J -M Ghuysen and R Hakenbeck (Eds ), Hacrenal Cell Wall 0 1994 Elsevier Science B V All nghts reserved 535

CHAPTER 26

Resistance to glycopeptide antibiotics REGINE HAKENBECK

Max-Planck lnstitut fur Molekulare Genetik, Ihnestrasse 73, 0-14195 Berlin 33, Germany

1. Introduction

The history of glycopeptides represents a perfect example of the various circumstances that provoke medical and scientific interest in an antibiotic. Due to increased isolation of Staphylococcus aureus strains resistant against antibiotics such as erythromycin, tetracy- clin and penicillin in the early 1950s, a large-scale screening program aimed at the identi- fication of antibiotics with high anti-staphylococcal activity was initiated. As a result, the first glycopeptide-producing microorganism Streptomyces orientalis was found in a soil sample from the jungle in Borneo. The compound isolated from the fermentation broth (early lots of which were named ‘Mississippi mud’ due to its appearance, later it was named vancomycin) was shown to be highly active against Gram-positive bacterial spe- cies [ 1,2]. Laboratory-induced levels of staphylococcal resistance against vancomycin were almost negligible [3,4] and resistance remained of no clinical significance for a long time. Vancomycin was soon introduced clinically, but problems with toxicity and the in- troduction of B-lactamase-resistant penicillins in the early 1960s caused a decline in the use of vancomycin. The appearance of methicillin-resistant staphylococci and other, mul- tiply resistant Gram-positive organisms revived interest in glycopeptide antibiotics espe- cially since improved purification procedures largely reduced early toxicity problems of vancomycin [5,6]. Other glycopeptides were used as animal feed additives (see [7] and refs. therein), whereas ristocetin, isolated for the first time from Nocurdiu luridu [8] in the 1950s, causes aggregation of blood platelets [9,10] and could therefore not be used thera- peutically. Recently, teicoplanin (produced by Actinoplanes teichomyceticus nov. sp. [ 1 11) has been introduced for the treatment of serious infections in human due to resistant Gram-positive bacteria or in cases of p-lactam allergy. The emergence of resistance to glycopeptide antibiotics in staphylococci and enterococci confronts scientists with a bac- terial defence mechanism which is unexpected and had been considered to be highly im- probable. It results in a severe clinical problem and a demand for new antibacterial agents.

2. Structure of glycopeptides

The structures of vancomycin and teicoplanin (Fig. 1) reveal several features common to all members of the glycopeptide group [7,12]. They contain a central heptapeptide back-

536

F A C T O R R 1

2

3 7'"-

h c o -

-c 0-

OH

Fig. 1. Structures of (a) teicoplanin and (b) vancomycin.

537

bone with five highly conserved amino acids. The amino acids are linked via several phe- nolic acid residues, resulting in the formation of complex tetra- or tricyclic structures. The compounds have seven aromatic rings (except vancomycin which has five) carrying vari- ous substituents. The variable presence of chlorine groups poses interesting biosynthetic questions. Attached to the core structure, the aglycone, are a variable number of some- times unusual sugars or amino sugars which are not essential for antibiotic activity. The source of these sugars is largely unknown. In the teicoplanin complex, an amino sugar carries the various fatty acids characteristic for each member, rendering the molecule more hydrophobic than vancomycin [ 13,141.

3. Mode of action

It is important to realize that the early biochemical studies on the mode of action of gly- copeptides were carried out before the structure of these antibiotics was known. The elu- cidation of the spatial arrangement of glycopeptides then confirmed and illustrated the unique interaction between these compounds and their target, a specific bacterial cell wall component. Much of this work has been reviewed in several articles [7,12,15 and refs. therein], and the following summarizes the major aspects.

