Highly conjugative pMG1-like plasmids carrying Tn1546-like transposons that encode vancomycin resistance in Enterococcus faecium

Abstract

A total of 12 VanA-type vancomycin-resistant enterococci, consisting of 10 Enterococcus faecium isolates and two Enterococcus avium isolates, were examined in detail. The vancomycin resistance conjugative plasmids pHTalpha (65.9 kbp), pHTbeta (63.7 kbp), and pHTgamma (66.5 kbp) were isolated from each of three different E. faecium strains. The plasmids transferred highly efficiently between enterococcus strains during broth mating and were homologous with pMG1 (Gm(r); 65.1 kb).

FIG. 1.

Agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs and hybridization with the pHTα probe. Southern hybridization was performed with the digoxigenin-based nonradioisotope system of Boehringer GmbH (Mannheim, Germany), and all procedures were based on the manufacturer's manual and standard protocols (34). (A1) Agarose gel electrophoresis of NdeI-digested plasmid DNAs isolated from vancomycin-resistant E. faecium or E. avium (VRE) isolates. (A2) The gel was Southern blotted and hybridized to pHTα. Lanes of panels A1 and A2: 1, HindIII-digested lambda DNA; 2, NdeI-digested pMG1; 3, NdeI-digested pHTα; 4 to 15, NdeI-digested plasmid DNAs from the strains FH1, FH2, FH3, FH4, FH5, FH6, FH7, FH8, FH9, FH10, FH11 and FH12, respectively. (B1) Agarose gel electrophoresis of NdeI-digested pHTα, pHTβ, and pHTγ plasmid DNA isolated from each transconjugant of FH1, FH4, and FH7, respectively. (B2) The gel was Southern blotted and hybridized to the pHTα probe. Lanes of panels B1 and B2: 1, HindIII-digested lambda DNA; 2, pMG1; 3, pHTα; 4, pHTβ; 5, pHTγ.

FIG. 2.

Physical map of the vancomycin resistance conjugative plasmid pHTβ (63.7 kb) and its relation to pHTα (65.9 kb) or pHTγ (66.5 kb). To determine the DNA sequence of the 2.2-kbp fragment of pHTα and the 2.8-kbp fragment of pHTγ and to confirm that these fragments had inserted into the NdeI A and NdeI B fragments of pHTβ, respectively, random fragments of the region of the 2.2-kb fragment or of the 2.8-kb fragment were cloned and sequenced as previously described (38). pHTα resulted from the insertion of the 2.2-kb fragment of IS232 into the region of NdeI fragment A of pHTβ. pHTγ resulted from the insertion of the 2.8-kb fragment of the group II intron into the region of NdeI fragment B of pHTβ. DNA sequence and PCR analysis were carried out to analyze the VanA determinant as described previously (1, 11, 16). The VanA-type determinant of pHTβ was encoded on the transposon Tn1546 or a closely related transposon. The location of the VanA determinant of each plasmid was determined by Southern analysis, PCR, and comparison of the restriction map covering the region of the VanA determinant with that of Tn1546.