Research Article
01 September 2002

Possible Connection between a Widely Disseminated Conjugative Gentamicin Resistance (pMG1-Like) Plasmid and the Emergence of Vancomycin Resistance in Enterococcus faecium

ABSTRACT

A total of 640 vancomycin-resistant Enterococcus faecium (VRE) isolates, which were obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were used in this study. Of the 640 strains, 611 and 29 were VanA and VanB VRE, respectively, based on PCR analysis. Four hundred ninety-two (77%) of the strains exhibited resistance to concentrations of gentamicin from 64 μg/ml (MIC) to more than 1,024 μg/ml (MIC). The gentamicin resistance of each of 261 (53%) of the 492 gentamicin-resistant strains was transferred to E. faecium at a frequency of about 10−5 to 10−6 per donor cell in broth mating. More than 90% of vancomycin resistances of the 261 strains cotransferred with the gentamicin resistances to E. faecium strains by filter mating. The conjugative gentamicin resistance plasmids were identified and were classified into five types (A through E) with respect to their EcoRI restriction profiles. The transfer frequencies of each type of plasmid between E. faecium strains or Enterococcus faecalis strains were around 10−3 to 10−5 per donor cell or around 10−6 to 10−7 per donor cell, respectively, in broth mating. Type A and type B were the most frequently isolated, at an isolation frequency of about 40% per VRE isolate harboring the gentamicin resistance conjugative plasmid. The plasmids did not show any homology in Southern hybridization with the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. The EcoRI or NdeI restriction fragments of each type of plasmids hybridized to the conjugative gentamicin resistance plasmid pMG1 (65.1 kb), which was originally isolated from an E. faecium clinical isolate, and transfer efficiently in broth mating.
The isolation of vancomycin-resistant enterococci (VRE) was first reported in the United Kingdom (A. H. Uttley, C. H. Collins, J. Naidoo, and R. C. George, Letter, Lancet i:57-58, 1988) and in France (29) in 1988. Shortly after the first reports were made, VRE were detected in hospitals in the United States (35). Since then, VRE have emerged with unanticipated rapidity and, especially in the United States, are now encountered in most hospitals (31).
Among the acquired glycopeptide resistances of VanA and VanB, VanA resistance has been predominantly isolated from both within and outside the health care environment, from animals, and from the general environment (11, 12; J. Bates, J. Z. Jordens, and J. B. Selkon, Letter, Lancet 342:490-491, 1993). Most VRE isolates from the health care environment in the United States have multiple-drug resistance, including high-level gentamicin resistance and ampicillin resistance. One of the major factors that have contributed to the dissemination of VRE in the United States and Europe is now evident. In the United States, it is likely that excessive use of glycopeptide antibiotics in the health care environment resulted in the selective increase of VRE in the human intestine (24, 31), which subsequently spread by nosocomial transmission. In Europe, it is strongly suggested that the use of avoparcin as a growth promoter in animal feed has resulted in the selective increase of VRE in animal intestines, and these VRE subsequently appear in the human community (41; Y. Ike et al., Letter, Lancet 353:1854, 1999; M. A. Schouten, A. Voss, and J. A. A. Hoogkamp-Korstanje, Letter, Lancet 349:1258, 1997; A. E. van den Bogaard, L. B. Jensen, and E. E. Stobberingh, Letter, N. Engl. J. Med. 337:1558-1559, 1997). In both cases, the direct selective pressure of glycopeptides is the largest contributor to the selective increase in VRE in different habitats.
Besides the direct selective pressure of antibiotics for increasing selectively in drug-resistant bacteria, the genetic transfer system of an organism is essential to the spread of drug resistance in the organism. In Enterococcus faecium, it has been reported that the transferable plasmid or mobile genetic elements encode drug resistance determinants. VanA-type resistance determinant is encoded on transposon Tn1546 borne by nonconjugative (2, 29) or conjugative plasmids that transfer in enterococci by mating on a solid surface (filter mating) (30). The VanB determinant of E. faecium is encoded on a large mobile genetic element of conjugative transposons such as Tn1547 (34), Tn1549 (20), and Tn5382 (4).
Little is known about systems of efficient plasmid transfer in E. faecium. Previously, we described the isolation of the pheromone-independent gentamicin resistance conjugative plasmid pMG1 (65.1 kb), which transfers efficiently among enterococcus strains during broth mating and was isolated from an E. faecium clinical isolate in Japan (26). In this report we describe the study of VRE clinical isolates derived from a hospital in the United States and show the wide dissemination of a gentamicin resistance plasmid that transferred in broth matings, like pMG1. We also show that these plasmids may contribute to the efficient dissemination of vancomycin-resistance determinants in enterococcus strains.