Glycopeptide antibiotics are too large ( > I kDa) to cross the outer membrane of Gram- negative organisms and are hence effective only against Gram-positive bacteria. Initial experiments suggesting that vancomycin interferes with cell wall synthesis were reported by Reynolds and Jordan who showed that vancomycin-treated bacteria accumulate UDP- N-acetylmuramyl-peptides [ 16,171. Vancomycin and ristocetin form a 1 : 1 complex with UDP-MurNAc-pentapeptide [18], and bind not only to bacterial cells but also to cell walls [ 19-2 I]. The specificity of this binding was elucidated by Perkins and collabora- tors, including the important observations that ristocetin or vancomycin binding could be reversed by addition of peptides ending with acyl-D-Ala-D-Ala [22], and that prerequisites of this binding were the D-configuration of both alanine residues and a free terminal car- boxyl group [23-261.

A key step in elucidating the interaction of glycopeptides with the target site was the determination of the three-dimensional structure of a vancomycin degradation product, CDP-1, and its complex with acetyl-D-Ala-D-Ala by X-ray analysis [27]. Further struc- tural refinement and stereochemical details were obtained by applying mass spectrometry and 'H NMR methods, and from analysis of nuclear Overhauser effects [7,12]. Figure 2 illustrates the hydrogen bond interactions between the aglycone of a glycopeptide and its target peptide, and a space-filling model of the complex derived from computer analysis is represented in Fig. 3. The acyl-D-Ala-D-Ala terminus fits tightly into a clef? of the anti- biotic ristocetin or aridicin; vancomycin which is a more flexible molecule undergoes a large conformational change on binding to target peptides, and the bound form closely resembles the ristocetidaridicin complex.

Ward [28] and Johnston and Neuhaus [29] have suggested that the main target peptide of glycopeptides is represented by the growing glycan chain rather than the lipid inter- mediate. In the presence of glycopeptides, transpeptidase and D,D-carboxypeptidase ac- tivities that depend on accessibility of C-terminal D-Ala-D-Ala residues will be prevented.

538

Fig. 2. Representation of an aridicin-like Ac+-Lys-DAla-DAla complex showing interpeptide hydrogen bonds (from 1121).

This has been shown for the soluble D,D-carboxypeptidase of Streptomyces albus G [30] and for Escherichia coli enzymes [3 1,321. It is also conceivable that a transglycosylase reaction may be inhibited by glycopeptides by steric hindrance although the disaccharide portion of the muropeptide is not masked in the glycopeptide complex [33]. This is in agreement with early in vitro studies where murein synthesis (due to polymerization of uncrosslinked murein) was blocked by vancomycin and ristocetin [34,35], and later ex- periments where the polymerization reaction was studied directly [36]. Interference with multiple critical steps in murein assembly may well explain why it was impossible to reach significant resistance levels in the laboratory. Resistance to glycopeptides due to alterations in the target site was therefore believed to be highly unlikely; it would require alterations in the specificity of a whole set of enzymes involved in biosynthesis of the murein layer.

539

4. Phenotypes of glycopeptide resistant bacteria

Several Gram-positive species are intrinsically constitutively resistant to glycopeptides. This includes high level resistant species like actinomycetes (the glycopeptide producers), and leuconostoc, pediococcus and lactobacillus species [37-42]. Low level intrinsically resistant strains are Enterococcus gallinarum and Enterococcus casselrjlavus [4445]. Although still uncommon, the clinical isolation of those strains is reported more fie- quently [46 and refs. therein]. Glycopeptide resistant strains of other Gram-positive spe- cies have rarely been observed [47 and refs. therein, 481, but they have not been investi- gated in further detail.

Fig. 3. Computer-generated model showing space-filling representation of (A) aridicin aglycone and (9) aridicin aglycone-diacctyl-L-Ala-(y)-~Gln-L-Lys(Ac)-~Ala-DAla complex. Structures were obtained using

distance geometry with subsequent energy minimization (from [ 121).

540

Since the late 1980s, glycopeptide-resistant coagulase-negative staphylococci [49-5 1 and refs. therein] and enterococci [52 and refs. therein] have been isolated in clinical set- tings on several occasions. Resistance is considered to be an acquired property; the inci- dence of glycopeptide resistant strains is still relatively low. Recently, glycopeptide resis- tant E. faecium were also found in environmental samples such as waste water of sewage treatment plants, demonstrating the spread of the resistant determinants [53]. In none of these cases was inactivation of the antibiotic reported.

