Glycopeptide resistance in enterococci

R E V I E W S

Glycopeptide resistance in enterococci Michel Arthur, Peter Reynolds and Patrice Courvalin This review is dedicated to the memory of Sandra Handwerger.

Glycopeptide resistance in enterococci results from the production of peptidoglycan precursors with low affinity for these antibiotics. The mobility of the resistance genes by transposition and conjugation and the ability of the resistance proteins to interfere with synthesis of normal precursors in different hosts indicate that dissemination into other bacterial species should be anticipated.

Synthesis of the target and basis for resistance The D-Ala residues that interact with glycopeptides are inlycopeptide antibiotics corporated into peptidoglycan such as vancomycin and precursors as a dipeptide synteicoplanin inhibit pepthesized by D-alanine:D-alanine tidoglycan synthesis and are ligases e (Fig. 3). The substrate used to treat severe infections specificity of ligases determines caused by Gram-positive bacthe nature of the residues interia. Glycopeptides do not corporated at the carboxyinteract with cell wall bioterminal end of peptidoglycan M. Arthur* and P. Courvalin are in the Unit8 des synthetic enzymes but form precursors and the possibility Agents AntibactSriens, Centre National de la complexes with peptidoglycan for subsequent interaction with Recherche Scientifique EP J0058, lnstitut Pasteur, precursors and prevent their 75724 Paris, Cedex 15, France; P. Reynoldsis in the glycopeptides at the cell surincorporation into the wall ~. face (Table 1). Dept of Biochemistry, University of Cambridge, Cambridge, UK CB2 IQW, but temporarily at the Consequently, the activity of Inducible resistance to high Institut Pasteur. *tel: +33 1 40 61 35 88, glycopeptides is not deterlevels of vancomycin and teicofax: +33 1 45 68 83 19, mined by the affinity of target planin (VanA phenotype) in e-mail: Michel.Arthur@pasteur.# enzymes for the molecules but enterococci is mediated by by the substrate specificity of Tn1546 or closely related elthe enzymes that determine the structure of peptido- ements 3. The transposon encodes a dehydrogenase glycan precursors. Acquisition of glycopeptide resist- (VanH) that reduces pyruvate to D-lactate (D-Lac), ance results from the transfer of mobile genetic elements and a ligase of broad substrate specificity (VanA) that that encode enzymes for synthesis of low-affinity catalyses the formation of an ester bond between D-Ala precursors and elimination of the high-affinity pre- and D-Lac4,s (Table 1). The resulting D-Ala-D-Lac cursors normally produced by the host. We shall first depsipeptide replaces the dipeptide D-AIa-D-AIa in the review the mode of action and the mechanism of re- pathway of peptidoglycan synthesis (Fig. 3). The subsistance, as exemplified by VanA-type glycopeptide stitution eliminates a hydrogen bond critical for antiresistance mediated by transposon Tn1546 which is biotic bindings (Fig. 1 and Table 1). The normal comwidely spread in enterococci. The diversity, origin plement of cell wall biosynthetic enzymes encoded by and evolution of glycopeptide resistance will then be the host chromosome tolerates the substitution of discussed. D-Lac for D-AIa6. However, penicillin-binding proteins (PBPs) that catalyse the final transpeptidation reacTarget of glycopeptides tions in the assembly pathway may display different Inhibition of cell wall synthesis by glycopeptides results activity for D-Ala- and D-Lac-terminating precursors from the formation of complexes between the anti- which would account for modification of the extent biotics and the carboxy-terminal D-alanine (D-AIa) of cross-linking7,8. Induction of glycopeptide resistance residues of peptidoglycan precursors ~(Fig. 1). Glyco- is associated with an increase in susceptibility to 13peptides do not penetrate into the cytoplasm and inter- lactams in certain strains of enterococci9. It has been action with the target can only take place after trans- proposed that PBP5 cannot perform cross-linking of location of the precursors bound to the undecaprenol D-Lac-terminating precursors 9. Consequently, other lipid carrier to the outer surface of the cytoplasmic PBPs would be required for cell wall synthesis leading membrane. Binding of the drugs at this step blocks to an increase in the susceptibility to ]3-lactams since incorporation of disaccharide pentapeptide subunits these PBPs are inhibited by lower concentration of into nascent peptidoglycan by transglycosylation ]3-1actams than PBP5 (Ref. 9). and leads to accumulation of cytoplasmic precursors (Fig. 2a). Binding of glycopeptides to peptide stems Elimination of D-Ala-containing precursors ending in D-Ala-D-Ala, if present in nascent peptido- Coproduction of precursors ending in D-Ala or D-Lac glycan, is also expected to inhibit the reactions does not result in resistance 1°. Under these conditions, catalysed by D,D-transpeptidases and D,D-carboxy- binding of glycopeptides to D-Ala-D-Ala-containing peptidases (Fig. 2b). precursors bound to the lipid carrier at the external