MATERIALS AND METHODS

Bacteria, plasmids, and media.

The laboratory strains and plasmids used in this study are listed in Table 1. A total of 640 vancomycin-resistant E. faecium clinical isolates were used in this study. They were obtained from different patients who had been admitted to the University of Michigan Medical School Hospital, Ann Arbor, between 1994 and 1999. Of the 640 isolates, strains numbered from 1 to 45, from 46 to 104, from 105 to 164, from 165 to 350, from 351 to 642, and from 643 to 730 were isolated in 1994, 1995, 1996, 1997, 1998, and between January and June of 1999, respectively. Enterococcus strains were grown in Todd-Hewitt broth (THB) (Difco Laboratories, Detroit, Mich.) throughout this study. Mueller-Hinton (MH) broth and MH agar were used for the sensitivity disk agar-N (Nissui, Tokyo, Japan) assay to test the MICs of antimicrobials. Agar plates were prepared by adding 1.5% agar to broth medium. All bacterial strains were grown at 37°C. The MICs of the antimicrobials were determined according to the criteria of the National Committee for Clinical Laboratory Standards using MH agar (33). Overnight cultures of the strains grown in MH broth were diluted 100 times with fresh broth. One loopful (5 μl; about 5 × 103 to 104 cells) of each dilution was transferred to agar plates containing the relevant drug. The antimicrobials used to test the MICs for VRE isolates were vancomycin, teicoplanin, gentamicin, ampicillin, kanamycin, streptomycin, tetracycline, and minocycline.

Mating procedures.

Broth matings were performed as previously described (26) with a donor/recipient ratio of 1:10. Overnight cultures of 5 μl of the donor and 50 μl of the recipient were each added to 0.5 ml of fresh THB, and the mixtures were incubated at 37°C with gentle agitation for the appropriate times and then vortexed. Unless otherwise described, the mating time of broth mating was 3 h. Portions of the mixed cultures were then transferred to plates of solid media with appropriate selective antibiotics. Colonies were counted after 48 h of incubation at 37°C. Mating on a solid surface was performed on agar plates. The mating mixture of donor and recipient was made as described above, and 10 μl of the mixed culture was spread onto THB agar without drug. The plates were then incubated overnight (18 h) at 37°C. After the incubation, the bacteria grown on the agar plates were scraped off and transferred to 1 ml of fresh broth, and then 0.1 ml of the suspension was inoculated onto appropriate selective agar plates. Filter matings were performed as previously described (18) with a donor/recipient ratio of 1:4. Overnight cultures were prepared, 0.05 ml of the donor and 0.2 ml of the recipient were added to 4.5 ml of fresh THB, and the cells were then trapped on a membrane filter (Millipore, Bedford, Mass.). The cells on the filters were incubated at 37°C for appropriate times and were then suspended in 1 ml of THB. Appropriate dilutions of the mixture were transferred to plates of solid medium containing selective antibiotics. Throughout the mating experiments, the antibiotic concentration used for the selection of gentamicin- or vancomycin-resistant transconjugants was 100 or 12.5 μg/ml, respectively. The antibiotic concentrations for the selection of rifampin- and fuscidic acid-resistant recipient strains or streptomycin- and spectinomycin-resistant recipient strains were 25 and 25 μg/ml or 500 and 250 μg/ml, respectively.

Isolation and manipulation of plasmid DNA.

Plasmid DNA was isolated by the alkaline lysis method (36). Plasmid DNA was treated with restriction enzymes and subjected to agarose gel electrophoresis for the analysis of DNA fragments, etc. Restriction enzymes were obtained from Nippon Gene (Toyama, Japan), New England Biolabs, Inc., and Takara (Tokyo, Japan) and were used in accordance with the suppliers' specifications.

Southern hybridization.