In enterococci, three classes of glycopeptide resistance VanA, VanB and VanC have been distinguished phenotypically on the basis of the resistance level, cross-resistance between vancomycin and teicoplanin, and whether resistance is inducible (Table I ) [44,54]. Class C strains, represented by E. gallinarum, show low level intrinsic, constitu- tive resistance to vancomycin and not to teicoplanin [4345]. E. faecium and E. faecalis strains with an acquired, inducible resistance are either of class A or class B. The high- level resistant class A phenotype includes resistance to teicoplanin; only this resistance has been demonstrated to be transferable; class B strains achieve only a moderate resis- tance level to vancomycin [55-61]. The incidence of class B strains may be underesti- mated because this phenotype is difficult to detect by disk diffusion assays [61].

5. Biochemical and genetic basis of glycopeptide resistance in enterococci

5.1. Class A vancomycin resistance

5.1.1. Transfer of vancomycin resistance determinants The presence of plasmids transferring class A resistance has been documented in several E. jaecium strains [55,56,62], whereas transfer was unrelated to plasmid DNA in other strains of E. faecium or E. faecalis [57,58,60]. Four of the plasmids were distinct in terms of size, restriction profiles, other resistance determinants and transfer properties, but ap- parently related at the DNA sequence level [55,56]. The host range differed considerably, one plasmid being readily transferred into a variety of Gram-positive species including

'I'AULL I Glycopeptidc rcbistance in enterococci

Relevant property vanA vanH vanC

Resistance MIC @g/ml)

Vancomycin 'I'eicoplanin

Transferability lnducihility (van/tei) Resistance protein (kDa) Species

Acquired

264 216

+I(+) 40 E faecrum E faecalis

+

Acquired Intrinsic

16-32 8-16 0.5 0.5

+ I- -I- 39 5 nd E ,faecrum E. gallinarum E, .faecalr.s

54 1

E. faecium, E. jaeca lis, Streptococcus pyogenes, Streptococcus lactis, and Listeria monocytogenes. Resistance was inducible in the transconjugants to varying degrees. Attempts to transfer this plasmid to B. subtilis or S. aureus failed. The plasmid pIPSl6 was not self-transferable but could be introduced into a S. sanguis strain via transfor- mation. A DNA fragment from pIP8 16 carrying a resistance determinant was cloned into a Gram-positive/Gram-negative shuttle vector [63]. When introduced into E. faecalis and B. thuringiensis (but not in E. coli or B. subtilis), it also conferred inducible glycopeptide resistance.

Sequence homology between DNA of a large number of highly resistance E. faecium isolates and a PIPS 16 probe encoding a vancomycin resistance determinant strongly sug- gested that the resistance phenotype is due to dissemination of a gene specifically associ- ated with resistant strains rather than of a bacterial clone or a single plasmid [54]. Typing of a number of strains including environmental samples confirmed the unrelatedness of highly resistant E. faecium [53]. Recently, sequence determination of the regions that flank the vancomycin resistance gene cluster (see below) of plP816 revealed that the van gene cluster in pIP816 is carried by a 10.8 kb transposon designated Tn1546 with similar- ity to transposons of the Tn3 family. Evidence was obtained of elements related to Tn1546 in other highly resistant enterococci, and further results indicated that transposi- tion plays a role in dissemination of the vancomycin resistance gene cluster [64]. The origin of the van resistance transposon is still unknown. So far, the van genes have been detected only in enterococci, but transposition of the vancomycin transposon into a broad range plasmid may change the situation dramatically. Astoundingly, experiments have been carried out under laboratory conditions showing that indeed conjugative transfer of glycopeptide resistance from E. faecalis to S. aureus is possible [65].

