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 in susceptible bacteria (11, 35). The substitution prevents the formation of complexes between glycopeptides and peptidoglycan precursors that are responsible for the inhibition of cell wall synthesis (14). Resistance by this mechanism is mediated by two types of gene clusters, vanA and vanB, which encode related proteins (21). Gene clusters related to vanA confer high-level resistance to vancomycin and teicoplanin (3). In contrast, enterococci belonging to the vanB hybridization class remain susceptible to teicoplanin because the VanS_B sensor kinase does not trigger induction of the resistance genes in response to this antibiotic (13, 21). Coproduction of peptidoglycan precursors ending in D-Ala and D-Lac leads to low-level vancomycin resistance in certain VanB-type strains that express the resistance genes at a low level (7).

LY333328 is a semisynthetic N-alkylated glycopeptide active on VanA- and VanB-type enterococci (16, 28). The mechanisms of action of LY333328 and the structurally related LY191145 are believed to be identical (1, 2). These two glycopeptides differ in that LY333328 has a chlorobiphenyl N-alkyl substitution on the vancosamine sugar, whereas LY191145 has a chlorophenyl moiety (19). In solution, the affinities of LY191145 and vancomycin for peptidoglycan precursor analogues with a C-terminal D-Lac residue are similarly low (2). However, the interaction of LY191145 with D-Lac-ending precursors may be significantly increased in vivo, since the drug binds to the membrane and dimerizes (2, 15).

This study was undertaken to assess the risk of emergence of resistance to LY333328 in VanA- and VanB-type enterococci. The activity of LY333328 against a collection of glycopeptide-resistant strains constructed in vitro that overproduce the resistance proteins or harbor mutations in the vanS_B sensor and host D-Ala:D-Ala ligase genes was studied.

	MATERIALS AND METHODS

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Strains and growth conditions. The origins and properties of the strains are described in Table 1. Enterococcus faecalis JH2-2 and BM4110 are derivatives of strain JH2 that are resistant to fusidic acid and rifampin or to streptomycin, respectively. Strains were grown in brain heart infusion (BHI) broth and agar (Difco Laboratories, Detroit, Mich.) at 37°C. Vancomycin (10 µg/ml) was added to the medium for growth of glycopeptide-dependent strains. The MICs of LY333328 (Eli Lilly & Co., Saint-Cloud, France), vancomycin (Eli Lilly & Co.), and teicoplanin (Hoechst-Marion-Roussel, Levallois-Perret, France) were determined by the method of Steers et al. (33) with 10⁵ CFU per spot on BHI agar after 24 h of incubation at 37°C. Since there are as yet no formally recognized breakpoints for LY333328, resistance is defined in this work as an increase in the MIC of the glycopeptide in comparison to that for the glycopeptide-susceptible host E. faecalis JH2-2. Glycolic acid, D,L-lactate lithium salt, D,L-2-hydroxybutyrate sodium salt, and D,L-2-hydroxyvalerate sodium salt (Sigma, Saint-Quentin Falavier, France) were added to the medium as 1 M neutralized (pH 7.0) solutions. Economic constraints imposed the use of D,L-2-hydroxy acids, although the VanA ligase uses exclusively D-2-hydroxy acids as substrates (18). For selection of spontaneous mutants, strains were grown overnight in broth and concentrated by centrifugation, and ca. 5 × 10⁹ CFU was plated on agar containing appropriate antibiotics.