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Copyright © 1996 Elsevier Sciencc Ltd. All rights reserved. 0966 842X/96/$15.{}0

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Fig. 1. Structure of the glycopeptide: peptidyI-D-Ala-B-Ala complex. (a) Binding of a glycopeptide to the D-Ala-o-Ala extremity of peptidoglycan precursors involves five hydrogen interactions indicated by dashed lines. The NH group of the amide D-Ala-o-Ala linkage that is substituted by an oxygen in D-Ala-D-Lac-containing peptidoglycan precursors is indicated in red. The methyl (CHs) side chain of the carboxyterminal D-Ala that is substituted by a hydroxymethyl (CH2OH) side chain in precursors ending in D-Ala-D-Ser is indicated in blue. (b) Space-filling models showing the bulky glycopeptide molecule bound to the precursor. Formation of the complex is thought to inhibit transglycosylases by steric hindrance, and penicillin binding proteins (PBPs) by directly preventing interaction with the carboxy-terminal D-Ala residues. Adapted, with permission, from Ref. 41.

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Fig. 2. (a) Reaction catalysed by the transglycosylases at the outer surface of the cytoplasmic membrane. The new subunit is depicted in red, the existing nascent peptidoglycan in blue. (b) Reactions catalysed by the penicillin-binding proteins (PBPs). The first step involves nucleophilic attack of the carboxyl group of the penultimate D-Ala residue by the hydroxyl group of the active site serine, leading to formation of the acyl enzyme and release of the carboxy-terminal D-Ala. Note that acyl enzymes derived from D-Ala- and D-Lac-ending precursors are identical. If the PBP has O,D-carboxypeptidase activity, the awl enzyme is hydrolysed and tetrapeptide is released. If the PBP has transpeptidase activity, there is nucleophilic attack of the acyl enzyme by the NH2 group of the amino-terminal amino acid in the interpeptide bridge (X) leading to the formation of a peptide bond. The latter reaction results in peptidoglycan cross-linking. The amino acids of the interpeptide bridge are linked to the E-NH2 group of lysine and differ between species 4°. The interpeptide bridge of Enterococcus faecalis and E. faecium consists of one D-asparagine or two L-Aia residues, respectivelyTM.

transcription of an operon encoding VanH, VanA and VanX. Several phosphotransfer reactions are catalysed in vitro by purified VanR and VanS, including ATPdependent autophosphorylation of a histidine residue in VanS, transfer of the phosphate group to an aspartate residue of VanR and spontaneous hydrolysis of phospho-VanR TM. The latter reaction is stimulated by Vans TM. Because phosphorylation increases the affinity of VanR for the DNA of the promoter of the vanHAX operon, it is likely that the actual transcriptional activator is the phosphorylated form of VanR, as found for related two-component regulatory systems ~s. This would imply that the VanS sensor displays increased kinase and/or reduced phosphatase activity in response to the presence of glycopeptides in the culture medium. The nature of the signal recognized by VanS remains unknown. The fact that moenomycin, a transglycosylase inhibitor that is structurally unrelated to glycopeptides, also induces glycopeptide resistance suggests that accumulation of peptidoglycan precursors rather than direct interaction with the drug could be responsible for induction .6.

