Abstract

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.

Highlights

  • Therapeutic usage of antibiotics has invariably led to emergence of resistance in target pathogens and members of commensal flora submitted to the same selective pressure

  • We have shown that bypass of the penicillin-binding proteins (PBPs) by the recently described ␤-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldtfm) can lead to high level resistance to glycopeptides and ␤-lactams

  • Complete elimination of D-Ala5, a residue essential for glycopeptide binding, was possible because Ldtfm uses the energy of the L-Lys3-D-Ala4 peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala4-D-Ala5 bond

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Summary

EXPERIMENTAL PROCEDURES

Spontaneous mutants G1 to G9 were obtained from E. faecium D344S [18] by using nine serial selection steps on agar containing increasing concentrations of glycopeptides. Mutants M6 to M9 were obtained from E. faecium M512 [18] by four serial selection steps on agar containing vancomycin at 16 (M6), 64 (M7), and 1,000 (M8) ␮g/ml or teicoplanin at 128 ␮g/ml (M9). Preparation and Analysis of Cytoplasmic Peptidoglycan Precursors—Bacteria were grown to an optical density at 650 nm of 0.35 in 400 ml of brain heart infusion broth and treated with bacitracin (200 ␮g/ml) for 5 min. Goat anti-rabbit IgG coupled to peroxidase (SouthernBiotech, Birmingham, AL) was used as secondary antibody, and Ldtfm was detected by chemiluminescence (ECL kit; Pierce, Amersham Biosciences)

RESULTS
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DISCUSSION
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