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Journal of Bacteriology logoLink to Journal of Bacteriology
. 2003 Dec;185(23):7024–7028. doi: 10.1128/JB.185.23.7024-7028.2003

Highly Conjugative pMG1-Like Plasmids Carrying Tn1546-Like Transposons That Encode Vancomycin Resistance in Enterococcus faecium

Haruyoshi Tomita 1, Koichi Tanimoto 2, Satoshi Hayakawa 3, Kyoko Morinaga 1, Kohji Ezaki 3, Hisaji Oshima 3, Yasuyoshi Ike 1,2,*
PMCID: PMC262697  PMID: 14617670

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 pHTα (65.9 kbp), pHTβ (63.7 kbp), and pHTγ (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 (Gmr; 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-host-range and pheromone-responsive plasmids (4, 5, 9). The other type consists of the broad-host-range pAMβ1 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 (Gmr; 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 10−5 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.

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid Genotype or phenotype Description; source or reference
Strains
    E. faecium BM4147 (pIP816 Vanr) van 25
    E. faecalis FA2-2 rif fus Derivative of JH2; 7
    E. faecalis JH2SS str spc Derivative of JH2; 37
    E. faecium BM4105RF rif fus Derivative of plasmid-free E. faecium BM4105; 3
    E. faecium BM4105SS str spc Derivative of plasmid-free E. faecium BM4105; 3
Plasmids
    pMG1 Gmr 65.1-kb conjugative plasmid from E. faecium strain; 20
    pG200 Gmr pMG1-like conjugative plasmid from VRE; 39
    pG445 Gmr pMG1-like conjugative plasmid from VRE; 39
    pG566 Gmr pMG1-like conjugative plasmid from VRE; 39
    pG700 Gmr pMG1-like conjugative plasmid from VRE; 39
    pG120 Gmr pMG1-like conjugative plasmid from VRE; 39
    pAD1 hly/bac uvr 59.6-kb pheromone-responsive conjugative plasmid from DS16; 7, 19, 37
    pPD1 bac 59-kb pheromone-responsive conjugative plasmid from E. faecalis 39-5; 15, 38, 41
    pAM373 tet 36-kb pheromone-responsive conjugative plasmid; 6
    pAMβ1 erm 26.5-kb broad-host-range conjugative plasmid from DS5; 8, 24
    pIP501 erm cat 39.2-kb broad-host-range conjugative plasmid; 2, 13, 18

TABLE 2.

Characterizations of vancomycin-resistant enterococcia

Species Strain no. Patientb Antimicrobial drug resistance level (MIC, μg/ml)c
Drug resistance pattern Vanr plasmid type harbored in strain
Apc Erm Gen Kan Str Tet Tei Van
E. faecium FH1 A 256 >128 2 >1,024 1,024 >256 32 256 Apc Erm Kan Str Tet Tei Van α
E. faecium FH2 B 256 >128 2 >1,024 512 >256 64 256 Apc Erm Kan Str Tet Tei Van β
E. faecium FH3 C 2 4 2 64 64 1 64 512 Tei Van β
E. faecium FH4 D 128 >128 2 >1,024 256 >256 64 256 Apc Erm Kan Tet Tei Van β
E. faecium FH5 E 64 >128 1 >1,024 1,024 >256 128 512 Apc Erm Kan Str Tet Tei Van β
E. faecium FH6 F 64 >128 2 >1,024 1,024 >256 64 256 Apc Erm Kan Str Tet Tei Van β
E. faecium FH7 G 128 >128 >256 >1,024 1,024 32 128 512 Apc Erm Gen Kan Str Tet Tei Van γ
E. faecium FH8 H 128 >128 >256 >1,024 >1,024 2 128 512 Apc Erm Gen Kan Str Tei Van γ
E. faecium FH9 I 128 >128 >256 >1,024 >1,024 128 128 512 Apc Erm Gen Kan Str Tet Tei Van γ
E. faecium FH10 J 128 >128 >256 >1,024 >1,024 128 128 >1,024 Apc Erm Gen Kan Str Tet Tei Van γ
E. avium FH11 C 16 0.25 2 128 64 16 16 256 Apc Tet Tei Van β
E. avium FH12 E 16 0.25 1 64 64 256 16 512 Apc Tet Tei Van β
a