TRENDS

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Relationship between the vanA and vanB gene clusters

Acquired resistance to glycopeptides in enterococci is mediated by two classes of related gene clusters that confer inducible resistance to high levels of vancomycin and teicoplanin (vanA) or inducible resistance to various levels of vancomycin (vanB) (Fig. 4). In the latter case, the strains remain susceptible to teicoplanin since this antibiotic is not an inducer ~°'lr. Both types of gene clusters mediate glycopeptide resistance by synthesis of peptidoglycan precursors ending in D-Lac ~8,m. Comparison of the vanA and vanB gene clusters indicates a high level of amino acid sequence identity in the dehydrogenases (VanH and VanHB), ligases (VanA and VanB) and D,D-dipeptidases lr {VanX and VanXB; Fig. 4). The VanA and VanB ligases display similar catalytic properties 2° (Table 1 ). In contrast, the VanRVanS and VanRB-VanSB two-component regulatory systems are only distantly related and the putative sensor domains of Vans and VanSb are not related in sequence, raising the possibility that these proteins sense the presence of glycopeptides by different mechanisms.

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(~

Penicillin-binding protein ............. Lipid carrier (undecaprenol) Amino acid(s) in interpeptide bridges N-acetylglucosamine N-acetylmuramic acid L-Ala D-Glu L-Lys D-Ala • • D-AlaorD-Lac a ~" _

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Fig. 3. Peptidoglycan synthesis in glycopeptide-resistant enterococci. The D-Ala residues that interact with vancomycin are incorporated into peptidoglycan precursors as a dipeptide synthesized by D-alanine:D-alanine ligases (Ddl). Formation of the complex between glycopeptides and peptidoglycan precursors at the cell surface prevents transglycosylation and leads to the accumulation of cytoplasmic peptidoglycan precursors. VanR and VanS act as a signal transducing system that couples inhibition of peptidoglycan synthesis to induction of the transcription of the vanHAX operon in the cytoplasm. Production of the corresponding enzymes turns on the pathway for synthesis of D-Lac-containing precursors (VanH and VanA) and turns off the pathway for synthesis of D-Ala-ending precursors by elimination of D-Ala-D-Ala (VanX). Analysis of the structure of the wall from resistant Enterococcus faecium and E. faecalis did not reveal any peptide stems ending in D-Lac, indicating that this residue is probably cleaved by the P,D-carboxypeptidases encoded by the host chromosome or by the resistance gene cluster 7,8 (vanYor vanYB). Resistance proteins are indicated in red and D-Lac in green.

The vanA and vanB clusters encode a D,D-carboxypeptidase (VanY or VanYB) but the proteins are not closely related and the genes are located at different positions of the clusters. VanZ (teicoplanin resistance) and VanW (unknown function), encoded respectively by the vanA and vanB clusters, do not seem to have counterparts in the other cluster. These observations and the variations in the base composition within the clusters suggest that genes from different origins have been recruited. Mobility of vanA and vanB gene clusters The vanA gene cluster was originally detected on the nonconjugative transposon Tn1546, which belongs to the Tn3 family 3. VanA-type resistance in clinical isolates of enterococci is mediated by genetic elements closely related to Tn1546 that are generally carried by self-transferable plasmids 2~ and, occasionally, by the host chromosome as part of larger conjugative elements 22. Tn1546-1ike elements are highly conserved except for the presence of insertion sequences that have transposed into intergenic regions not essential for the expression of glycopeptide resistance 22. Conjugal transfer of plasmids that have acquired Tn1546-1ike elements by transposition appears to be responsible for the spread of glycopeptide resistance in enterococci 3. Gene clusters related to vanB are generally carried by large (90-250 kb) elements that are transferable by conjugation from chromosome to chromosome 23. One

