Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate

Abstract

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanXB dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.

FIG. 1

Schematic representation of the right (integrase-encoding) end of Tn5382 and the left end of Tn916 (42). The positions of the vanX_B (Tn5382) and tet(M) (Tn916) genes are shown. The approximate positions of orf6, -9, and -10 from Tn916 are indicated. The number of base pairs cited in this region represents the number of bases between the termination codon of vanX_B and the start codon of orf7-VB in the upper diagram and the number of bases between the stop codon of tet(M) and orf7 in the lower diagram. Investigators have postulated that orf6, -9, and -10 are transcriptionally related to the tet(M) gene (42, 43). The positions of the individual ORFs and their relative sizes are indicated. Directions of transcription of the ORFs are indicated by the arrows above the ORFs. Amino acid identities between the ORFs of the two transposons are listed between them. The relative GC contents of Tn5382 and the flanking sequences are indicated at the top. Sequence alignments were established by using the Blastn and Blastx basic alignment search tool (1).

FIG. 2

Sequences of the termini of Tn5382 (line A) and comparison with the ends of Tn916 (line B). Identical nucleotides are indicated by vertical lines between Tn5382 and Tn916 (line B). Identical nucleotides are indicated by vertical lines between Tn5382 and Tn916. The boxed sequences represent the 11-bp imperfect inverted repeats of Tn5382. Arrows indicate the direct repeats within the ends of Tn916. Boldface underlining represents the regions of Tn916 protected by the integrase enzyme in DNase protection assays.

FIG. 3

Position of Tn5382 relative to the pbp5 structural gene in E. faecium C68. The stop codon of pbp5 (sequence in uppercase) is indicated by the double underline. The target sequence (and presumed target duplication after insertion) is boxed.

FIG. 4

Relationships between the putative target sequences within E. faecium C68 and E. faecium D366 and the circularized form of Tn5382 prior to insertion. Both target sites exhibit significant homology with the ends of the transposon, suggesting that they may represent hot spots for Tn5382 insertion. The joint region in this diagram is represented by a series of Ns, since we have no knowledge of the flanking regions prior to this episode of circularization.

FIG. 5

(A) Schematic representation of strategy to amplify the putative joint region of Tn5382, based on previous strategies for amplification of similar regions of classic conjugative transposons. P1 and P2 represent the two outward-directed primers used to synthesize the amplification product. (B) Joint PCR product generated from genomic DNA isolated from E. faecium C68. Lane 1, φX174 digested with HaeIII (size standard); lane 2, PCR product from E. faecium C68. See the text for details of primers and joint sequences.

FIG. 6

PFGE of SmaI-digested genomic DNA from C68 (lane A), CV133 (lane C), CV142 (lane F), and GE-1 (lane I). Lanes B, D, G, and J, hybridizations of Southern transfers of lanes A, C, F, and I, respectively, with a 2.1-kb EcoRV fragment internal to the vanH_B-vanB-vanX_B operon. Lanes E, H, and K, hybridization of the gel with a PCR amplification product of a region internal to the pbp5 gene. Blots were not stripped and reprobed; duplicate lanes from the same gel were blotted. The additional gel lanes are not shown to conserve space. Lane L, Megabase II size standard (Bethesda Research Laboratories), with the corresponding band sizes (in kilobases) at the right. Hybridizations were performed and filters were washed under high-stringency conditions.