Southern hybridization was performed with the digoxigenin (DIG)-based nonradioisotope system of Boehringer GmbH (Mannheim, Germany), and all procedures were based on the manufacturer's manual and standard protocols (36). Hybridization was performed overnight at 42°C in the presence of 50% formamide. The probe for vanA was generated by PCR amplification of DNA from the VanA-type E. faecium FN1 (N. Fujita, M. Yoshimura, T. Komori, K. Tanimoto, and Y. Ike, Letter, Antimicrob. Agents Chemother. 42:2150, 1998) using the PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany). The nucleotide sequences of the primer pair were as follows: 5′-ATGAATAGAATAAAAGTTGCAATAC and 5′-CCCCTTTAACGCTAATACGAT for vanA ligase (32) and 5′-CCCGAATTTCAAATGATTGAAAA and 5′-CGCCATCCTCCTGCAAAA for vanB ligase (32). Signals were detected with the DIG chemiluminescence detection kit (Boehringer GmbH). CSPD (Boehringer GmbH) was used as a substrate for alkali phosphatase conjugated to the antidigoxigenin antibody.

RESULTS

Drug resistance of VRE isolates.

Six hundred and forty VRE isolates were examined for drug resistance as described in Materials and Methods. Many of the strains exhibited high-level resistance to various antibiotics and multiple drug resistance (Table 2). There were bipolar distributions of the MICs of antibiotics except for vancomycin in the VRE strains. The distribution of the MICs of vancomycin, teicoplanin, gentamicin, and ampicillin for the 640 VRE strains are shown in Fig. 1. The 640 vancomycin-resistant E. faecium strains were resistant to vancomycin at levels equal to or greater than 64 μg/ml. Five hundred seventy (89%) of the strains were resistant to teicoplanin at levels equal to or greater than 16 μg/ml. Four hundred and ninety-two (77%) of the strains exhibited resistance to concentrations of gentamicin from 64 μg/ml (MIC) to more than 1,024 μg/ml (MIC), and 608 (95%) isolates exhibited resistance to concentrations of ampicillin from 16 to 512 μg/ml, depending on the strain. About 90 and 70% of the strains were resistant to kanamycin at concentrations of more than 1,024 μg/ml and had a resistance to streptomycin equal to or greater than 512 μg/ml, respectively (data not shown). About 60% of the strains exhibited MICs of tetracycline and minocycline equal to or less than 0.5 μg/ml, and the remainder of the strains exhibited MICs of tetracycline or minocycline between 0.5 and 128 μg/ml or 0.5 and 64 μg/ml, respectively (data not shown).
The DNAs of the VRE strains were analyzed by PCR for the presence of the vancomycin resistance gene with each of the vanA- and vanB-specific primers. Of the 640 strains, 611 strains gave rise to the expected 1,029-bp product with the primers specific for the vanA gene, indicating that the strains were VanA-type VRE (15, 16). Of the 611 VanA-type VRE, 570 strains were resistant to teicoplanin at levels equal to or greater than 16 μg/ml, and for 41 strains the MICs of teicoplanin were less than 16 μg/ml. Of the 640 strains, 29 strains, for which the MICs of teicoplanin were equal to or less than 0.5 μg/ml gave rise to the expected 457-bp product with primers specific for the vanB gene, indicating that the strains were VanB-type VRE (15, 32).

Transferability of high-level gentamicin resistance of VRE isolates.

Of the 640 VRE, 492 (77%) isolates exhibited resistance to concentrations of gentamicin from 64 μg/ml (MIC) to more than 1,024 μg/ml (MIC). The transferability of the gentamicin resistance trait from each of the 492 gentamicin-resistant strains to E. faecium BM4105RF was examined by mating in broth or on a solid surface overnight at 37°C. The gentamicin resistance of each of 261 (53%) of the 492 strains was transferred at a frequency of about 10−5 to 10−6 per donor cell, and about 10−2 to 10−4 per donor cell, respectively, in broth mating and mating on a solid surface. The gentamicin resistance of each of 86 (17%) of the 492 strains was transferred at a frequency of about 10−4 to 10−5 per donor cell by mating on a solid surface and was not transferred at a frequency of less than 10−7 per donor cell by broth mating. Of the 492 gentamicin-resistant VRE strains, 145 strains (29%) did not transfer the gentamicin resistance, even by filter mating, at a frequency of less than 10−8 per donor cell.