5.1.2. The role of VanA and VanH In several strains, subinhibitory concentrations of vancomycin induced the synthesis of a membrane-associated protein that appeared to be required for expression of vancomycin resistance [57,58,60]. The vanA gene encoding the inducible resistance protein was iden- tified on the 34 kb plasmid pIP816 of E. faecium BM4147 using a two-stage cloning pro- cedure based on the use of a Gram-positive/Gram-negative shuttle vector [66]. First, EcoRl restriction fragments of plP816 were cloned and transformed into E. coli, and characterized by restriction profiles. Recombinant plasmids were then cloned into an E. coli strain containing a conjugative plasmid which could mobilize the shuttle vector derivatives. These constructs served as donors in mating experiments with different Gram-positive species which were tested for expression of glycopeptide resistance [63]. A 1.7-kb derivative encoded a 40-kDa protein in E. coli designated VanA. The deduced amino acid sequence of the vanA gene revealed a high degree of homology to D-Ala-D- Ala ligases of E. coli and S. Wphimurium, and vanA could transcomplement an E. coli mutant with a thermosensitive D-Ala-D-Ala ligase activity [66]. The VanA protein puri- fied from E. coli contained ligase activity of altered substrate specificity. It preferentially catalyzed the ester bond formation between D-Ala and other residues such as D-2-hydroxy acids [67].

A gene located upstream from vanA encodes a dehydrogenase VanH that has D-keto acid reductase activity, and can provide the D-2-hydroxyacid substrate of VanA [68,69].

542

VanH shows some sequence similarity with D-lactate dehydrogenases found in many lac- tobacilli and leuconostoc, i.e. bacteria with intrinsic vancomycin resistance. When D-lac- tate was added to the culture medium of a strain with an insertionally inactivated vanH gene, it restored the vancomycin resistance phenotype, suggesting that the depsipeptide D- alanyl-D-lactate is the in vivo product of VanA activity [70]. A novel murein precursor was found after induction of vancomycin resistance [71]. Analysis of the structure of murein precursors confirmed that D-laCtate is the substrate of VanA in vivo [72-741.

5.1.3. The van gene cluster In addition to vanA and vanH, three other genes contribute to vancomycin resistance. This five gene cluster appears to be sufficient for murein synthesis in the presence of van- comycin [75]. The distal part includes vanA, vanH and a third gene vunY of unknown function. These genes are regulated at the transcriptional level by a two-component regu- latory system vans-vanR which is located in the proximal part of the cluster. These two proteins are homologous to the two families of signal-transducer/response-regulator pro- teins (for review, see [76]) and are probably involved in transmitting a signal in response to the presence of vancomycin outside the membrane [75]. Insertional inactivation of the regulator component VanR suppressed vancomycin resistance completely [75,77].

A sixth gene vanY which is located downstream of the vanY gene was not required for vancomycin resistance in the genetic construct used, although it is inducible in response to the addition of glycopeptide antibiotics [78]. In addition to release of D-Ala, VanY also hydrolyzed muropeptide terminating with D-lactate, i.e. the precursor synthesized in vancomycin-induced cells [79]. It thus corresponds to the inducible D,D-carboxypeptidase described earlier in other strains [80,8 11 . No similarity to known D,D-carboxypeptidases was detectable at the sequence level [78], and the enzyme did not display transpeptidase or P-lactamase activity. Its physiological role remains to be clarified.

S.2. Class B and class C vancomycin resistance

VanB strains produce a 39.5 kDa membrane protein termed VanB [59] which is immu- nologically unrelated to the 39-40 kDa protein described in class A strains [46,82]. Internal fragments of the vanB genes of an E. faecalis and E. faecium strain, respectively, could be amplified using degenerate oligonucleotides that had been shown to prime am- plification of the vanA gene and other D-Ala-D-Ala ligases [83]. Comparisons of the E. faecalis sequence confirmed that VanB was highly similar to VanA (77% identity) and to the other ligases tested (<40%); similarities of the N-terminal sequence of VanB to D- Ala-D-Ala ligases had been noted before [82]. VanB related sequences were detected by hybridization in a number of E. faecium and E. faecalis strains displaying the vanB phe- notype but with different vancomycin resistance levels, indicating that a single class of resistance determinants is represented in class B strains [84]. Consistent with the pro- posed role of the VanB protein are reports on murein precursor analyses of a high and a low vancomycin-resistant strain that demonstrate the appearance of a new peak after van- comycin induction [71]. An inducible carboxypeptidase activity was also found in class B strains [81], but its role in the glycopeptide resistance mechanism is still unknown.

