Moderate-Level Resistance to Glycopeptide LY Mediated by Genes of the vana and vanb Clusters in Enterococci

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1999, p Vol. 43, No /99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. Moderate-Level Resistance to Glycopeptide LY Mediated by Genes of the vana and vanb Clusters in Enterococci MICHEL ARTHUR, 1 FLORENCE DEPARDIEU, 1 * PETER REYNOLDS, 2 AND PATRICE COURVALIN 1 Unité des Agents Antibactériens, Institut Pasteur, Paris, Cedex 15, France, 1 and Department of Biochemistry, University of Cambridge, Cambridge, CB2 1 QW, United Kingdom 2 Received 8 March 1999/Returned for modification 23 April 1999/Accepted 1 June 1999 Three of five natural plasmids carrying a wild-type vana gene cluster did not confer LY glycopeptide resistance on Enterococcus faecalis JH2-2 (MIC 2 g/ml). The two remaining plasmids conferred resistance to the drug (MIC, 8 g/ml). The vanb gene cluster did not confer resistance to LY333328, since this antibiotic was not an inducer. Mutations in the vans B sensor gene that allowed induction by teicoplanin or constitutive expression of the vanb cluster led to LY resistance (MIC, 8 to 16 g/ml). Overproduction of the VanH, VanA, and VanX proteins for D-alanyl-D-lactate (D-Ala-D-Lac) synthesis and D-Ala-D-Ala hydrolysis was sufficient for resistance to LY (MIC, 16 g/ml). Mutations in the host D-Ala:D-Ala ligase contributed to LY resistance in certain VanA- and VanB-type strains, but the MICs of the antibiotic did not exceed 16 g/ml. Addition of D-2-hydroxybutyrate in the culture medium of mutants that did not produce the VanH D-lactate dehydrogenase led to incorporation of this D-2-hydroxy acid at the C-terminal ends of the peptidoglycan precursors and to LY resistance (MIC, 64 g/ml). The vanz gene of the vana cluster conferred resistance to LY (MIC, 8 g/ml) by an unknown mechanism. These data indicate that VanA- and VanB-type enterococci may acquire moderate-level resistance to LY (MIC < 16 g/ml) in a single step by various mechanisms. 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). LY is a semisynthetic N-alkylated glycopeptide active on VanA- and VanB-type enterococci (16, 28). The mechanisms of action of LY and the structurally related LY are believed to be identical (1, 2). These two glycopeptides differ in that LY has a chlorobiphenyl N-alkyl substitution on the vancosamine sugar, whereas LY has a chlorophenyl moiety (19). In solution, the affinities of LY and vancomycin for peptidoglycan precursor analogues with a C-terminal D-Lac residue are similarly low (2). However, the interaction of LY 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 * Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 28, rue du Dr. Roux, Paris Cedex 15, France. Phone: (33) (1) Fax: (33) (1) fdepard@pasteur.fr. Present address: Biochimie Structurale et Cellulaire, CNRS EP1088, Université Paris-Sud, Orsay Cedex, France. resistance to LY in VanA- and VanB-type enterococci. The activity of LY against a collection of glycopeptideresistant 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 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 g/ml) was added to the medium for growth of glycopeptide-dependent strains. The MICs of LY (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 10 5 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 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 g/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 C 18 reversephase column with 0.05 M ammonium acetate (ph 5.3) as the eluant at a flow 1875