of these elements (250 kb) was found to contain the composite transposon Tn1547 (64kb) delineated by insertion sequences belonging to the IS256 family 24. Plasmid-borne vanB-related gene clusters have also been detected in clinical isolates of enterococci2L Intrinsic resistance Enterococci belonging to the species Enterococcus gallinarum, E. casseliflavus and E. flavescens are intrinsically resistant to low levels of vancomycin but remain susceptible to teicoplanin. Resistance results from t h e production of peptidoglycan precursors ending in D-serine 1~26 (Table 1). Substitution of D-Ala by D-Ser at the carboxyl terminus of peptidoglycan precursor analogues lowers the affinity of the precursors for vancomycin with a relatively smal] change in the affinity for teicoplanin z7. Intrinsically resistant enterococci appear to produce two types of ligases that synthesize D-Ala-D-Ser and D-AIa-D-AIa2L Production of peptidoglycan precursors ending in D-Ser was reported to be inducible by vancomycin at least in certain strains 18. Intrinsic resistance to high leve]s of vancomycin and teicoplanin due to production of peptidoglycan precursors ending in D-Lac is frequent in lactic acid bacteria including certain species belonging to the genera Lactobacillus, Leuconostoc and PediococcuslS.2L Comparison of the amino acid sequences of D-alanine: D-lactate and D-alanine:D-serine ligases from these organisms has not revealed any close relationship with the VanA or VanB ligases 3°.

Table 1. Substrate specificity of ligases and affinity of glycopeptides for the corresponding precursors Enzyme

Substrate (carboxy-terminus)

Km(mM)

I~/Km (M-iS-1)

1.2

14000

Decrease in affinity

Ddl (E. coil)

D-Ala X=NH2 Y=CH3 (D-Lac is not a substrate)

VanA

D-Ala D-Lac D-HBut

X=NH 2 X=OH X=OH

Y=CH 3 Y=CH3 Y=CH2CH3

38 7.1 0.6

129 220 3000

Not applicable >lO00-fold No d e t e c t a b l e binding

VanB

D-Ala D-Lac D-HBut

X=NH 2 X=OH X=OH

Y=CH 3 Y=CH 3 Y=CH2CH 3

34 11 3.0

120 41 84

Not applicable >lO00-fold No d e t e c t a b l e binding

VanC

D-Ser

X=NH 2

Y=CH20H

Not d e t e r m i n e d

Not applicable

Not d e t e r m i n e d

7-fold

The ligases catalyse peptide or ester bond formation between D-Ala and D-amino acids (X=NH2) or D-2-hydroxy acids (X=OH), respectively (D-HBut, D-2-hydroxybutyrate). Differences in specificity also involve the side chain (Y) of the substrate. These differences account for resistance, since substitution of NH in a peptide bond by 0 in an ester bond prevents formation of one of the five hydrogen interactions, whereas bulky side chains destabilize the complex 5,27.38.

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41

45

45

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29

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47

vanYB vanW

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51

49

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(b) Regulation

Synthesis of D-Ala-D-Lac

Hydrolysis of precursors

Response regulator (34%) (VanR or VanRB)

Dehydrogenase (67%) (VanH or VanHB)

D,D-Dipeptidase (VanX or VanXB)

Sensor (VanS or VanSB)

Ligase (VanA or VanB)

(23%)

(76%)

(71%)

D,D-Carboxypeptidase (30%) (VanY or VanYa)

Fig. 4. Comparison of the vanA and vanB gene clusters. (a) Transposon Tn1546 (10851 bp) carries the vanA gene cluster and is delineated by 38-bp imperfect inverted repeats represented by arrowheads (IRL and IRR).Transposon Tn1547 (65 kb) carries the vanBgene cluster and is delineated by insertion sequences IS256-1ike and IS16 in direct orientation (represented by boxes). Open arrows indicate open reading frames (ORF1, transposase; ORF2, resolvase). The percentage of guanosine plus cytosine is indicated below each open reading frame. (b) Amino acid identity (%) between homologous proteins encoded by the vanA and the vanB gene clusters.