543

The vanC gene of E. gallinarum was cloned with the help of the same pair of oligonu- cleotides used for identification of the vanB gene fragment; it encodes a D-Ala-D-Ala li- gase related protein [85]. The vanC probe was specific for E. gallinarum; no homology with DNA to E. casselijlavus strains was detected which express a glycopeptide resis- tance phenotype very similar to that of E. gallinarum [86].

6. Concluding remarks

In all three classes of glycopeptide resistance, a D-Ala-D-Ala ligase with modified sub- strate specificity appears to be an important component of the resistance mechanism. The relatedness of other genes involved in resistance is not known but may account for the different modes of inducibility. In all cases, resistance is most likely achieved by the syn- thesis of a novel murein subunit which will not complex with glycopeptides, allowing maturation of the murein network in the presence of those antibiotics. The impact of the change in murein composition on other enzymes acting on the murein network, especially penicillin-binding proteins (PBPs), is not known in the induced resistance phenotype, nor has the specificity of PBPs in intrinsically resistant species been analyzed. PBPs can in- teract with Ac,-L-LyS-D-Ala-D-Lac and other esters and thioesters [87,88], and reaction constants of the PBPs are probably as diverse as with /?-lactams. Different PBPs may be responsible for transpeptidation of muropentapetide and the D-lactate-containing homo- logue, respectively. Such a change in the role of individual PBPs due to glycopeptide treatment would also result in a shift in /?-lactam sensitivity as has been proposed by Al- Obeid et al. [89] to account for the striking synergy between glycopeptides and certain /?- lactam antibiotics of low- and high-level vancomycin resistant enterococci [44,90].

Acknowledgements

I thank P. Courvalin and L. Gutmann for providing manuscripts prior to publication, M. Achtmann for critical reading of the manuscript, and F. Parenti for contributing Fig. 1.

References

1.

2. 3. 4. 5 . 6. 7. 8.

9 . 10. I I . 12.

McCormick, M.H., Stark, W.M., Pittenger, G.E., Pittenger, R.C. and McGuire, J.M. (1956) Antibiot. Ann. 1955-1956.606-611. Griffith, R.S. (1981) Rev. Infect. Dis. 3, S200-S204. Ziegler, D.W.. Wolfe, R.N. and McGuire, J.M. (1956) Antibiot. Ann. 1955-1956, 612-618. Griffith, R.S. and Peck, Jr. , F.B. (1956) Antibiot. Ann. 1955-1956, 6 1 9 6 2 2 . Riley, Jr. , H.D. (1970) Med. Clin. N. Am. 54, 1277-1289. Farber, B.F. and Moellering, R.C. (1983) Antimicrob. Agents Chemother. 23, 138-141. Barna, J.C J . and Williams, D.H. (1984) Annu. Rev. Microbiol. 38, 339-357. Grundy, W.E., Sinclair, A.C., Theriault, R.J., Goldstein, A.W., Rickher, C.J., Warren, Jr. , H.B., Oliver, T.J. and Sylvester, J.C. (1957) Antibiot. Ann. 1956-1957, 6 8 7 4 9 2 . Coller, B.S. and Gralnick, H.R. (1977) J . Clin. Invest. 60,301-312. Howard, M.A. and Firkin, B.G. (1971) Thromb. Diath. Haemorrh. 26, 362-369. Parenti, F . , Beretta, G., Berti, M. and Arioli, V. (1978) J . Antibiot. 31,276-281. Jeffs, P.W. and Nisbet, L.J. (1988) in: P. Actor, L. Daneo-Moore, M. Higgins, M.R.J. Salton and G.D.