The isolation of VanA-type VRE from clinical sources is still rare in Japan (i.e., fewer than 30 cases) (16, 33; N. Fujita, M. Yoshimura, T. Komori, K. Tanimoto, and Y. Ike, Letter, Antimicrob. Agents Chemother. 42 :2150, 1998; Y. Ike, K. Tanimoto, Y. Ozawa, T. Nomura, S. Fujimoto, and H. Tomita, Letter, Lancet 353:1854, 1999). The presence of VanA VRE was examined in the feces of a total of 1,699 inpatients obtained by the microbiology division of the clinical microbiology of the university hospital of Fujita Health University School of Medicine, Aichi, Japan, between 1 August 1999 and 31 March 2001.

b

All strains were isolated from feces of patients. E. faecium FH3 and E. avium FH11, and E. faecium FH5 and E. avium FH12, were isolated from patient C and patient E, respectively. Each of the other strains was isolated from a different patient.

c

Abbreviations: Apc, ampicillin resistance; Gen, gentamicin resistance; Kan, kanamycin resistance; Str, streptomycin resistance; Tet, tetracycline resistance; Tei, teicoplanin resistance; Van, vancomycin resistance. The drug resistance levels of ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, teicoplanin, and vancomycin were equal to or greater than 16, 64, 1,024, 512, 8, 16, and 64 μg/ml, respectively. Enterococcus strains were grown in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) throughout this study. Mueller-Hinton broth and Mueller-Hinton agar for the sensitivity disk agar-N (Nissui, Tokyo, Japan) assay were used to test the MICs of antimicrobials. The MICs of the antimicrobials were determined according to the criteria of the National Committee for Clinical Laboratory Standards using Mueller-Hinton agar (32).

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 pHTα 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, α, β, and γ, with respect to the restriction profiles that hybridized to the type α plasmid pHTα (Fig. 1, A2) (Table 2). The pHTβ and pHTγ plasmids, which were type β and γ plasmids, respectively, were identified from the transconjugants of strains FH4 and FH7, respectively (Fig. 1, B1) (Table 2). Each type of plasmid DNA encoded the VanA gene by PCR analysis with the vanA-specific primer (data not shown) (11, 12, 29). pHTα DNA hybridized to all NdeI and EcoRI fragments of each type of plasmid DNA (Fig. 1, B2). DNA from the conjugative plasmid pMG1 (Gmr; 65.1 kbp) hybridized to specific NdeI or EcoRI fragments (data not shown). Each type of plasmid transferred at a frequency of around 10−3 to 10−5 per donor cell between E. faecium BM4105 or around 10−6 to 10−7 per donor cell between E. faecalis JH2 strains during broth mating.

FIG. 1.

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.

The restriction maps of the vancomycin resistance plasmids.

The restriction maps of pHTα (65.9 kbp), pHTβ (63.7 kbp), and pHTγ (66.5 kbp) were constructed (Fig. 2). The molecular sizes of the NdeI A fragment of pHTα and the NdeI B fragment of pHTγ were 18.2 and 13.3 kbp, respectively, which were 2.2 and 2.8 kbp larger than the NdeI A fragments (16 kbp) and NdeI B fragments (10.5 kbp) of pHTβ, respectively.

FIG. 2.

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.

The nucleotide sequences showed that the 2.2-kbp (2,156-bp) fragment of pHTα contained two open reading frames of 1,236 bp (412 amino acids) and 759 bp (253 amino acids), which were homologous with the IS232-mediating transposase and the transposition helper protein, respectively (28). The nucleotide sequence of the 2.8-kb (2,748-bp) fragment of the pHTγ plasmid was homologous with that of the group II intron that encodes a reverse transcriptase consisting of 638 amino acids (22, 23, 30, 31). The nucleotide sequences around the 2.2-kbp fragment of the NdeI A fragment of pHTα were completely identical to the nucleotide sequence of the NdeI A fragment of the pHTβ plasmid. Likewise, the nucleotide sequences around the 2.8-kbp fragment of NdeI B framgent of pHTγ were completely identical to that of the NdeI B fragment of the pHTβ plasmid. These results indicated that pHTβ might be the original or wild-type plasmid, and the 2.2-kbp fragment and the 2.8-kbp fragment were inserted into the NdeI A and NdeI B fragments of the pHTβ plasmid, respectively.

Analysis of the pMG1 traA gene.

The traA gene of pMG1, which encodes a 287-amino-acid protein, is involved in the tra gene system for conjugation and is specific to pMG1 (36). Each plasmid was examined to determine whether traA was conserved in each of these plasmids by sequence analysis of the PCR product for traA.