Analysis of peptidoglycan precursors. Extraction and analysis of peptidoglycan precursors was performed essentially as described previously (27). Enterococci were grown in BHI broth (120 ml) supplemented with 0.5% yeast extract, vancomycin, and D,L-2-hydroxy acids at the concentrations specified in Table 2. At mid-exponential phase (optical density at 600 nm = 1), ramoplanin (3 µg/ml) was added, and incubation was continued for 20 min. Bacteria were harvested by centrifugation at 12,000 × g for 2 min at 4°C, resuspended in water, and treated with 7% trichloroacetic acid for 15 min at 0°C in a final volume of 2 ml. The extract was centrifuged (at 48,000 × g for 1 min at 4°C), and the supernatant fraction containing the cytoplasmic peptidoglycan precursors was collected. Trichloroacetic acid was neutralized by the addition of solid sodium bicarbonate, and salt was removed from the extract by gel filtration of 0.5-ml samples on a Sephadex G-10 column (28 by 1 cm). The eluate was monitored at 252 nm, and cell wall precursors eluted immediately after the void volume of the column. Twenty-microliter samples of the fraction containing the cell wall precursors were analyzed by high-performance liquid chromatography on a C₁₈ reverse-phase column with 0.05 M ammonium acetate (pH 5.3) as the eluant at a flow rate of 0.8 ml per min and with the application of two consecutive methanol gradients in the same buffer (0 to 2.5% between 5 and 45 min; 2.5 to 7.5% between 45 and 60 min). Elution was continued for a further 15 min. The elution times for the precursors were as follows: for UDP-N-acetylmuramyl-L-Ala--D-Glu-L-Lys (UDP-MurNAc-tripeptide), 6 min; for UDP-N-acetylmuramyl-L-Ala--D-Glu-L-Lys-D-Ala-D-Ala (UDP-MurNAc-tetra-D-Ala), 22 min; for UDP-N-acetylmuramyl-L-Ala--D-Glu-L-Lys-D-Ala-D-Lac (UDP-MurNAc-tetra-D-Lac), 39 min; for UDP-N-acetylmuramyl-L-Ala--D-Glu-L-Lys-D-Ala-glycolic acid (UDP-MurNAc-tetra-Gly a.), 28 min; for UDP-N-acetylmuramyl-L-Ala--D-Glu-L-Lys-D-Ala-D-2-hydroxybutyrate (UDP-MurNAc-tetra-D-HBut), 57 min; and for UDP-N-acetylmuramyl-L-Ala--D-Glu-L-Lys-D-Ala-D-2-hydroxyvalerate (UDP-MurNAc-tetra-D-HVal), 68 min. The relative proportions of these peptidoglycan precursors were determined from the integrated peak areas, and the results were expressed as percentages. UDP-MurNAc-tetrapeptide was not detected in significant amounts, since the strains analyzed did not produce the VanY D,D-carboxypeptidase.

	RESULTS AND DISCUSSION

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Activities of glycopeptides against wild VanA-type strains. The five clinical isolates of Enterococcus faecium harboring wild-type vanA gene clusters on natural plasmids were susceptible to LY333328 (MIC  4 µg/ml) (group A1 in Table 1). Three of the five corresponding plasmids did not confer resistance to LY333328 on E. faecalis JH2-2 (group A2). The MIC of LY333328 against strains harboring the two remaining plasmids was 8 µg/ml. Thus, the glycopeptide remained active or partially active in spite of high-level resistance to vancomycin and teicoplanin as reported elsewhere (16, 28). It is worth noting that the MICs of LY333328 for JH2-2, BM4281, and BM4316 are lower in broth (32).

Effect of coproduction of D-Ala- and D-Lac-ending peptidoglycan precursors on the activities of glycopeptides. We previously showed that constitutive high-level production of the VanH dehydrogenase, VanA D-Ala:D-Lac ligase, and VanX D,D-dipeptidase encoded by the vanRSHAX gene cluster present on a multicopy plasmid was necessary and sufficient for high-level vancomycin and teicoplanin resistance (JH2-2/pAT80; group A3 in Table 1) (7). In this strain, production of peptidoglycan precursors ending in D-Ala was not detectable (2%) (7). Vancomycin and teicoplanin remained partially active against strains harboring one to five chromosomal copies of the vanRSHAX gene cluster, since these strains coproduced D-Ala- and D-Lac-ending precursors (7). The increase in the copy number of the resistance genes (from 1 to 5) was associated with an increase in the relative proportion of D-Lac-ending precursors (from 51 to 96%) (7). The vancomycin resistance level increased as a function of increased replacement of D-Ala by D-Lac at the C-terminal ends of peptidoglycan precursors (7). Resistance to teicoplanin required more complete elimination of D-Ala-ending precursors (7).