544

Shockman (Eds.). Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function, American Society for Microbiology, Washington, DC, pp. 509-530.

13. Parenti, F. (1986) J . Hosp. Infect. 2 (Suppl. A), 79-83. 14. Somma, S., Gastdldo, 1,. and Corti, A. (1984) Antimicrob. Agents Chemother. 26. 91 7-923. 15. Perkins, H.R. (1987) in: D.J Tipper (Ed ), Antibiotic Inhibitors of Bacterial Cell Wall Biosynthesis,

Pergamon Press, New York, pp. I 15- 132. 16. Jordan, D.C. (1961) Biochcm. Riophys. Res. Commun. 6 , 167-1 70. 17. Reynolds, P.E. (1961) Biochim Biophys. Acta 52,403405. 18. Chatter-jee. A . N . and Perkins, 1I.R. (1966) Biochem. Biophys. Res. Commun. 24, 489494. 19. Best, G K and Durham, N.N. (1965) Arch. Biochem. Biophys. 1 1 I , 685-691. 20. Sinha, R.K. and Neuhaus. F.C. (1968) J. Ractcriol. 96, 374-382. 21. Perkins, H.R. and Nieto, M. (1970) Biochem. J . 116, 83-92. 22. Nieto, M.. Perkins, H.R. and Reynolds. P.E. (1972) Biochem. J. 126, 139-149. 23. Perkins, H.R. (1969) Riochem. J. I I I , 195-205. 24. Nieto, M. and Perkins. H.R. (1971) Biochem. J. 123, 773-787. 25. Nieto, M. and Perkins, H.R. (1971) Biochcm. J . 123,789-803. 26. Nieto, M. and Perkins, H.R. (1971) Biocheni. 1. 124, 845-852. 27. Shcldrick. G M., Jones. P.G., Kennard, O., Williams, D.11. and Smith, G.A. (1978) Nature 271,

28. Ward, J.B. (1974) Biochem J. 141,227-241, 29. Johnston, L.S. and Neuhaus, F.C. (1975) Biochemistry 14.2754-2760. 30. I.eyh-Bouille. M.. Ghuysen. J -M., Nieto. M., Perkins, H K., Schleifer, K.11. and Kandler. 0. (1970)

Biochemistry 9,297 1-2975. 31. Izaki, K. and Strominger, J.L. (1968) J. Biol. Chem. 243. 3193-3201. 32. Rogdanovsky, D., Hricas, E. and DcxIC. P. (1969) C.R. Acad. Sci.. Series D 269. 390-393. 33. Reynolds. P.E. (1989) Eur. J . Clin. Microbiol. Infect. Dis. 8, 943-950. 34. Anderson, J.S., Matsuhashi. M., Haskin. M.S. and Stromingcr, J.L. (1965) Proc. Natl. Acad. Sci. USA

35. Anderson, J.S., Matsuhashi. M., Haskin, M.S. and Strominger. J.L. (1967) J. Hiol. Chem. 242,

36. 37. 38. 39. 40. 41, 42. 43. 44. 45.

46.

47. 48. 49.

50. 5 I . 52.

53 54.

223-225.

53,881-889.