The nucleotide sequence and the deduced amino acid sequence of the open reading frame in 945-bp PCR products analyzed in pHTα, pHTβ, and pHTγ were completely identical to those of traA of pMG1, with the exception of eight nucleotide substitutions and six amino acid substitutions (i.e., V19F, S23N, R26S, V84M, A102V, and K237E). The nucleotide sequence and the deduced amino acid sequence of the gentamicin resistance pMG1-like plasmids (39) pG200, pG445, pG560, pG700, and pG120 were completely identical to those of pMG1 traA.

Based on the differences observed in the nucleotide sequence of traA, these results indicated that the traA gene of pMG1 was conserved in pMG1-like plasmids and that there was no direct connection between the gentamicin resistance pMG1 plasmid (including pMG1-like plasmids) and the vancomycin resistance pHT plasmids.

Incompatibility of vancomycin resistance plasmids and pMG1 and Southern analysis with other reported plasmids.

The transfer frequency of each of the vancomycin resistance plasmids to the recipient cell carrying pMG1 was lower than that when the recipient was plasmid free (Table 3). All transconjugants were vancomycin resistant (conferred by the incoming plasmid), but they had lost gentamicin resistance (encoded by the resident plasmid). These results indicate that each of the vancomycin resistance plasmids and pMG1 were incompatible. Southern analysis showed that the pHTβ plasmid did not contain any sequence homologous with those of the pheromone-responsive plasmids (Table 1) (4-7, 10, 15, 19, 38, 41) and the broad-host-range plasmids (Table 1) (2, 8, 13, 18) (data not shown).

TABLE 3.

Transfer frequencies of vancomycin resistance plasmids from donor strains to recipients carrying the pMG1 plasmida

Plasmid from donor cells of E. faecium BM4105SS Transfer frequency in broth mating with recipient E. faecium BM4105RF carrying:
Transfer frequency in filter mating with recipient E. faecium BM4105RF carrying:
pMG1 None pMG1 None
pHTα <1 × 10−7 1 × 10−4 2 × 10−3 >1 × 100
pHTβ <1 × 10−7 3 × 10−4 5 × 10−3 >1 × 100
pHTγ <1 × 10−7 2 × 10−4 3 × 10−3 >1 × 100
a

The mating experiments were carried out as previously described (14, 20). The mating times of broth mating and filter mating were 3 and 18 h, respectively. The transconjugants were examined after 48 h of incubation of the selective agar plates at 37°C. Throughout the mating experiments, the antibiotic concentration used for the selection of gentamicin- or vancomycin-resistant transconjugants was 100 or 12.5 μ/ml, respectively. The selection of rifampin- and fuscidic acid-resistant recipient strains was carried out at a concentration of 25 μg/ml each, while selection of streptomycin- and spectinomycin-resistant recipient strains was carried out at concentrations of 500 and 250 μg/ml, respectively.

Gentamicin and kanamycin resistance determinants on pMG1.

pMG1 was examined to determine whether the gentamicin and kanamycin resistance determinants also reside on a transposon. The nucleotide sequence revealed that the EcoRI B fragment of pMG1 encoded a Tn4001-like transposon (4,523 bp) (17, 26). The composite transposon Tn4001 (4,566 bp) carries the gentamicin and kanamycin resistance gene aacA-aphD, which is flanked by two 1,324-bp inverted repeats, IS256L and IS256R (26). The nucleotide sequence of the Tn4001-like transposon was completely identical to that of the original Tn4001 transposon, except that the resistance gene aacA-aphD was flanked by two 1,324-bp (IS256) direct repeats and there was deletion of a 43-bp sequence upstream from the end of IS256R.

Conclusions.

The pheromone-independent gentamicin resistance plasmid pMG1 and pMG1-like plasmids are found in E. faecium and are widely disseminated in vancomycin-resistant E. faecium isolates in the United States (39). The data shown in this report suggest that pMG1-like plasmids without any resistance gene or any other selectable determinant must be prevalent in E. faecium, and there is the possibility that a mobile genetic element encoding drug resistance or another determinant might insert onto them. As shown by this study, there is now evidence that in addition to gentamicin and kanamycin resistance transposon Tn4001-like elements, vancomycin resistance transposon Tn1546-like elements and other mobile genetic elements, such as IS232 and the group II intron, are capable of insertion onto pMG1-type plasmids.

Nucleotide sequence accession numbers. The nucleotide sequence data reported here have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AB091473, AB105542, and AB105543

Acknowledgments

This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology [Tokuteiryoiki, Kiban (B)] and Japanese Ministry of Health, Labor, and Welfare (H15-Shinko-9).

We thank Elizabeth Kamei for helpful advice.

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