INTRODUCTION

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Enterococcus faecalis is increasingly implicated as an important cause of nosocomial infections, particularly in surgical intensive care units (17, 21). The natural tolerance of E. faecalis strains for antibiotics that interfere with cell wall synthesis has led to the current use of synergistic and bactericidal combinations of a beta-lactam or a glycopeptide with an aminoglycoside, usually gentamicin (16). The emergence and spread of E. faecalis strains with multiple antibiotic resistance, including resistance to penicillins, glycopeptides, and high levels of aminoglycosides, have greatly reduced the efficacy of such combinations. For this reason, nosocomial and severe E. faecalis infections continue to be a major medical problem (20).

The bifunctional enzyme, 6'-acetyltransferase-2"-phosphotransferase, which mediates high-level gentamicin resistance (Gm^r) in enterococci and streptococci is encoded by the fused aac6-aph2 gene (6). This gene is plasmid borne in most gentamicin-resistant strains of E. faecalis (11, 18, 22) and other enterococcal strains studied so far (23, 25) and has been reported to be situated, on these plasmids, on elements similar to the  or  forms of Tn4001, originally identified in Staphylococcus aureus (15). The  element is a form of Tn4001 that contains a tandem duplication of IS256, whereas the  form does not. The Gm^r determinant is carried on plasmids by Tn4001-like elements in strains of E. faecalis (7, 8, 22) and Enterococcus avium (23). There are other plasmid-borne structures, including the Tn4001-truncated elements in E. faecalis (22), Enterococcus hirae (23), and Enterococcus raffinosus (23), and, in E. faecalis (8), the hybrid element Tn4001-IS257, originally found in S. aureus (2). The Gm^r determinant has been found to be carried on the chromosome in two E. faecalis (19, 24), one group B Streptococcus (strain B128) (1), and several Streptococcus mitis (13) clinical isolates. The chromosomal transposons so far reported that carry the Gm^r determinant are Tn3706 (5.8 kb), the Tn4001-like  element in B128 (10), and, in E. faecalis, Tn5384 (26.0 kb), which consists in part of a Tn4001-like  structure (19), and Tn924 (27.0 kb), a novel type of transposable element (24).

The purpose of the present report was to provide information on the genetic and molecular basis of high-level gentamicin resistance in E. faecalis strains isolated in France. Twenty-four E. faecalis clinical isolates were examined with respect to the conjugative transfer, location, and structure of the genetic elements carrying the Gm^r determinant.

	MATERIALS AND METHODS

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The 24 independent wild-type E. faecalis clinical strains used in this study (see Table 1) were isolated from the blood and urine of patients with septicemia, endocarditis, or genital and urinary tract infections. In addition to being resistant to high levels of gentamicin-kanamycin (MIC >2,000 µg/ml), most of them were also resistant to chloramphenicol, erythromycin, and tetracycline-minocycline. In mating experiments, carried out on membrane filters (9), the wild-type strains, used as donors, were crossed with E. faecalis recipient strain JH2-2 (12), which is resistant to rifampin and fusidic acid, and the resulted transconjugant clones were crossed with E. faecalis recipient strain BM133 (11), which is resistant to high levels of streptomycin. The antibiotics used were 1,000 µg of gentamicin per ml for the selection of transconjugants and 25 µg of fusidic acid per ml plus 100 µg of rifampin per ml or 2,000 µg of streptomycin per ml for the counterselection of the donors.

Isolation of cellular and plasmid DNA from E. faecalis strains, digestion by restriction enzymes (EcoRI, HaeIII, HincII, HindIII, and ScaI), gel electrophoresis, DNA blotting, DNA-DNA hybridization (under stringent conditions), and labeling of the probes with [-³²P]dCTP were carried out as previously described (14). The probes used for hybridization were pSF815A, containing the aac6-aph2 gene (Gm^r probe) (6); pIP1551, containing a 468-bp DNA fragment of IS256 which is a part of Tn4001 (IS256 probe) (see Fig. 1) (4); and pIP1644, containing a 629-bp DNA internal fragment of IS257 (IS257 probe) (5).

Macrorestriction analysis of the SmaI-digested cellular DNA of the wild-type strains and pulsed-field gel electrophoresis were done as we described recently (22).