2 1876 ARTHUR ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. MICs of LY333328, vancomycin, and teicoplanin against various VanA- and VanB-type strains Strain and characteristics MIC ( g/ml) of: LY a VM b TE c E. faecalis JH E. faecalis BM Group A1: E. faecium clinical isolates harboring Tn1546 or related elements on natural plasmids BM4147(pIP816) 2 1, HM1071(pIP837) 4 1, HM1072(pIP838) 2 1, HM1073(pIP839) HM1074(pIP840) 4 1, Source or reference Group A2: E. faecalis harboring Tn1546 or related elements on natural plasmids BM4110/pIP , JH2-2/pIP JH2-2/pIP JH2-2/pIP JH2-2/pIP Group A3: E. faecalis JH2-2 with vanrshax of Tn1546 at various copy numbers per chromosome BM4299 (1 copy) BM4300 (1 copy) BM4301 (2 copies) BM4302 (5 copies) JH2-2/pAT80 (20 copies) 16 2, Group A4: Glycopeptide-dependent derivative of E. faecalis JH2-2/pIP837 JH2-2/pIP Vm D Te D derivative of JH2-2/pIP837 d 16 2, This study Group A5: E. faecalis JH2-2 harboring combinations of genes from Tn1546 in multicopy plasmid pat29 pat80 (vanrshax) 16 2, pat382 (vanrshaxy) 8 1, pat398 (vanrshaxyz) 8 1, pat393 (P 2 vany) pat396 (P 2 vanz) e Group B1: E. faecalis BM4110 harboring transposon Tn1547 on pip964 and mutations in ddl and vans B BM4305 VanB (i) 1 1, BM4320 Vm D Ddl(S 319 -I) (i) NA 2,048 NA 13 BM4322 Vm D Te D Ddl(S 319 -I) VanS B (A 30 -G) (i) 16 2,048 2, BM4344 Vm r Te r VanS B (A 30 -G) (i) 2 1,024 1, BM4346 Vm r Te r VanS B (D 168 -Y) (i) 16 1,024 2, BM4347 Vm r Te r VanS B (T 237 -K) (c) 8 1,024 2, BM4348 Vm Het Te Het VanS B (Y 426 -ter) (i) , BM4323 Vm r Te r Ddl(S 319 -I) VanS B (Y 426 -ter) (c) 16 1,024 2, a LY, LY333328; NA, not applicable, since the strain did not grow in the absence of vancomycin. b VM, vancomycin. c TE, teicoplanin. d D, dependent. e c, constitutive; Het, heterogeneous; i, inducible; ter, termination. 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-Nacetylmuramyl-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,Dcarboxypeptidase. RESULTS AND DISCUSSION 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 LY (MIC 4 g/ml) (group A1 in Table 1). Three of the five corresponding plasmids did not confer resistance to LY on E. faecalis JH2-2 (group A2). The MIC of LY against strains harboring the two remaining plasmids was 8 g/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 LY for JH2-2, BM4281, and BM4316 are lower in broth (32).

3 VOL. 43, 1999 IN VITRO ACTIVITY OF GLYCOPEPTIDE LY 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-Lacending 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). Qualitatively, the results obtained for teicoplanin and LY were similar, since increases in the MICs of the drugs were detectable only for strains harboring 5 or 20 copies of the vanrshax gene cluster per chromosome (group A3 in Table 1). Quantitatively, high-level expression of the resistance genes in JH2-2/pAT80 conferred lower-level resistance to LY than to teicoplanin. Although incorporation of D-Ala-D-Ala into the peptidoglycan precursors of JH2-2/pAT80 is not detectable (7), this observation does not exclude the possibility that binding of LY to minute amounts of D-Ala-ending precursors could be responsible for the residual activity. In order to explore this possibility, we screened for mutants with increased resistance to glycopeptides that could potentially have reduced D-Ala:D-Ala ligase activity (13, 22, 31, 34). Mutants derived from JH2-2/pAT80 were not obtained on agar containing 16 g of LY333328/ml. A vancomycin-dependent derivative of JH2-2/pIP837 harboring a wild-type vana gene cluster on a natural plasmid was obtained on agar containing 1,024 g of vancomycin/ml (group A4 in Table 1). The MICs of vancomycin, teicoplanin, and LY for the mutant were increased two- to fourfold over those against the parental strain. Taken together, these data indicate that extensive replacement of D-Ala-ending precursors by D-Lac-ending precursors can be responsible for an eightfold increase in the MIC of LY (from 2 to 16 g/ml). No further increase in the level of LY resistance was obtained by this mechanism. This observation is in agreement with the proposal that binding of LY to D-Lac-ending precursors could be responsible for the inhibition of peptidoglycan synthesis (1, 2). Role of the VanY and VanZ accessory proteins in LY resistance. Previous analyses indicated that the VanY D,Dcarboxypeptidase 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 LY resistance (group A5 in Table 1). Expression of vanz alone under the control of the heterologous P 2 promoter led to four- and eightfold increases in the MICs of LY and teicoplanin, respectively (group A5 in Table 1). These results indicate that resistance to LY can be due to two mechanisms, as previously found for teicoplanin (6, 7). The first involves