related, two being indistinguishable (data not shown). A similar analysis indicated that 10 of the 14 E. faecium were clonally unrelated. Two of the three isolates from Villejuif (BM4460 and BM4462) were indistinguishable, and the two isolates from the same hospital in Paris in 1999 (BM4471 and BM4472) might be clonally related (data not shown).

Association of vanB2 gene cluster with Tn1549

The VanB-type strain collection was analysed by specific amplification of the left end of Tn1549 (Table 2, orf 14–15) and by Southern hybridization using Tn1549 and vanB amplicons as probes (Table 2). A PCR product of the left end of Tn1549 with the expected size of 264 bp was obtained with the DNA from all the strains (data not shown). Co-hybridization of the vanB and Tn1549 probes to the same SmaI fragments was observed for all strains (data not shown). Strain V583 of the vanB1 subtype was negative for Tn1549 PCR and hybridization (data not shown). These data indicated that the vanB2 gene cluster was associated with Tn1549.

Location of vanB2-containing Tn1549

Recent reports have shown that the putative Tn5382 conjugative transposon can be located downstream from a pbp5 gene encoding ampicillin resistance in distinct E. faecium from the USA3,9 and Europe.2 In contrast, in two enterococci, Tn1549 has been shown to be inserted into the pheromone conjugative plasmid pAD1.5 The vanB2-containing E. faecium were examined for pbp5–Tn1549/5382 linkage by amplification of a 1079 bp region between pbp5 and the left end of the transposon (Table 2). DNA from six E. faecium exhibiting various levels of resistance to ampicillin gave rise to a pbp5–Tn1549 amplicon of the expected size (Table 1). We did not detect the additional 2 kb fragment between pbp5 and the left end of the transposon as described for the Scottish E. faecium isolates.10

The I-CeuI-digested DNA from the 18 strains was successively hybridized with 16S rRNA (rrs) and vanB probes (Table 2). The rrs probe hybridized with all I-CeuI-generated fragments from all strains. In contrast, the vanB probe hybridized with a single I-CeuI fragment resolved in the gel for 12 strains and produced a strong signal with the DNA that remained in the well, but did not hybridize with the fragments resolved in the gel for the remaining six strains (Table 1; data not shown). These observations indicate that Tn1549 was inserted in the chromosome of the first 12 E. faecium isolates and was plasmid borne in the six other isolates, including the three related E. faecalis. Moreover, a positive signal with a probe specific to pAD1 (Table 2) was detected only with DNA from E. faecalis BM4465, BM4466 and BM4467 (Table 1; data not shown).

VanB-type resistance among enterococci in France is predominantly associated with the vanB2 gene cluster and appears to have spread by horizontal dissemination of the vanB2 operon in association with Tn1549-like elements.


    Acknowledgements
 
We thank P. Y. Allouch, J. L. Avril and A. Bouvet for their gifts of strains. This work was supported in part by a Bristol-Myers Squibb Unrestricted Biomedical Research Grant in Infectious Diseases and by the Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires.


    Footnotes
 
* Present address. Faculty of Health Sciences, School of Medicine, Yamaguchi University, Ube, Japan. Back

§ Present address. Laboratoire de Bactériologie, Hôpital Dupeytren, Limoges, France. Back

Corresponding author. Tel: +33-1-45-68-83-18; Fax: +33-1-45-68-83-19; E-mail: galimand{at}pasteur.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Arthur, M., Reynolds, P. & Courvalin, P. (1996). Glycopeptide resistance in enterococci. Trends in Microbiology 4, 401–7.[ISI][Medline]

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