Role of the VanY and VanZ accessory proteins in LY333328 resistance. Previous analyses indicated that the VanY D,D-carboxypeptidase and the VanZ protein contribute to vancomycin and teicoplanin resistance in a host that coproduces peptidoglycan precursors ending in D-Ala and D-Lac (4, 6, 7). Introduction of vanY or of both vanY and vanZ in pAT80 (vanRSHAX) did not result in an increase in the level of LY333328 resistance (group A5 in Table 1).

Activity of LY333328 against a VanB-type E. faecalis strain and teicoplanin-resistant or glycopeptide-dependent derivatives. E. faecalis BM4305 harbors transposon Tn1547 on plasmid pIP964. The wild-type copy of the vanB cluster located in Tn1547 did not confer resistance to LY333328 (group B1 in Table 1). A vancomycin-dependent mutant of BM4305 with the ddl(S₃₁₉-I) mutation in the host D-Ala:D-Ala ligase gene did not grow in the presence of LY333328 or teicoplanin, indicating that these glycopeptides did not induce expression of the vanB D-Ala:D-Lac ligase gene (see reference 13 for a discussion). The double mutant BM4322 [vanS_B(A₃₀-G) ddl(S₃₁₉-I)] grew in the presence of vancomycin, teicoplanin, and LY333328. Thus, the A₃₀-G substitution in VanS_B allowed induction by teicoplanin and LY333328. The vanS_B(A₃₀-G) mutation alone led only to a twofold increase in the MIC of LY333328 in comparison to wild-type BM4305 (VanB). In contrast, the vanS_B(D₁₆₈-Y) and vanS_B(T₂₃₇-K) mutations were sufficient for higher-level resistance to LY333328 (MIC, 16 and 8 µg/ml, respectively). These mutations were previously shown to lead to inducible [vanS_B(D₁₆₈-Y)] or constitutive [vanS_B(T₂₃₇-K)] expression of resistance to vancomycin and teicoplanin (13). Likewise, the vanS_B(Y₄₂₆-ter) mutation responsible for the emergence of heterogeneous resistance to vancomycin and teicoplanin conferred resistance to LY333328 (MIC = 8 µg/ml). Combination of the vanS_B(Y₄₂₆-ter) and ddl(S₃₁₉-I) mutations in BM4323 led to a further twofold increase in the MIC of LY333328. These results indicate that VanB-type strains may simultaneously acquire resistance to teicoplanin and LY333328 in a single step. The emergence of resistance to these glycopeptides can be due to three alterations of the VanS_B sensor that lead to inducible, constitutive, or heterogeneous expression of the resistance genes. The MIC of LY333328 did not exceed 16 µg/ml for any of the mutants studied, even though certain mutants harbored combinations of mutations in vanS_B and ddl. Quantitatively, the level of resistance against LY333328 was significantly lower than that against teicoplanin.

Incorporation of various D-2-hydroxy acids at the C-terminal extremity of peptidoglycan precursors of JH2-2/pAT83. Insertional inactivation of the vanH dehydrogenase gene of pAT80 (vanRSHAX) was reported to lead to glycopeptide susceptibility (10). The addition of D-Lac in the culture medium restores the glycopeptide resistance of the resulting vanH null mutant JH2-2/pAT83 (vanRSHaphA-1AX) (9). This indicates that D-Lac penetrates into the cytoplasm and is incorporated in the depsipeptide D-Ala-D-Lac by the VanA ligase (9). The addition of various D-2-hydroxy acids was tested in this system to determine whether incorporation of homologues of D-Lac into the peptidoglycan precursors could confer high-level resistance to LY333328 (Table 2). The D-2-hydroxy acids had various side chains, including a hydrogen atom (H) for glycolic acid, a methyl group (CH₃) for D-lactate, an ethyl group (CH₂-CH₃) for D-2-hydroxybutyrate, and an isopropyl group [CH(CH₃)₂] for D-2-hydroxyvalerate. These D-2-hydroxy acids were previously shown to be substrates in vitro for the VanA ligase, with the exception of glycolic acid, which was not tested (18). Replacement of D-Lac by the D-2-hydroxy acids could potentially reduce the binding of glycopeptides to peptidoglycan precursors by steric hindrance (D-2-hydroxybutyrate and D-2-hydroxyvalerate) or by suppressing the hydrophobic interaction with the methyl side chain of D-Lac (glycolic acid) (17, 18, 29).