Isolation of the gentamicin resistance conjugative plasmids.

Of the 261 strains that transferred gentamicin resistance by broth mating, 60 strains of VanA VRE were selected at random, and their conjugative plasmids were analyzed. Plasmid DNA was isolated from a representative transconjugant generated from matings using each of the 60 strains. The DNA was digested with EcoRI and examined by agarose gel electrophoresis. A number of transconjugants harbored several plasmids based on a number of different fragments. Plasmid DNAs were studied for homology with the plasmid pMG1 (Gmr) (65.1 kb). The plasmid pMG1 hybridized to specific EcoRI fragments from plasmids of each of the 60 transconjugants. The plasmids isolated from 52 of 60 transconjugants were classified into five types (type A to E) with respect to the EcoRI restriction profiles that hybridized to pMG1 DNA. The plasmids isolated from the remaining 8 transconjugants exhibited different EcoRI restriction profiles that hybridized to pMG1 DNA. Of the 52 strains, a total of 25, 22, 2, 2, and 1 strain(s) harbored the type A, B, C, D, or E plasmid, respectively. Figure 2 shows representative results of Southern hybridization of the plasmids isolated from transconjugants harboring type A or type B plasmid.
Each type of plasmid DNA was identified from the transconjugant by repeated transfer experiments between E. faecium BM4105RF and E. faecium BM4105SS by short mating (30-min mating) in either one or two strains of each type. The transconjugant harboring each type of plasmid was resistant only to gentamicin, and each type of plasmid did not encode the VanA determinant based on PCR analysis or Southern analysis with vanA-specific primer or vanA-specific probe, respectively (data not shown). Each type of transferable plasmid DNA was studied to determine the EcoRI or NdeI restriction profiles and homology with pMG1 by Southern analysis. The pMG1 DNA probe hybridized to all EcoRI fragments of type A, B, and C plasmid DNA, and hybridized to specific EcoRI fragments of type D and E plasmid DNA, with the exception of the fragment of about 0.8 kbp (Fig. 3A). The pMG1 DNA probe hybridized to all NdeI fragments of each type of plasmid DNA (Fig. 3B).

Conjugative transfer of gentamicin resistance plasmid.

Each type of gentamicin resistance plasmid was examined for conjugative transfer in broth or filter mating. As shown in Table 3, each of the plasmids examined transferred between E. faecium strains, between Enterococcus faecalis strains, and between E. faecium and E. faecalis strains by broth mating. The transfer frequencies between E. faecium strains or between E. faecalis strains were around 10−3 to 10−5 per donor cell or around 10−6 to 10−7 per donor cell, respectively, by broth mating. The transfer frequencies to E. faecium recipient strains were about 2 or 3 orders higher than the E. faecalis recipient strain. The gentamicin resistance plasmid transferred highly efficiently to the E. faecium recipient strain at a frequency of more than 100 per donor cell and to the E. faecalis recipient strain at a frequency of around 10−2 to 10−3 per donor cell by filter mating.

Conjugative cotransfer of vancomycin resistance with gentamicin resistance.

Transferability of the vancomycin resistance trait of VRE strains was examined between the donor strain of each of the VRE strains and the recipient strain E. faecium BM4105RF by broth mating or on a solid surface. Of the 261 VRE strains that were resistant to gentamicin and transferred the gentamicin resistance trait at a frequency of around 10−5 to 10−6 per donor cell by broth mating, 255 strains (97.7%) transferred vancomycin resistance at a frequency of around 10−5 to 10−6 per donor cell on a solid surface. Typical results showing the transferability between wild-type strains to the laboratory strain E. faecium BM4105RF by filter mating are shown in Table 4.
Of the 86 VRE strains that were resistant to gentamicin and transferred gentamicin on a solid surface and did not transfer in broth mating, 77 strains (90%) transferred vancomycin resistance at a frequency of around 10−8 to 10−6 per donor cell on a solid surface.
Of the 145 VRE strains that were resistant to gentamicin and did not transfer the gentamicin resistance trait even on a solid medium, 70 strains (48.5%) transferred vancomycin resistance at a frequency of 10−8 to 10−7 per donor cell by filter mating on a solid surface.
Of the 148 VRE strains that were not resistant (MIC ≤ 8 μg/ml) to gentamicin, 64 (43%) transferred vancomycin resistance at a frequency of around 10−8 to 10−7 per donor cell on a solid surface.
These results implied that when the conjugative gentamicin resistance plasmid is present in a vancomycin-resistant E. faecium, the likelihood of being able to transfer vancomycin resistance is enhanced.

