INTRODUCTION

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Acquired resistance to glycopeptides in enterococci is due to production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac), instead of the dipeptide D-Ala-D-Ala present in susceptible bacteria (4, 30, 31). The substitution prevents formation of complexes between glycopeptides and peptidoglycan precursors at the cell surface that are responsible for inhibition of cell wall synthesis (8, 22). Acquired glycopeptide resistance by this mechanism is conferred by two classes of genetic elements (vanA or vanB) that encode a dehydrogenase (VanH or VanH_B) and a ligase (VanA or VanB) for synthesis of D-Ala-D-Lac (3, 8, 10). Each element also encodes a D,D-dipeptidase (VanX or VanX_B) that limits synthesis of precursors containing the target of glycopeptides by hydrolyzing D-Ala-D-Ala produced by the host Ddl D-Ala:D-Ala ligase (23, 33).

Synthesis of the resistance proteins is regulated by two-component regulatory systems composed of a membrane-bound kinase that senses the presence of glycopeptides in the external medium (VanS or VanS_B) and a cytoplasmic response regulator (VanR or VanR_B) that activates a promoter for transcription of the resistance genes (1, 3, 6, 10, 15, 32). The sensor is thought to control the activity of the response regulator positively by phosphorylation and negatively by dephosphorylation. According to this model, the sensor acts as a kinase in inducing conditions leading to phosphorylation of the response regulator and promoter activation. In the absence of glycopeptides, the sensor acts as a phosphatase and prevents accumulation of the phosphorylated form of the response regulator.

The VanS sensor mediates induction in response to vancomycin and teicoplanin, leading to inducible expression of resistance to high levels of both glycopeptides (VanA phenotype) (2). In contrast, the majority of enterococci harboring vanB-type gene clusters are inducibly resistant to various levels of vancomycin but remain susceptible to teicoplanin since the VanS_B sensor triggers induction only in response to vancomycin (VanB phenotype) (2, 6, 10, 21). Teicoplanin-resistant mutants of VanB-type strains obtained in vitro harbor mutations in vanS_B that are responsible for various alterations in the regulation of the resistance genes. The mutants are inducibly or constitutively resistant to vancomycin and teicoplanin (Vm^r Te^r) or are heterogeneously resistant to these antibiotics (Vm^Het Te^Het) (6, 7, 13).

Emergence of teicoplanin resistance in VanB-type strains has also been observed in a patient (14) and in rabbits with experimental endocarditis treated with vancomycin or teicoplanin (5). In the latter model, the combination of gentamicin and teicoplanin was efficient since it reduced the number of surviving bacteria in the vegetations and prevented emergence of mutants resistant to teicoplanin (5). We undertook the present study to identify the conditions of emergence of mutants resistant to one or both components of the glycopeptide-gentamicin combination in VanB-type strains, in vitro and in the rabbit model of aortic endocarditis.

	MATERIALS AND METHODS

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Bacterial strains and media. Enterococcus faecalis JH2-2 (Vm^s Te^s) is susceptible to glycopeptides and -lactams and is intrinsically resistant to low levels of aminoglycosides (16). E. faecalis BM4281 (Vm^r Te^s) and BM4275 (Vm^r Te^s) harbor a 250-kb chromosomal genetic element conferring VanB-type resistance and were obtained by conjugal transfer of vancomycin resistance from clinical isolate Enterococcus faecium BM4120 (Vm^r Te^s) to JH2-2 (20). E. faecalis BM4309 (Vm^r Te^r), BM4310 (heterogeneously resistant to vancomycin and teicoplanin [Vm^Het Te^Het]), and BM4307 (vancomycin dependent [Vm^d]) are spontaneous in vitro mutants of BM4281 (5). Cultures and antibiotic susceptibility testing were performed in brain heart infusion (BHI) broth or agar (Difco Laboratories, Detroit, Mich.) at 37°C.

Amplification, cloning, and sequencing. The vanS_B and ddl genes were amplified by PCR with Pfu polymerase (Stratagene, La Jolla, Calif.) and oligodeoxyribonucleotide primers (Unité de Chimie Organique, Institut Pasteur, Paris, France) as described previously (7). The amplified genes were cloned into pUC18 and sequenced by the dideoxy chain termination method with T7 DNA polymerase (T7 sequencing kit; Pharmacia, Uppsala, Sweden) and [-³⁵S]dATP (Amersham Radiochemical Centre, Amersham, England).

Regulation of VanX_B D,D-dipeptidase synthesis. Bacteria were grown to an optical density at 600 nm of 0.7 in broth containing or not containing vancomycin or teicoplanin, treated with lysozyme, and lysed by sonication (2). The lysate was centrifuged at 100,000 × g for 45 min at 4°C, and the supernatant was assayed for D,D-dipeptidase activity by using D-Ala-D-Ala as a substrate and D-amino acid oxidase coupled to peroxidase for the indicator reactions (2).