Selection of m1 and m2 mutants. The D-2-hydroxy acids added to the culture medium of JH2-2/pAT83 competed with the endogenous sources of D-Ala and D-Lac, leading to coproduction of various peptidoglycan precursors (Table 2). In order to improve incorporation of the D-2-hydroxy acids, mutants of E. faecalis JH2-2/pAT83 were selected on agar containing 1 mM D,L-hydroxyvalerate and 8 µg of vancomycin/ml (mutant m1) or 50 mM glycolic acid and 64 µg of vancomycin/ml (mutant m2). Incorporation of D-Ala in the peptidoglycan precursors of mutants m1 and m2 was not detectable even in the absence of supplementation (Table 2). As expected, these mutants expressed vancomycin resistance in the medium devoid of D-2-hydroxy acid. The mutations in m1 and m2 did not allow increased incorporation of D-2-hydroxyvalerate, D-2-hydroxybutyrate, or glycolic acid to the detriment of D-Lac. Further attempts to obtain mutants that expressed resistance only in media containing D-2-hydroxybutyrate, D-2-hydroxyvalerate, or glycolic acid were unsuccessful (data not shown). Thus, mutations that result specifically in increased incorporation of D-2-hydroxy acids added to the culture medium were not obtained.

Characteristics of mutants m1 and m2. In the absence of supplementation of the culture medium, JH2-2/pAT83 accumulated low amounts of UDP-MurNAc-tripeptide (Table 2). This observation suggests that the combined pool of D-Ala-D-Ala and D-Ala-D-Lac was limiting, probably because the dipeptide was hydrolyzed by the VanX D,D-dipeptidase and the concentration of D-Lac was too low for efficient synthesis of the depsipeptide. In agreement with this notion, the tripeptide was not detected if the medium was supplemented with the D-2-hydroxy acids. Synthesis of D-Ala-D-Ala may be impaired in mutant m2; this would account for the absence of precursors ending in D-Ala and the accumulation of the tripeptide only in the absence of supplementation. A distinct mutation was present in m1, since large amounts of the tripeptide were detected under all conditions.

Conclusions. In agreement with previous studies (16, 28), acquisition of wild-type vanA and vanB gene clusters by E. faecalis had little or no effect on the in vitro activity of LY333328 (Table 1). However, VanA- and VanB-type enterococci may acquire resistance to this glycopeptide by various mechanisms. Production of precursors ending in D-Lac was sufficient for resistance to this glycopeptide if precursors terminating in D-Ala were completely eliminated (Table 1). This could be achieved by increased expression of the resistance genes or reduced production of D-Ala-D-Ala by the host ligase. Expression of the vanZ gene may also confer LY333328 resistance. Finally, cross-resistance to teicoplanin and LY333328 was acquired following various mutations in the vanS_B sensor gene of the vanB cluster. These observations indicate that emergence of LY333328 resistance should be anticipated, since mutations in ddl and in vanS_B are known to be selected under treatment and are present in natural populations of VanA- and VanB-type enterococci (12, 24, 31, 34). However, the levels of LY333328 resistance obtained by these mechanisms were moderate (MIC  16 µg/ml), probably because replacement of the C-terminal D-Lac residue of peptidoglycan precursors by another D-2-hydroxy acid is required for higher-level resistance to this glycopeptide. A high dosage of LY333328 or the combination of this glycopeptide with an aminoglycoside might be able to prevent the emergence of LY333328 resistance, since the LY333328-gentamicin combination is synergistic against vancomycin-resistant E. faecium (36) and the teicoplanin-gentamicin combination prevents the emergence of teicoplanin resistance in VanB-type enterococci (12).