DNA-DNA hybridization.

Two types of conjugative plasmids in enterococci have been reported and well analyzed. One type of plasmid is able to transfer at relatively low frequencies on a solid surface, such as during filter mating (8). These plasmids usually have a broad host range. Macrolide-lincosamide-streptogramin B resistance plasmid pIP501 (3, 17, 23) and pAMβ1 (28) are representative.
The other type of plasmid is mainly found in E. faecalis and is a pheromone-responsive plasmid (7, 8, 13, 14) which transfers between E. faecalis strains at a high frequency of 100 to 10−2 per donor cell within a few hours of broth mating. Among these plasmids, the pheromone-related conjugation systems are well studied for pAD1 (7, 8, 10, 25), pCF10 (6, 14, 22), pPD1 (19, 40, 42), and pAM373 (9), which confer responses to the sex pheromones cAD1, cCF10, cPD1, and cAM373, respectively. There is homology between the genes involved in the regulation of the pheromone response of these plasmids.
Type A and B plasmid DNAs were each studied for homology with that of the pheromone-responsive plasmids pAD1, pPD1, and pAM373 and broad-host-range plasmids pIP501 and pAMβ1. Each of the type A or B plasmid DNA probes did not hybridize with any EcoRI fragments from these pheromone-responsive plasmids and broad-host-range plasmids and did hybridize with all EcoRI fragments of the type A, type B, and pMG1 plasmid DNAs. These results indicate that the type A or type B plasmid did not contain any sequence homologous with the pheromone-responsive plasmids and the broad-host-range plasmids (data not shown).

Restriction endonuclease digestion patterns of the VRE chromosomal DNA.

Pulsed-field gel electrophoresis was used to compare the clinical isolates of gentamicin-resistant VRE harboring the conjugative plasmid. The VRE strains used were clinical isolates corresponding to the transconjugants shown in Fig. 2 that harbored type A and type B gentamicin resistance conjugative plasmids, respectively. The patterns of a total of 14 strains harboring type A conjugative plasmids showed eight different patterns (data not shown). Of these 14 strains, seven strains showed identical bands or differed by one or two bands, indicating that these strains were identical or were related (37, 38). Another seven strains showed different patterns. The patterns of the 14 strains harboring the type B conjugative plasmid showed six different patterns (data not shown). Of these 14 strains, two groups of two strains and one group of six strains showed identical patterns or differed by one or two bands, indicating that strains of each group were identical or were related (37, 38). The other four strains showed different patterns. These results indicated that the gentamicin resistance conjugative plasmid could disseminate to different E. faecium strains.