In vitro susceptibility testing and selection of mutants. Antibiograms were performed by disk-agar diffusion with disks containing 30 µg of vancomycin, 30 µg of teicoplanin, or 500 µg of gentamicin (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). The MICs of vancomycin (Eli Lilly & Co., Saint-Cloud, France), teicoplanin (Marion Merrell Dow, Levallois-Perret, France), and gentamicin (Unilabo, Levallois Perret, France) were determined by the method of Steers et al. (28) with 10⁵ CFU per spot on BHI agar after 24 h of incubation. The MICs of gentamicin were determined by using ca. 1.25-fold dilution of the antibiotic (i.e., 10, 8, 6.4, 5, 4, etc., µg/ml) instead of the usual 2-fold dilution.

Experimental endocarditis. Polyethylene catheters were inserted through the right carotid artery into the left ventricle of New Zealand White rabbits (2.0 to 2.5 kg) (11). Twenty-four hours after catheter insertion, the rabbits were inoculated by the ear vein with approximately 5 × 10⁸ CFU of E. faecalis BM4275 (Vm^r Te^s) in 1 ml of 0.9% NaCl. The catheter was left in place throughout the experiment. Forty-eight hours after inoculation, animals received intramusculary twice daily for 5 days vancomycin (50 mg/kg of body weight), teicoplanin (20 mg/kg after a loading dose of 40 mg/kg), gentamicin (3 mg/kg), or combinations of gentamicin plus vancomycin or teicoplanin. These regimens were previously shown to lead to peak and trough antibiotic serum levels, respectively, of 57 ± 5.5 and 7.0 ± 1.5 µg/ml for vancomycin, 63 ± 23 and 25 ± 10 µg/ml for teicoplanin, and 7.5 ± 2.0 and <0.2 µg/ml for gentamicin (5). Control animals were left untreated. Animals were killed by intravenous injection of pentobarbital. At the time of sacrifice, the heart was removed and the chambers on the left side were examined to confirm vegetative endocarditis. All vegetations from single rabbits were excised, pooled, weighed, and homogenized in 1 ml of sterile distilled water. Vegetation homogenates were plated on agar to count surviving bacteria and on agar containing teicoplanin (16 µg/ml) or gentamicin at twice the MIC to enumerate mutants after 48 h of incubation. The MICs of gentamicin were determined for bacteria recovered from the plate containing this antibiotic. The phenotype of the mutants selected on teicoplanin was determined by disk-agar diffusion.

Statistics. Variance analysis followed by the Fisher test for multiple comparisons was used to compare the bacterial counts in vegetations from animals treated with various regimens (27). Comparisons of bacterial counts from animals with or without mutants and treated with the same regimen were performed by the Mann-Whitney U test. The Fisher exact test was used for comparisons of the proportions of animals with vegetations retaining resistant mutants. A P value of <0.05 was considered significant.

	RESULTS AND DISCUSSION

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Properties of the teicoplanin-resistant mutants. We previously reported the in vitro selection of spontaneous mutants of E. faecalis BM4281 (Vm^r Te^s), in particular BM4309, which was homogeneously resistant to vancomycin and teicoplanin (Vm^r Te^r), BM4310, which was heterogeneously resistant to these antibiotics (Vm^Het Te^Het), and BM4307, which required vancomycin for growth (vancomycin-dependent phenotype [Vm^d]) (5). In the present study, we characterized these mutants for the presence of mutations in the vanS_B sensor and ddl D-Ala:D-Ala ligase genes (Table 1). Regulation of the glycopeptide resistance genes was also studied by determination of VanX_B D,D-dipeptidase activity in crude extracts from bacteria grown under various inducing conditions.

In vitro activity of gentamicin. BM4281 (Vm^r Te^s) and the teicoplanin-resistant mutants BM4309 (Vm^r Te^r), BM4310 (Vm^Het Te^Het), and BM4360 (Vm^r Te^r) were significantly more susceptible to gentamicin (MIC = 5 µg/ml) than the parental strain JH2-2 (MIC = 16 µg/ml) (Table 1). Two lines of evidence indicate that expression of the glycopeptide resistance genes was not responsible for increased susceptibility to gentamicin. VanB-type BM4281 (Vm^r Te^s) was more susceptible to gentamicin than JH2-2 although the resistance genes were tightly regulated in BM4281, as indicated by the absence of D-Ala-D-Lac-ending peptidoglycan precursors and of VanX_B D,D-dipeptidase activity in the absence of induction (2). The MICs of gentamicin were variable in 15 independent transconjugants obtained by mating between VanB-type clinical isolate E. faecalis BM4120 (Vm^r Te^s) and JH2-2 (20, 21). The MIC of gentamicin was 5 µg/ml for 5 transconjugants, including BM4281 (Vm^r Te^s), and 16 µg/ml for JH2-2 and the remaining 10 transconjugants, such as BM4275 (Vm^r Te^s) (Table 1 and data not shown). Thus, increased activity of gentamicin may not be due to acquisition of the vanB gene cluster, since it did not depend upon expression of the glycopeptide resistance genes and was not detected in all of the transconjugants that had received the same vanB cluster. Chromosomal markers were recently reported to be cotransferred with conjugative elements in E. faecalis (7), suggesting that certain VanB transconjugants may have acquired genetic information responsible for increased activity of gentamicin in addition to the vanB element.