synthesis of D-Ala-D-Lac by the VanH dehydrogenase and the VanA ligase and elimination of D-Ala-ending precursors by the VanX and VanY D,D-peptidases. The second mechanism does not involve an alteration of the assembly pathway of UDP-MurNAc-tetra-D-Ala (6) and requires only the VanZ protein. Independently, the two mechanisms conferred similar levels of resistance to LY Combination of both mechanisms did not increase the MIC of the glycopeptide. Activity of LY 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 LY (group B1 in Table 1). A vancomycin-dependent mutant of BM4305 with the ddl(s 319 -I) mutation in the host D-Ala:D-Ala ligase gene did not grow in the presence of LY 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 (A 30 -G) ddl(s 319 -I)] grew in the presence of vancomycin, teicoplanin, and LY Thus, the A 30 -G substitution in VanS B allowed induction by teicoplanin and LY The vans B (A 30 -G) mutation alone led only to a twofold increase in the MIC of LY in comparison to wild-type BM4305 (VanB). In contrast, the vans B (D 168 -Y) and vans B (T 237 -K) mutations were sufficient for higher-level resistance to LY (MIC, 16 and 8 g/ml, respectively). These mutations were previously shown to lead to inducible [vans B (D 168 -Y)] or constitutive [vans B (T 237 -K)] expression of resistance to vancomycin and teicoplanin (13). Likewise, the vans B (Y 426 -ter) mutation responsible for the emergence of heterogeneous resistance to vancomycin and teicoplanin conferred resistance to LY (MIC 8 g/ml). Combination of the vans B (Y 426 -ter) and ddl(s 319 -I) mutations in BM4323 led to a further twofold increase in the MIC of LY These results indicate that VanB-type strains may simultaneously acquire resistance to teicoplanin and LY 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 LY did not exceed 16 g/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 LY 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 (vanrsh apha-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 LY (Table 2). The D-2-hydroxy acids had various side chains, including a hydrogen atom ( H) for glycolic acid, a methyl group ( CH 3 ) for D-lactate, an ethyl group ( CH 2 -CH 3 ) for D-2-hydroxybutyrate, and an isopropyl group [ CH(CH 3 ) 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

4 1878 ARTHUR ET AL. ANTIMICROB. AGENTS CHEMOTHER. E. faecalis strain c Hydroxy acid in culture medium (concn, mm) d TABLE 2. Cytoplasmic cell wall precursors of E. faecalis JH2-2 and derivatives Tripeptide % Cytoplasmic peptidoglycan precursor a Glycopeptide MIC ( g/ml) b Pentapeptide Pentadepsipeptide ending in: Lac HBut HVal Glycolic acid LY VM TE JH2-2 No addition JH2-2/pAT83 No addition D,L-Lactate (50) ,048 4 D,L-2-Hydroxybutyrate (50) D,L-2-Hydroxyvalerate (10) Glycolic acid (50) JH2-2/pAT83 m1 e No addition ,048 4 D,L-Lactate (50) , D,L-2-Hydroxybutyrate (50) ,096 1,024 D,L-2-Hydroxyvalerate (10) , JH2-2/pAT83 m2 No addition D,L-Lactate (50) , D,L-2-Hydroxybutyrate (50) , D,L-2-Hydroxyvalerate (10) , Glycolic acid (50) ,048 4 a Tripeptide, UDP-MurNAc-tripeptide; pentapeptide, UDP-MurNAc-tetra-D-Ala; pentadepsipeptide, UDP-MurNAc-tetra-D-2-hydroxy acid; Lac, D-lactate; HBut, D-2-hydroxybutyrate; HVal, D-2-hydroxyvalerate. b LY, LY333328; TE, teicoplanin; VM, vancomycin. c Plasmid pat83 harbors the vanrshax gene cluster with vanh inactivated by insertion of the apha-1 kanamycin resistance gene. Spontaneous mutants of E. faecalis JH2-2/pAT83 were selected on medium containing 1 mm D,L-hydroxyvalerate and 8 g of vancomycin/ml (mutant m1) or 50 mm glycolic acid and 64 g of vancomycin/ml (mutant m2). d For analysis of peptidoglycan precursors, the medium also contained 4 g of vancomycin/ml, except for JH2-2/pAT83 grown in the absence of D,L-2-hydroxy acid (vancomycin, 0.5 g/ml) and JH2-2 (no vancomycin added). e Glycolic acid was not tested because JH2-2/pAT83 m1 did not grow in 24 h in the presence of this compound at 50 mm. interaction with the methyl side chain of D-Lac (glycolic acid) (17, 18, 29). Incorporation of D-Lac into the precursors of JH2-2/pAT83 was detected even in the absence of supplementation of the culture medium (Table 