DISCUSSION

The data shown in this report indicate that many VRE clinical isolates (about 80% of VRE isolates) from a major teaching hospital in the United States have high-level gentamicin resistance and that the gentamicin resistance determinant of at least half of the gentamicin-resistant strains is encoded on conjugative plasmids that efficiently transfer in broth mating at a frequency of about 10−3 to 10−5 per donor cell and transfer efficiently on a solid surface at a frequency of about 100 to 10−1 per donor cell. The conjugative plasmids were classified into five types (A through E) with respect to their EcoRI restriction profiles. Types A and B were the most frequently isolated, at an isolation frequency of about 40% per VRE isolate harboring the gentamicin resistance conjugative plasmid. The EcoRI or NdeI restriction fragments of each type of plasmid hybridized to the plasmid pMG1, indicating that each type of plasmid was similar to pMG1. pMG1 does not show any homology in Southern hybridization with that of the pheromone-responsive plasmids of E. faecalis (7, 8, 13, 14) or broad-host-range plasmids such as pAMβ1 and pIP501 of enterococcal plasmids (26). Each of the type A and type B plasmid DNAs also did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and the broad-host-range plasmids. These results indicate that each type of plasmid was similar to pMG1 with respect to the efficient transferability in broth mating and the nonhomology with the pheromone-responsive plasmids and broad-host-range plasmids.
Some VanA-type VRE isolates exhibited low-level teicoplanin resistance (i.e., MIC ≤ 8 μg/ml). The mechanism of low-level teicoplanin resistance of each of these VanA-type VRE isolates is not yet known. There are reports that amino acid substitutions in the VanS protein or defects in vanZ of the VanA-type determinant, which consists of VanRSHAXYZ genes, result in low-level teicoplanin resistance (1, 21). The MICs of gentamicin resistance were distributed between 64 and >1,024 μg/ml. The MICs of 64 and 128 μg/ml were relatively low for gentamicin resistance. The gentamicin resistance of many of the strains for which the MICs were 64 or 128 μg/ml were transferred to recipient E. faecium BM4105 strains, and the MICs of gentamicin for the transconjugants were more than 512 μg/ml. These indicate that the gentamicin resistance levels of the clinical isolates depend on each of the isolates.
Systems of efficient plasmid transfer are not well known among gram-positive bacteria in general. However, enterococci possess potent and unique abilities to transfer plasmids among themselves, and some of these transfer to other genera (7, 8, 27, 37). Before the identification of the conjugative plasmid pMG1, two types of conjugative plasmids by which enterococci naturally transfer genetic elements were known and were well characterized in enterococci. One type consists of the narrow-host-range and pheromone-responsive plasmids (7, 8, 13). These plasmids transfer between E. faecalis at high frequencies (100 to 10−2 per donor cell) in broth and on solid surfaces and also in vivo (7, 8, 13). The other type represent broad-host-range plasmids (e.g., pAMβ1 or pIP501) that transfer on a solid surface at low frequency (17, 23, 28). Transfer of these plasmids requires stable contact between donor and recipient cell on a solid surface.
The identification of pMG1 shows the existence of a new system of plasmid conjugative transfer in enterococci that differs from other known conjugative plasmids. At first, pMG1 was thought to be unique to the E. faecium clinical isolate from Japan. However, as described above, gentamicin resistance conjugative plasmids that transfer efficiently in broth mating were isolated at a high frequency from E. faecium clinical isolates in the hospital in the United States, and Southern analysis implied that these plasmids were similar to pMG1. The gentamicin resistance cotransferred at a high frequency with vancomycin resistance. These results implied that the gentamicin resistance plasmid might cotransfer vancomycin resistance plasmids, which might be conjugative or nonconjugative. The mechanism of cotransfer with gentamicin and vancomycin resistance is not yet known. Although this study is limited to the isolates of one hospital, these results imply that pMG1-like plasmids are widely disseminated among E. faecium and may contribute significantly to the spread of other resistance traits, notably vancomycin resistance.