	RESULTS

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Conjugative transfer of the Gm^r marker and plasmid isolation. Each of the 24 wild-type E. faecalis strains was mated with JH2-2. The results are presented in Table 1. The Gm^r determinant transferred by conjugation from the nine -hemolytic wild-type strains (1 to 4, 13 to 16, and 23) at high frequency (1 × 10¹ to 2 × 10³ transconjugants per donor cell), and in each cross, cell aggregates were observed on the mating filters (9). The frequency of transfer of the Gm^r marker was low (1 × 10⁵ to 3 × 10⁶) for 8 (6 to 9 and 19 to 22) of the 15 nonhemolytic wild-type strains; no detectable transconjugants were obtained (transfer frequency <10⁹) for the remaining seven strains (5, 10 to 12, 17, 18, and 24). Analysis of the transconjugants for the presence of unselected antibiotic resistance markers showed that the clones obtained by using as donors the wild-type strains 7 to 9, 13, and 19 to 22 were resistant to only gentamicin-kanamycin. Each of these clones carried a single plasmid, estimated to be 65.0 to 80.0 kb, after digestion with HindIII. The transconjugants that were resistant to gentamicin-kanamycin and also to chloramphenicol, erythromycin, or both carried at least two large plasmids. None of the transconjugant clones carried the tetracycline-minocycline marker. The Gm^r plasmids retransferred into BM133 from the nonhemolytic transconjugants, whether obtained from -hemolytic or from nonhemolytic wild-type strains, at low frequencies (10⁵ to 10⁶) and without the formation of cell aggregates on mating filters.

Location of the aac6-aph2 gene and of IS256 and mapping of the structures carrying the Gm^r determinant. Plasmid and cellular DNA isolated from all the wild-type strains was digested with the restriction enzymes used to characterize Tn4001-like structures: HaeIII, HincII, HindIII, ScaI, and ScaI plus HindIII (double digestion). The digested DNA was subjected to gel electrophoresis and to DNA-DNA hybridization experiments using the gene aac6-aph2 as well as IS256 and IS257 as probes.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Restriction maps of the structures carrying the Gmr marker. (A) Tn4001-truncated type I element carried by the plasmids from strains 1 to 5, 7 to 10, and 13; lack of IS256 in the right-hand flanking region of Tn4001. (B) Tn4001-truncated type II element carried by the plasmids from strains 18 to 22; lack of IS256 in the right-hand flanking region of Tn4001 and a partial deletion of IS256 in its left-hand flanking region. (C) Tn4001-truncated type III element, carried by the plasmids from strains 6, 14 to 16, and 23; lack of IS256 in both right- and left-hand flanking regions of Tn4001. (D) Tn4001-like  element borne on the chromosome of strains 11 to 13, 17, and 24. The open horizontal arrows indicate the direction of transcription of the gene aac6-aph2 and of IS256. Restriction endonuclease abbreviations: Ha, HaeIII; Hc, HincII; Hd, HindIII; Sc, ScaI. The location of the probe IS256 is indicated by a thick black line.

	DISCUSSION

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The results presented here suggest that the conjugative transfer frequency of the Gm^r plasmids carried by the -hemolytic wild-type or transconjugant E. faecalis strains may have been high because these plasmids cotransferred along with the -hemolysin plasmids carried by these strains. In fact, the Gm^r plasmids studied here, when they occur in nonhemolytic strains, either are nonconjugative or transfer at low frequency; apparently, the conjugative transfer of these plasmids is not mediated by bacterial sex pheromones (3).

Structures that carry the aac6-aph2 gene in E. faecalis and other enterococcal strains are highly diverse (8, 19, 22-24). The Gm^r plasmids from E. faecalis strains isolated in France (this study) carry either Tn4001-truncated type I elements, as reported for several Gm^r plasmids harbored by E. faecalis strains isolated in Romania (22) and for pIP1701 from an E. raffinosus strain isolated in Portugal (23), or Tn4001-truncated type II and III structures which, to our knowledge, have not yet been described. In addition to the truncated structures I, II, and III, pICC8, the Gm^r plasmid from an E. hirae strain isolated in Romania (23), carries an element characterized by the lack of IS256 in the left-hand flanking region of the aac6-aph2 gene, designated now as a Tn4001-truncated type IV structure.

The Tn4001-truncated elements were detected in this study only on the Gm^r plasmids (20 strains). By contrast, the Tn4001-like  element has been found to be carried only on the chromosome (four strains). The chromosomal location of this transposon may serve to stabilize it while the location on a plasmid may contribute to its instability, as a great variety of truncated forms of Tn4001 were found on the plasmids examined here. Certain regions of the Tn4001 transposon may have undergone natural molecular rearrangements, generating the truncated structures that we observed. As we examined the DNA of the wild-type strains, these rearrangements did not occur in the course of mating experiments, although they may have occurred during in vivo conjugative transfer prior to the isolation of the wild-type strains.

Except for the Tn4001-truncated type II structure, the other three types of elements, detected in the present study (Table 2 and Fig. 1), have been found in both epidemiologically related and unrelated strains. The spread of high-level gentamicin resistance in E. faecalis isolates in France could be due to the dissemination of such elements in unrelated strains as well as to the spread of endemic and epidemic gentamicin-resistant strains. However, for about 15 years the incidence of high-level gentamicin resistance has been stable (<10%) in France. It is possible that the genetic instability of the structures carrying the Gm^r determinant on plasmids in E. faecalis, associated with a decreased selective pressure, may act as a natural regulation mechanism to maintain the incidence of gentamicin resistance in France at a low but stable level.