2). However, D-Lac was not present in sufficient amounts to compete efficiently with D-Ala, thus leading to the coproduction of D-Ala- and D-Lac-ending precursors. The MIC of vancomycin was increased only twofold in comparison to that for JH2-2 (Table 2). Supplementation of the culture medium of JH2-2/pAT83 with D-Lac and D-2-hydroxybutyrate resulted in high-level resistance to vancomycin and moderate-level resistance to teicoplanin and LY Peptidoglycan precursors ending in D-Ala were not synthesized at a detectable level ( 2%). The D-2-hydroxybutyrate added to the culture medium was efficiently used by VanA, since 96% of the peptidoglycan precursors contained this D-2-hydroxy acid. Incorporation of D-2-hydroxyvalerate and glycolic acid was less efficient (36 and 49% of the precursors, respectively). Supplementation of the medium with D-2-hydroxyvalerate reduced but did not abolish the incorporation of D-Ala into the precursors (from 19 to 6%). This was associated with low-level resistance to vancomycin and susceptibility to teicoplanin and LY Similar amounts of peptidoglycan precursors ending in D-Ala were detected in the presence (18%) or absence (19%) of glycolic acid. Accordingly, supplementation of the culture medium with this acid did not lead to glycopeptide resistance. 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 g of vancomycin/ml (mutant m1) or 50 mm glycolic acid and 64 g 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. Supplementation of the culture medium with D-2-hydroxybutyrate, D-2-hydroxyvalerate, or glycolic acid led to the incor-

5 VOL. 43, 1999 IN VITRO ACTIVITY OF GLYCOPEPTIDE LY poration of these D-2-hydroxy acids in the peptidoglycan precursors of mutants m1 and m2 and to increased resistance to vancomycin, teicoplanin, and LY (Table 2). The levels of resistance to teicoplanin and LY were higher for production of precursors ending in D-2-hydroxybutyrate than for production of the natural precursors terminating in D-Lac. Of note, the MIC of LY obtained for mutants m1 and m2 grown in the presence of D-2-hydroxybutyrate corresponds to the highest MIC obtained in this study (64 g/ml). These observations suggest that the binding of LY to peptidoglycan precursors is reduced by steric hindrance if the methyl side chain of D-Lac is replaced by an ethyl group. Whether residual activity of LY is actually due to binding of the drug to peptidoglycan precursors is not known. As discussed by Ge et al. (23), experiments support the hypothesis that membrane anchoring and dimerization can significantly increase D-Ala-D-Ala binding avidity, but there is little evidence that these features enhance binding to D-Ala-D-Lac sufficiently to explain the antibacterial activity of the substituted glycopeptide against resistant bacteria. These authors showed that vancomycin derivatives carrying a chlorobiphenyl substituent, such as LY333328, remain active even in the absence of an intact peptidoglycan precursor binding pocket. Moreover, the chlorobiphenyl-substituted carbohydrate moiety of the molecule was sufficient to inhibit transglycosylation and bacterial growth, albeit at high concentrations (on the order of 100 g/ml). The finding that the peptide binding pocket is not essential for biological activity suggests that substituted glycopeptides have an additional mode of action. Our results indicate that the activity of LY against VanA-type enterococci is partly due to binding to residual D-Ala-ending precursors and suggest that this glycopeptide might interact with D-Lac-ending precursors, since replacement of D-Lac by D-2- hydroxybutyrate increased the level of resistance. LY might inhibit the growth of enterococci producing D-2-hydroxybutyrate-ending precursors by a different, unknown mechanism that appears to require relatively high concentrations of the drug (64 g/ml [Table 2]), in agreement with the data presented by Ge et al. (23). The VanA ligase has a broad substrate specificity (18). Detection of peptidoglycan precursors ending in D-2-hydroxybutyrate, D-2-hydroxyvalerate, and glycolic acid (Table 2) indicates that various depsipeptides synthesized by VanA can be added to UDP-MurNAc-tripeptide. Furthermore, the increase in glycopeptide resistance associated with production of these precursors strongly suggests that they can be used for peptidoglycan synthesis. Glycopeptide-resistant enterococci produce peptidoglycan precursors ending in D-Lac or D-Ser (17, 18). Further evolution of these resistance pathways leading to incorporation of other substituents in the C-terminal positions of peptidoglycan precursors could occur if the bacteria gain the capacity to synthesize these compounds. 