3 180-3 190. van I-leijenoort, Y., Derrien, M. and van tleijenoort, 1. (1978) FEBS Lett. 89, 141-144. I.erner, P.I. (1974) Antimicrob. Agents Cheniother. 5 . 302-309. Baycr, A S , Chow, A.W., Belts, D. and Guze, L.B. (1 978) Am. J. Med. 64, 808-8 13. Vescovo, M.. Morelli, L. and R o t t m i , V. (1982) Appl. Environ. Microbiol. 43, 50-56 Orberg, P K. and Sandine. W.E. (1984) Appl. Environ. Microbiol. 48, I129--1133. Colnian, G. and Efstratiou. A. (1987) J . Hosp. Infcct. 10. 1-3. Buu-Hoi, A,. Brangcr, C. and Acar, J.F. (1985) Antimicrob. Agents Chcmother. 28.458460. Swenson. J.M., I l i l l . R.C. and Thornsberry, C. (1989) J. Clin Microbiol. 27,2140-2142. Shlaes, D.M.. Etter. L. and Gutmann, L. (1991) Antimicrob. Agents Chemother. 35, 776-779. Vincent. S., Knight, R G , Green. M., Sahm. D F. and Shlaes. D.M. (1991) J . Clin. Microbiol. 29, 2335-2337. Nicas, T.I., Cole. C.T.. Preston, D.A , Schabcl, A.A. and Nagarajan, II. (1989) Antimicrob. Agents Chemother. 33. 1477-1481. Watanakunakorn, C. (1981) Rev. Infcct. Dis. 3, S2IO-S215. Shlaes, D M., Marino. J . and Jacobs, M.K. (1984) Antimicrob. Agents Chemother. 25, 527-528. Goldstein, I:.. Cotitrot, A,. Sieffer, A. and Acar, J.F. (1990) Antimicrob. Agents Chemother. 34. 899-900. Moore, E.P. and Speller. D.C.E. (1988) J. Antimicrob. Chemother. 21, 4 17424. Schwalbe, R.S., Staplcton. J .T. and Gilligan, P.11. (1987) N. Gng. J. Mcd. 316, 927-931. 1,eclercq. R., Dutka-Malen, S. , hisson-Noel. A,, Molinas. C.. Derlot. E.. Arthur. M.. Ouval, J . and Courvalin. P. (1992) Clin. Infect. Ilis. 15, 495-501. Klarc, I. . Ileicr, 11.. Claus. H and Witte. W. (1992) FEMS Microbiol Lett. 106. 23-30 Dutka-Malcn. S.. I.eclercq, R., Coutnnt, V.. Duval, J . and Courvalin, P (1990) Antimicrob. Agcnls Chemothcr. 34. 1875-1 879.

545

55. 56.

57.

58.

59.

60.

61

62. 63.

64. 65. 66. 67.

68. 69

70.

71.

72. 73.

74. 75. 76.

77. 78. 79.

80. 81.

82.

83 84 85 86

87. 88. 89.

90

Leclercq, R., Derlot, E., Duval, J . and Courvalin, P. (1 988) N . Engl. J. Med. 3 19, 157-1 6 1. Leclercq. R , Dcrlot, E., Weher, M., Duval, J. and Courvalin, 1’. (1988) Antimicrob. Agents Chemother. 33.10-15. Nicas, T.I., Wu, C.Y.E., llobbs. Jr., J .N. , Preston, D.A and Allen, N.E. (1989) Antimicrob. Agents Chemother. 33, 1121-1 124. Shlaes, D.M , Bouvct, A., Devine, C., Shlaes, J.H., Al-Obeid, S. and Williamson, R. (1989) Antimicrob. Agents Chemother. 33. 198-203. Williamson, R.. Al-Obeid. S , Shlaes, J.H., Goldstein, F.W. and Shlaes, D.M (1989) J. Infect. Dis., 159,

Shlaes, D.M., Al-Obeid. S., Shlaes, J.11.. Biosivon, A. and Williamson, R. (1989) J . Antimicrob. Chemother. 23, 503-508. Sahm, D.F., Kissingcr, J., Ciilmore. M S.. Murray, P.R., Mulder, R., Solliday, J. and Blarke, B. (1989) Antiniicrob. Agents Chemother. 33, 1588-1 591 Handwerger, S., I’ucci, M.J. and Kolokathis. A. (1990) Antimicrob. Agents Chemother. 34,358-360. Brisson-Nod, A.. Dutka-Malen, S., Molinas, C., Leclercq, R. and Courvalin, P. (1990) Antimicrob. Agents Chemother. 34.924-927. Arthur, M.. Molinas, C., Dcpardieu, I . and Courvalin, P. (1993) J. Bacteriol. 175. 117-127. Noble, W . C , Virani, Z. and Cree, R.G.A. (1992) FEMS Microbiol. Lett. 93, 195-198. Dutka-Malen. S., Molinas, C , Arthur, M. and Courvalin, P. (1990) Mol. Gen. Genet. 224, 364-372. Bugg, T.D.H.. Dutka-Malen, S , Arthur. M., Courvalin, P. and Walsh, C.T. (1991) Biochemistry 30,