MICs of gentamicin determined in the presence of glycopeptides. The MICs of gentamicin for BM4281 (Vm^r Te^s) (5 µg/ml) and BM4275 (Vm^r Te^s) (16 µg/ml) were reduced 2.5- and 32-fold, respectively, when 10 µg of vancomycin per ml was added to the medium (Table 1). Vancomycin or teicoplanin at 10 µg/ml decreased by at least 2.5-fold the MICs of gentamicin against the teicoplanin-resistant mutants derived from BM4281 (Vm^r Te^s). Thus, the gentamicin-glycopeptide combinations were more active than gentamicin alone in spite of resistance to the glycopeptide present in the combination (Table 1).

Selection of spontaneous gentamicin-resistant mutants. Mutants resistant to gentamicin were obtained at frequencies of 10⁶ to 10⁷ on agar containing gentamicin at concentrations of twice the MICs (Table 1). The MIC of gentamicin was increased two- to eightfold and the phenotype was stable after three serial subcultures in the absence of the antibiotic. The aspects of the colonies of the mutants and of the parental strains were similar. The generation times of JH2-2 and of two gentamicin-resistant derivatives of this strain were indistinguishable. Thus, acquisition of gentamicin resistance was not associated with a severe growth defect.

In vitro bactericidal activity of glycopeptide-gentamicin combinations. The gentamicin-vancomycin combination initially killed BM4281 (Vm^r Te^s) and BM4275 (Vm^r Te^s), but the number of CFU increased after 6 h (Fig. 1), leading to an overall increase in the number of CFU after 24 h of incubation (Table 2). Bacteria incubated with the combination for 24 h formed colonies on agar containing gentamicin and vancomycin (Table 2). The MICs of gentamicin against these bacteria were increased two- to sixfold in comparison with BM4281 (Vm^r Te^s) and BM4275 (Vm^r Te^s) (data not shown). Thus, the gentamicin-vancomycin combination selected mutants with increased resistance to gentamicin that were also resistant to the combination. Of note, an initial phase of killing followed by growth was previously observed for VanB-type strains incubated in the presence of vancomycin and streptomycin (19, 25), suggesting that mutants could also be selected by this combination.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Time-kill curves. Bacteria were incubated in BHI broth in the absence of antibiotic (control) or in broth containing 4 µg of gentamicin per ml (Gent 4), 4 µg of gentamicin per ml and 10 µg of vancomycin per ml (Gent 4 + Vm 10), or 4 µg of gentamicin per ml and 10 µg of teicoplanin per ml (Gent 4 + Te 10). Surviving bacteria were enumerated on agar plates after 0, 3, 6, and 24 h of incubation at 37°C.

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Experimental endocarditis. Various 5-day antibiotic regimens were tested in the rabbit model of endocarditis against E. faecalis BM4275 (Vm^r Te^s), which did not display increased susceptibility to gentamicin in comparison to JH2-2 (Vm^s Te^s) (Table 1). The efficacy of glycopeptides and gentamicin, alone or in combination, was analyzed in terms both of reduction of bacterial counts in the vegetations and of selection of mutants resistant to these antibiotics (Fig. 2).

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Efficacy of various antibiotic regimens for treatment of experimental endocarditis due to E. faecalis BM4275 (Vmr Tes). Vegetation homogenates were plated on agar to enumerate surviving bacteria and on agar containing teicoplanin (16 µg/ml) or gentamicin (32 µg/ml) to enumerate mutants resistant to teicoplanin or gentamicin, respectively. Log10 CFU per gram of vegetation are shown for individual rabbits. For the gentamicin, teicoplanin, and gentamicin-plus-vancomycin regimens, animals were assigned to two groups based on the presence or absence of mutants. The mean ± standard deviation of log10 CFU per gram was calculated for the two groups and for all rabbits.

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Conclusions. Combination of a glycopeptide with an aminoglycoside is the reference treatment for severe enterococcal infections if the patient is allergic to -lactams or if the strain is highly resistant to the latter antibiotics (18, 31). Efficacy of the combination is abolished, both in vitro and in animal models, by high-level resistance to vancomycin and teicoplanin mediated by gene clusters related to vanA (9, 17, 26). In contrast, combination of an aminoglycoside and teicoplanin may be efficient against VanB-type strains since the bacteria remain susceptible to teicoplanin. Indeed, combinations of teicoplanin and gentamicin (5) or streptomycin (19) were reported to be active against VanB-type enterococci in vitro and in experimental endocarditis, leading to the sterilization of cardiac vegetations in 25% (5) and 75% (19) of rabbits.