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 LY (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 LY resistance. Finally, cross-resistance to teicoplanin and LY was acquired following various mutations in the vans B sensor gene of the vanb cluster. These observations indicate that emergence of LY 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 LY resistance obtained by these mechanisms were moderate (MIC 16 g/ml), probably because replacement of the C-terminal D-Lac residue of peptidoglycan precursors by another D-2-hydroxy acid is required for higherlevel resistance to this glycopeptide. A high dosage of LY or the combination of this glycopeptide with an aminoglycoside might be able to prevent the emergence of LY resistance, since the LY 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). ACKNOWLEDGMENTS This work was supported in part by Eli Lilly & Co., France, and by a Bristol-Myers Squibb Unrestricted Biomedical Research Grant in Infectious Diseases. REFERENCES 1. Allen, N. E., J. N. Hobbs, Jr., and T. I. Nicas Inhibition of peptidoglycan biosynthesis in vancomycin-susceptible and -resistant bacteria by a semisynthetic glycopeptide antibiotic. Antimicrob. Agents Chemother. 40: Allen, N. E., D. L. LeTourneau, and J. N. Hobbs, Jr Molecular interactions of a semisynthetic glycopeptide antibiotic with D-alanyl-D-alanine and D-alanyl-D-lactate residues. Antimicrob. Agents Chemother. 41: Arthur, M., and P. Courvalin Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob. Agents Chemother. 37: Arthur, M., F. Depardieu, L. Cabanié, P. Reynolds, and P. Courvalin Requirement of the VanY and VanX D,D-peptidases for glycopeptide resistance in enterococci. Mol. Microbiol. 30: Arthur, M., F. Depardieu, G. Gerbaud, M. Galimand, R. Leclercq, and P. Courvalin The VanS sensor negatively controls VanR-mediated transcriptional activation of glycopeptide resistance genes of Tn1546 and related elements in the absence of induction. J. Bacteriol. 179: Arthur, M., F. Depardieu, C. Molinas, P. Reynolds, and P. Courvalin The vanz gene of Tn1546 from Enterococcus faecium BM4147 confers resistance to teicoplanin. Gene 154: Arthur, M., F. Depardieu, P. Reynolds, and P. Courvalin Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide-resistant enterococci. Mol. Microbiol. 21: Arthur, M., F. Depardieu, H. A. Snaith, P. E. Reynolds, and P. Courvalin Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors. Antimicrob. Agents Chemother. 38: Arthur, M., C. Molinas, T. D. H. Bugg, G. D. Wright, C. T. Walsh, and P. Courvalin Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 36: Arthur, M., C. Molinas, and P. Courvalin The VanS-VanR twocomponent regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J. Bacteriol. 174: Arthur, M., P. Reynolds, and P. Courvalin Glycopeptide resistance in enterococci. Trends Microbiol. 4: Aslangul, E., M. Baptista, B. Fantin, F. Depardieu, M. Arthur, P. Courvalin, and C. Carbon Selection of glycopeptide-resistant mutants of VanBtype Enterococcus faecalis BM4281 in vitro and in experimental endocarditis. J. Infect. Dis. 175: Baptista, M., F. Depardieu, P. Reynolds, P. Courvalin, and M. Arthur Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB-type enterococci. Mol. Microbiol. 25: Barna, J. C. J., and D. H. Williams The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu. Rev. Microbiol. 38: Beauregard, D., A. Maguire, D. Williams, and P. Reynolds Semiquantitation of cooperativity in binding of vancomycin-group antibiotics to vancomycin-susceptible and -resistant organisms. Antimicrob. Agents Chemother. 41: Biavasco, F., C. Vignaroli, R. Lupidi, E. Manso, B. Facinelli, and P. E.