FIG. 1.
FIG. 1. Distribution of MICs of antimicrobials used in this study. The MIC of various antimicrobials (vancomycin [A], teicoplanin [B], gentamicin [C], and ampicillin [D])for 640 isolates were examined by agar dilution methods as described in Materials and Methods.
FIG. 2.
FIG. 2. Agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs and hybridization with plasmid pMG1. (A1) Agarose gel electrophoresis of EcoRI-digested plasmid DNAs isolated from gentamicin-resistant E. faecium BM4105SS transconjugants harboring the type A gentamicin resistance conjugative plasmid. (A2) The gel was Southern blotted and hybridized to pMG1. (B1) Agarose gel electrophoresis of EcoRI-digested plasmid DNA isolated from gentamicin-resistant E. faecium BM4105SS transconjugants harboring the type B gentamicin resistance conjugative plasmid. (B2) The gel was Southern blotted and hybridized to pMG1. (A) Lanes: 1, HindIII-digested lambda DNA; lanes 2 to 15, EcoRI-digested plasmid DNA isolated from transconjugants of strains 161, 200, 210, 253, 300, 306, 311, 317, 376, 469, 494, 537, 581, and 692, respectively; lane 16, EcoRI-digested pMG1. (B) Lanes: 1, HindIII-digested lambda DNA; lanes 2 to 14, EcoRI-digested plasmid DNA isolated from transconjugants of strains 70, 87, 133, 166, 205, 247, 282, 411, 445, 526, 587, 619, 666, and 725, respectively; lane 15, EcoRI-digested pMG1.
FIG. 3.
FIG. 3. Agarose gel electrophoresis of restriction endonuclease-digested DNA of each type of gentamicin resistance conjugative plasmid and hybridization with pMG1. Agarose gel electrophoresis of EcoRI-digested plasmid DNA (A1) and the Southern hybridization with pMG1 (A2). Agarose gel electrophoresis of NdeI-digested plasmid DNA (B1) and Southern hybridization with pMG1 (B2). Lanes: 1, HindIII-digested lambda DNA; 2, pMG1; 3, pG200 (type A); 4, pG445 (type B); 5, pG566 (type C); 6, pG700 (type D); 7, pG120 (type E); 8, pAD1.
TABLE 1.
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmidGenotype or phenotypeDescription and/or reference(s)
Strains  
    E. faecium BM4147 (pIP816 Vanr)van29
    E. faecalis FA2-2rif fusDerivative of JH2; 10
    E. faecalis JH2SSstr spcDerivative of JH2: 39
    E. faecium BM4105RFrif fusDerivative of plasmid-free E. faecium BM4105; 5
    E. faecium BM4105SSstr spcDerivative of plasmid-free E. faecium BM4105; 5
Plasmids  
    pMG1Gmr65.1-kb conjugative plasmid from E. faecium strain; 26
    pAD1hly/bac uvr59.6-kb pheromone-responsive conjugative plasmid from DS16; 39
    pPD1bac59-kb pheromone-responsive conjugative plasmid from E. faecalis 39-5; 19, 42
    pAM373tet36-kb pheromone-responsive conjugative plasmid; 9
    pAMβ1erm26.5-kb broad-host-range conjugative plasmid from DS5; 28
    pIP501erm cat39.2-kb broad-host-range conjugative plasmid; 17
TABLE 2.
TABLE 2. Antimicrobial drug resistance pattern of vancomycin-resistant E. faecium isolates
Resistance patternaNo. of isolates (%) (n = 640)
Apc Gen Kan Str Tei Van245 (38.3)
Apc Gen Kan Str Tet Tei Van97 (15.2)
Apc Gen Kan Tei Van81 (12.7)
Apc Kan Str Tei Van50 (7.8)
Apc Gen Kan Tet Tei Van34 (5.3)
Apc Kan Str Tet Tei Van18 (2.8)
Apc Tei Van17 (2.7)
Apc Kan Str Van14 (2.2)
Apc Gen Kan Str Van8 (1.3)
Apc Str Tet Tei Van6 (0.9)
Apc Gen Kan Van6 (0.9)
Other 30 different patternsb64 (10)
a
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 (MICs) of ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, teicoplanin, and vancomycin were equal to or greater than 16, 64, 1024, 512, 8, 16, and 64 μg/ml, respectively.
b
The number of strains for each of 30 different patterns was less than six.