Arthur, M., Molinas, C., Dutka-Malen, S. and Courvalin, P. (1991) Gene 103, 133-134. Bugg, T.D.H., Wright, G.D., Dutka-Malen, S., Arthur, M., Courvalin P. and Walsh, C.T. (1992) Biochemistry 30, 10408-10415. Arthur, M., Molinas, C., Bugg, T.D.H., Wright, G.D., Walsh, C.T. and Courvalin, P. (1992) Antimicroh Agents Chemother. 36, 867-869. Billot-Klein, D., Gutmann. L., Collatz, E. and van Heijenoort, J . (1992) Antimicrob. Agents Chemother.

Ilandwerger, S., Pucci, M.J.. Volk, K.J . , Liu, J and Lee, M.S. (1992) J. Bacteriol. 174, 5982-5984. Allen. N.E., I-lohbs, Jr., J N., Richardson, J.M. and Riggins, R.M. (1992) FEMS Miocrobiol. Lett. 98. 109-1 16. Messcr, J . and Reynolds, P.E. (1992) FEMS Microbiol. Lett. 94, 195-200. Arthur, M., Molinas, C. and Courvalin, P. (1992) J . Bacteriol. 174, 2582-2591. Surctte, M.G. and Stock, J.B. (1994) in: J -M. Ghuysen and R. Hakenbeck (Eds.). Bacterial Cell Wall, New Comprehensive Biochemistry, Vol. 27, Elsevier, Amsterdam, pp. 465433. Handwerger. S., Discotto, L., Thanassi, J. and Pucci, M.J. (1992) FEMS Microbiol. Lett 92, 11-14. Arthur, M., Molinas. C. and Courvalin, P. (1992) Gene 120, I 1 1-1 14. Wright, G.D.. Molinas, C., Arthur, M., Courvalin, P. and Walsh, C.T. (1992) Antimicrob. Agents Chemother. 36, 1514-1518. Al-Oheid, S. , Collatz, E. and Gutmann, L. (1990) Antimicrob. Agents Chemother. 34,252-256. Gutmann, L., Billot-Klein, D., Al-Obeid, S., Klare, I . , Francoual, S., Collatz, E. and van Heijenoort, J. (1992) Antimicrob. Agents Chemother. 36, 77-80. Al-Ohcid. S.. Ciutmann, L.. Shlaes, D.M., Williamson, R. and Collatz, E. (1990) FEMS Microbiol. Lett.

Evers, S ~ Sahrn, D.1:. and Courvalin, P. (1993) Gene 124, 143-144. Quintiliaill. K., Evers, S. and Courvalm, P. (1993) J. Infccl. Dis 167, 1220-1223. Dutka-Malen. S., Molinas, C., Arthur, M. and Courvalin. P. (1992) Gene 112, 53-58, Lcclercq. I<.. Dutka-Malen, S.. Duval, J. and Courvalin, P. (1992) Antimicroh Agents Chcmother. 36, 2005-2008 Rasmusscn, J .R . and Strominger, J.L. (1978) I’roc. Natl h a d . Sci. USA 75. 84-88. Adam. M ~ Camblon. C., Christiaens, 1.. and Frere, J.-M. (1990) Biochcm. J . 270, 525-529. Al-Ohcid. S., I3illot-Klein. D.. van Heijenoort. J , Collatz, E and Gutmann, L. (1992) FEMS Microbiol.

I,eclercq, R. , Bingen, E.. Su. Q . H . , Lambert-Zechovski, N., Courvalin, P. and Duval. J. (1991) Antimicrob. Agents Chemother. 35.92-98.

1095-1 104.

20 17-202 I .

36, 1487-1490.

70. 101-106.

Lett. 91. 79-84.