Highly Conjugative pMG1-Like Plasmids Carrying Tn 1546 -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 pHT␣ (65.9 kbp), pHT␤ (63.7 kbp), and pHT␥ (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).Gene transfer systems are an essential requirement for the spread of drug resistance in microorganisms. In general, the systems of efficient plasmid transfer have not been well characterized for the gram-positive bacteria. However, enterococci possess potent and unique capabilities of transferring plasmids among themselves and to other genera (4,5,21,35). One type of enterococcal plasmid consists of the group of narrow-hostrange and pheromone-responsive plasmids (4,5,9). The other type consists of the broad-host-range pAM␤1 and pIP501 plasmids, which were originally isolated from Enterococcus faecalis (8,24) and Streptococcus agalactiae (13, 18), respectively, and transfer on a solid surface at low frequency (8,13,18,24,27,40).We have described the isolation of the pheromone-independent gentamicin resistance conjugative plasmid pMG1 (Gm r ; 65.1 kb) from an Enterococcus faecium clinical isolate in Japan (20). pMG1 transfers efficiently among enterococcus strains during broth mating. pMG1-like plasmids are widely disseminated in vancomycin-resistant E. faecium clinical isolates obtained from a hospital in the United States (39).In this report, we show that the VanA resistance encoded on a Tn1546-like transposon was mediated by a pMG1-like plasmid and that this vancomycin resistance pMG1-like plasmid was capable of highly efficient transfer among the enterococci.Drug resistance of VRE isolates and isolation of vancomycin resistance conjugative plasmids. The laboratory strains and plasmids used in this study are listed in Table 1. A total of 12 isolates of vancomycin-resistant enterococci (VRE) were used in this study ( Table 2). The vancomycin resistance of each strain transferred to E. faecium BM4105RF at a frequency of about 10 Ϫ5 per donor cell by mating in broth for 4 h at 37°C.The transconjugants of each strain acquired only vancomycin and teicoplanin resistance, indicating that the glycopeptide resistance was transferred during broth mating. Analysis of agarose gel electrophoresis of restriction fragments of plasmid DNAs of each strain showed many DNA bands, indicating that each of the strains harbored several plasmids (Fig. 1, A1). The conjugative vancomycin resistance plasmid pHT␣ was identified from the transconjugant of E. faecium FH1 by repeated transfer experiments between E. faecium BM4105 strains. The plasmids isolated from each of the strains were classified into three types, ␣, ␤, and ␥, with respect to the restriction profiles that hybridized to the type ␣ plasmid pHT␣ (Fig. 1, A2) ( Table 2). The pHT␤ and pHT␥ plasmids, which were typ...

Genetic Analysis of Transfer-Related Regions of the Vancomycin Resistance Enterococcus Conjugative Plasmid pHTβ: Identification of oriT and a Putative Relaxase Gene

“…The nucleotide next to the right end of the Tn1546-like transposon was designated as the first base pair of the plasmid. Transcription of the genes encoded on the pHT␤ plasmid was in a counterclockwise direction from the physical map described in our previous report (42). A homology search of 61 ORFs encoded on the pHT plasmid was performed by BLAST against the protein databases, and the results are shown in Table 2.…”

“…Recently, we reported the isolation of pMG1-like vancomycin resistance pHT plasmids from clinical Enterococcus faecium and Enterococcus avium strains in Japan (42). pHT plasmids, including pHT␣ (65.9 kbp), pHT␤ (63.7 kbp), and pHT␥ (66.5 kbp), are highly conjugative plasmids carrying Tn1546-like transposons (3) that encode vancomycin resistance (VanA).…”

“…The pHT plasmids are related to the gentamicin resistance conjugative plasmid pMG1 with respect to DNA hy-bridization, and they are thought to contain the same efficient conjugation system. The transfer gene traA, which is involved in the formation of mating aggregates, is conserved in all pMG1-like plasmids (36,42).…”

The Plasmidome of Firmicutes: Impact on the Emergence and the Spread of Resistance to Antimicrobials

“…Because many open reading frames of pHT and pMG1 plasmids do not show significant homology with any reported proteins, they used to be categorized as a new type of highly efficient conjugative plasmids with a MOB P family REL. This plasmid group is represented by relatives of pHT (pHTα, pHTβ, and pHTγ) and pMG1, which have greatly facilitated the dissemination of resistance to glycopeptides (Tn1546_vanA) and high-level resistance to aminoglycosides (Tn4001-like elements) among human E. faecium and E. avium isolates from the United States and Japan (123,124) and, to a lesser extent, European countries (7,70,71,122).…”

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

“…In some cases, plasmids with the various vancomycin resistance gene clusters have been recovered from enterococci (5)(6)(7)(8), although the prevalence of plasmid-encoded resistance in VRE has not been defined. Large plasmids often have intricate mechanisms by which they maintain themselves in the bacterial population.…”