The threat of dissemination of glycopeptide resistance

chickens is also

Glycopeptides, alone or in c o m b i n a t i o n w i t h amino-

glycosides, often constitute the only therapeutic treatment for multiresistant strains of staphylococci, streptococci and enter•cocci 3~,s2. The emergence and dissemination of high-level resistance to glycopeptides in enter•cocci in the past decade has resulted in clinical isolates resistant to all antibiotics of proven efficacy33,34. Although enter•cocci are not highly pathogenic, the incidence of glycopeptide resistance among clinical isolates is increasing and the enter•Questions for future research • How do the VanS and VanSB sensors recognize the presence of glycopeptides? *What is the origin of the vanA and vanB gene clusters? ,Why has resistance to glycopeptides by enzymatic detoxification never developed? • Is there a selective advantage associated with the production of D-Lac-ending peptidoglycan precursors in addition to glycopeptide resistance and why do lactobacilli, leuconostocs and pediococci use this pathway? • What is the mechanism of VanZ-mediated teicoplanin resistance? *Are the resistance proteins encoded by the vanA and vanB clusters potential targets for new antibiotics? • How do glycopeptide-producing organisms protect themselves against the action of these molecules?

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cocci have become important as nosocomial pathogens and as a reservoir of resistance genes 31,32. This is associated with increased difficulty in treating enterococcal infections and a risk of dissemination of glycopeptide resistance to more pathogenic bacteria such as staphylococci and streptococci (Table 2). There is no barrier to heterospecific expression or transfer of glycopeptide resistance genes among these bacteria in laboratory conditions, and the mobility of the v a n A and v a n B gene clusters by conjugation and transposition is expected to facilitate such transfer in natural conditions 3s,~6. The threat of dissemination of glycopeptide resistance in human pathogens has stimulated many debates on measures intended to limit the selective pressure resulting from the unnecessary use of glycopeptides in clinical medicine, especially oral administration of the drugs, since glycopeptides are not found in the intestinal tract following parenteral administration 3r. The use of the glycopeptide antibiotic avoparcin as a growth promoter for pigs and a cause for concern (Table 2).

Acknowledgements P.R. was supported by a Fellowshipfromthe Ciba FellowshipTrust. References 1 Reynolds,P.E. (1989) Eur. J. Clin. Microbiol. Infect. Dis. 8, 943-950 2 Wright,G.D. and Walsh,C.T. (1992) Acc. Chem. Res. 25, 468-4.73 3 Arthur,M. et al. (1993)J. Bacteriol. 175,117-127 4 Dutka-Malen,S. et al. (1990)Mol. Gen. Genet. 224, 364-372 5 Bugg,T.D.H. et al. (1991) Biochemistry 30, 10408-10415 6 Reynolds,P.E. et al. (1994) MoI. Mitt•biol. 13, 1065-1070 7 Boudewijn,L. et al. (1996) Antimicrob. Agents Chemother. 40, 863-869 8 Bill•t-Klein,D. et al. (1996) Biochem. J. 313, 711-715 9 A1-Obeid,S. et al. (1992) FEMS Microbiol. Lett. 91, 79-84 10 Arthur,M. et al. (1996) Mol. Mitt•biol. 21, 33-44 11 Arthur,M. et al. (1994) Antimicrob. Agents Chemother. 38, 1899-1903 12 Arthur,M. et al. (1995) Gene 154, 87-92 13 Arthur,M. et al. (1992)J. Bacteriol. 174, 2582-2591 14 Wright,G.D. et al. (1993)Biochemistry32, 5057-5063 15 Holman,T.R. et al. (1994) Biochemistry 33, 4625-4631 16 Handwerger,S. and Kolokathis,A. (1990) FEMS Microbiol. Lett. 70, 167-170 17 Evers, S. and Courvalin,P. (1996)J. Bacteriol. 178, 1302-1309 18 Bill•t-Klein,D. et al. (1994)J. Bacteriol. 176, 2398-2405 19 Evers, S. et al. (1994) Gene 140, 97-102 20 Meziane-Cherif,D. etal. (1994)FEBSLett. 354, 140-142 21 Leclercq,R. et al. (1988) New Engl. J. Med. 319, 157-161

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Table 2. Glycopeptide-resistant enterococci: increase in incidence and measures to prevent spread of resistance Source of GRE

Selective pressure likely to result in acquisition

Measures to avoid or reduce incidence of GRE

(a) Intensive use of glycopeptides:

(a) Limit use of glycopeptides to the treatment of severe, life-threatening infections, e.g. MRSA