TABLE 3.
TABLE 3. Transfer frequency of each type gentamicin resistance plasmid in Enterococcus
Type of Gmr plasmidDonorRecipientNo. of transconjugants/donor 
   Broth matingFilter mating
AE. faecium BM4105RF(pG200)E. faecium BM4105SS8 × 10−5> 1
 E. faecium BM4105RF(pG200)E. faecalis JH2SS1 × 10−78 × 10−4
 E. faecalis JH2SS(pG200)E. faecalis FA2-23 × 10−73 × 10−3
 E. faecalis JH2SS(pG200)E. faecium BM4105RF7 × 10−5> 1
BE. faecium BM4105RF(pG247)E. faecium BM4105SS1 × 10−3> 1
 E. faecium BM4105RF(pG247)E. faecalis JH2SS3 × 10−63 × 10−2
 E. faecalis JH2SS(pG247)E. faecalis FA2-21 × 10−61 × 10−2
 E. faecalis JH2SS(pG247)E. faecium BM4105RF3 × 10−4> 1
BE. faecium BM4105RF(pG445)E. faecium BM4105SS2 × 10−3> 1
 E. faecium BM4105RF(pG445)E. faecalis JH2SS3 × 10−62 × 10−2
 E. faecalis JH2SS(pG445)E. faecalis FA2-24 × 10−64 × 10−2
 E. faecalis JH2SS(pG445)E. faecium BM4105RF3 × 10−3> 1
CE. faecium BM4105RF(pG566)E. faecium BM4105SS4 × 10−5> 1
 E. faecium BM4105RF(pG566)E. faecalis JH2SS1 × 10−78 × 10−4
 E. faecalis JH2SS(pG566)E. faecalis FA2-23 × 10−74 × 10−3
 E. faecalis JH2SS(pG566)E. faecium BM4105RF9 × 10−5> 1
DE. faecium BM4105RF(pG700)E. faecium BM4105SS5 × 10−5> 1
 E. faecium BM4105RF(pG700)E. faecalis JH2SS2 × 10−72 × 10−3
 E. faecalis JH2SS(pG700)E. faecalis FA2-22 × 10−71 × 10−3
 E. faecalis JH2SS(pG700)E. faecium BM4105RF8 × 10−5> 1
EE. faecium BM4105RF(pG120)E. faecium BM4105SS7 × 10−5> 1
 E. faecium BM4105RF(pG120)E. faecalis JH2SS1 × 10−71 × 10−3
 E. faecalis JH2SS(pG120)E. faecalis FA2-21 × 10−72 × 10−3
 E. faecalis JH2SS(pG120)E. faecium BM4105RF2 × 10−5> 1
pMG1E. faecium BM4105SS(pMG1)E. faecium BM4105RF6 × 10−4> 1
 E. faecium BM4105SS(pMG1)E. faecalis FA2-22 × 10−43 × 10−3
 E. faecalis FA2-2(pMG1)E. faecalis JH2SS5 × 10−42 × 10−2
 E. faecalis FA2-2(pMG1)E. faecium BM4105SS4 × 10−5> 1
TABLE 4.
TABLE 4. Transfer frequency of gentamicin and vancomycin resistance of VRE strain harboring each type of gentamicin resistance plasmid
Type of Gmr plasmid harbored in VRE donor strainDonorRecipientNo. of transconjugants/donor cell   
   Gmr Vmr 
   Broth matingFilter matingBroth matingFilter mating
AVRE200E. faeciumBM4105RF3 × 10−53 × 10−4<1 × 10−75 × 10−8
 VRE210E. faeciumBM4105RF1 × 10−51 × 10−3<1 × 10−77 × 10−7
 VRE253E. faeciumBM4105RF1 × 10−52 × 10−4<1 × 10−72 × 10−8
BVRE247E. faeciumBM4105RF5 × 10−52 × 10−4<1 × 10−73 × 10−8
 VRE411E. faeciumBM4105RF1 × 10−51 × 10−3<1 × 10−74 × 10−5
 VRE445E. faeciumBM4105RF1 × 10−42 × 10−1<1 × 10−76 × 10−5
CVRE566E. faeciumBM4105RF1 × 10−53 × 10−2<1 × 10−75 × 10−6
DVRE700E. faeciumBM4105RF2 × 10−52 × 10−4<1 × 10−77 × 10−7
EVRE120E. faeciumBM4105RF1 × 10−54 × 10−4<1 × 10−72 × 10−6
 E. faecium BM4105SS(pMG1)E. faeciumBM4105RF7 × 10−41 × 100NTaNT
a
NT, not tested.

Acknowledgments

This work was supported by grants from the Japanese Ministry of Health, Labor, and Welfare, and the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
We thank Xinghua Ma for technical assistance and Elizabeth Kamei for helpful advice.

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cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 40Number 9September 2002
Pages: 3326 - 3333
PubMed: 12202574

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Received: 4 February 2002
Revision received: 21 April 2002
Accepted: 22 June 2002
Published online: 1 September 2002

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Haruyoshi Tomita
Department of Microbiology
Carl Pierson
Division of Microbiology, Clinical Laboratory, Medical School Hospital
Suk Kyung Lim
Department of Microbiology
Present address: National Veterinary Research and Quarantine Service, Manan-gu, Anyang kyonggido, Republic of Korea, 430-016.
Don B. Clewell
Department of Microbiology/Immunology, School of Medicine
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109
Yasuyoshi Ike [email protected]
Department of Microbiology
Laboratory of Bacterial Drug Resistance, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan

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