6 1880 ARTHUR ET AL. ANTIMICROB. AGENTS CHEMOTHER. Varaldo In vitro antibacterial activity of LY333328, a new semisynthetic glycopeptide. Antimicrob. Agents Chemother. 41: Billot-Klein, D., D. Blanot, L. Gutmann, and J. van Heijenoort Association constants for the binding of vancomycin and teicoplanin to N- acetyl-d-alanyl-d-alanine and N-acetyl-D-alanyl-D-serine. Biochem. J. 304: Bugg, T. D. H., G. D. Wright, S. Dutka-Malen, M. Arthur, P. Courvalin, and C. T. Walsh Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry 30: Chopra, I N-alkyl-substituted glycopeptide antibiotics. Exp. Opin. Investig. Drugs 6: Courvalin, P., and C. Carlier Transposable multiple antibiotic resistance in Streptococcus pneumoniae. Mol. Gen. Genet. 205: Evers, S., and P. Courvalin Regulation of VanB-type vancomycin resistance gene expression by the VanS B -VanR B two-component regulatory system in Enterococcus faecalis V583. J. Bacteriol. 178: Fraimow, H. S., D. L. Jungkind, D. W. Lander, D. R. Delso, and J. L. Dean Urinary tract infection with an Enterococcus faecalis isolate that requires vancomycin for growth. Ann. Intern. Med. 121: Ge, M., Z. Chen, H. R. Onishi, J. Kohler, L. L. Silver, R. Kerns, S. Fukusawa, C. Thompson, and D. Kahne Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala. Science 284: Hayden, M. K., G. M. Trenholme, J. E. Schultz, and D. F. Sahm In vivo development of teicoplanin resistance in a VanB Enterococcus faecium isolate. J. Infect. Dis. 167: Jacob, A. E., and S. J. Hobbs Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J. Bacteriol. 117: Leclercq, R., E. Derlot, J. Duval, and P. Courvalin Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N. Engl. J. Med. 319: Messer, J., and P. E. Reynolds Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci. FEMS Microbiol. Lett. 94: Nicas, T., M. Zeckel, and D. Braun Beyond vancomycin: new therapies to meet the challenge of glycopeptide resistance. Trends Microbiol. 5: Nieto, M., and H. R. Perkins Modifications of the acyl-d-alanyl-dalanine terminus affecting complex-formation with vancomycin. Biochem. J. 123: Quintiliani, R., Jr., and P. Courvalin Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281. Gene 172: Rosato, A., J. Pierre, D. Billot-Klein, A. Buu-Hoi, and L. Gutmann Inducible and constitutive expression of resistance to glycopeptides and vancomycin dependence in glycopeptide-resistant Enterococcus avium. Antimicrob. Agents Chemother. 39: Saleh-Mghir, A., A. Lefort, Y. Petegnief, S. Dautrey, J.-M. Vallois, D. Le Guludec, C. Carbon, and B. Fantin Activity and diffusion of LY in experimental endocarditis due to vancomycin-resistant Enterococcus faecalis. Antimicrob. Agents Chemother. 43: Steers, E., E. L. Foltz, B. S. Graves, and J. Riden An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. (Basel) 9: Van Bambeke, F., M. Chauvel, P. Reynolds, H. Fraimow, and P. Courvalin Vancomycin-dependent Enterococcus faecalis clinical isolates and revertant mutants. Antimicrob. Agents Chemother. 43: Wright, G. D., and C. T. Walsh D-Alanyl-D-alanine ligases and the molecular mechanism of vancomycin resistance. Acc. Chem. Res. 25: Zelenitsky, S. A., B. Booker, N. Laing, J. A. Karlowsky, D. J. Hoban, and G. G. Zhanel Synergy of an investigational glycopeptide, LY333328, with once-daily gentamicin against vancomycin-resistant Enterococcus faecium in a multiple-dose, in vitro pharmacodynamic model. Antimicrob. Agents Chemother. 43: Downloaded from on April 8, 2018 by guest