Hospitals" New York: 1 hospital

1989 1993

1case 361 cases

USA: intensive care

1989

0.4% b

(ii) In neutropenic patients

1993

13.6% b

(iii) For decontamination of gut

(i)

In immunosuppressed patients

Examples of the increased use of vancomycin: (i) 1976-1985; 23-fold increase in sales worldwide (ii) 1982-1995; 23-fold increase in value of sales in USA (iii) 1978-1992; 160-fold increase in use in 1 USA hospital

(iv) For prophylactic use USA: general

1993

8%b

European: general

1993

0.7-1.0% b

(b) Use of long-term, broad-spectrum (b) Improve hygiene to prevent ctonal spread antibiotics

Community (limited number of studies) UK: 2%b (184 people Not from previous treatment with glycopeptides: possibly from eating investigated) meat products Belgium: (i) 3.5% b (636 people investigated) (ii) 27% b (40 people investigate d) Germany: 12%b (100 people investigated) Animal (limited number of studies) 39 Broiler chickens (manure) Chicken carcasses Chickens from retail outlets Pigs Not isolated from cattle or sheep

Meat products 39 Minced pork: 5 of 13 samples from butchers in Germany

Any measure to reduce the reservoir of glycopeptide resistance genes (see above and below)

Inclusion of non-clinical glycopeptides in animal feed. NB During 1993 in Denmark, more than 19000 kg avoparcin was used in animal feed compared with 22 kg vancomycin in human medicine

Avoid use of avoparcin or other glycopeptides (cross-resistance to vancomycin and teicoplanin) as animal food additives for growth promotion. No definitive evidence for transfer of resistance from animal to human strains but use of avoparcin may increase the reservoir of glycopeptide resistance genes

Contamination with faecal material at slaughter

Improvement of hygiene in slaughter houses. Avoidance of contamination of meat products with faecal material

aExamples given of numerous investigations. bPercentages of enterococci examinedthat were resistant to glycopeptides. GRE, glycopeptide-resistantenterococci; MRSA, methicillin-resistant Staphylococcus aureus.

22 Handwerger, S. and Skoble,J. (1995)Antimicrob. Agents Chemother. 39, 2446-2453 23 Quintiliani, R., Jr and Courvalin, P. (1994) FEMS Microbiol. Lett. 119, 359-364 24 Quintiliani, R., Jr and Courvalin, P. (1996) Gene 172, 1-8 25 Woodford, N. et al. (1995)J. Antimicrob. Chemother. 35, 179-184 26 Reynolds, P.E. et al. (1994) Biochem. J. 301, 5-8 27 Billot-Klein,D. et al. (1994) Biochem. J. 304, 1021-1022 28 Navarro, F. and Courvalin, P. (1994) Antimicrob. Agents Chemother. 38, 1788-1793 29 Handwerger, S. et al. (1994)J. Bacteriol. 176, 260-264 30 Evers, S. et al. (1996)]. Mol. EvoI. 42, 706-712 31 Murray, B.E. (1990) Clin. Microbiol. Rev. 3, 46-65

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32 Woodford, N. et al. (1995) Clin. Microbiol. Rev. 8,585-615 33 Handwerger, S. et al. (1992) Clin. Infect. Dis. 14, 655-661 34 Handwerger, S. et al. (1993) Clin. Infect. Dis. 16, 750-755 35 Brisson-No~l,A. et al. (1990) Antimicrob. Agents Chemother. 34, 924-927 36 Noble, W.C. et al. (1992) FEMS Microbiol. Lett. 93,195-198 37 Van der Auwera, P. et aI. (1996) J. Infect. Dis. 173, 1129-1136 38 Nieto, M. and Perkins, H.R. (1971) Biochem. J. 123,789-803 39 Klare, I. et al. (1995) Microb. Drugs Resist. 1,265-272 40 Schleifer,K.H. and Kandler, O. (1972) Bacteriol. Rev. 36, 407-477 41 Jeffs, P.W. and Nisbet, L.J. (1988) in Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (Actor, P. et al., eds), pp. 509-530, American Societyfor Microbiology

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